Abstract: Bacteriophages (phages) are the viruses of bacteria and biofilms represent a frequent niche for bacteria, where they are embedded in extensive extracellular polymeric substances (EPS) and can be structured into complex microcolonies. As a consequence of the resulting spatial structure and heterogeneity, phage-bacterial interactions within biofilms can be more complicated than those between phages and planktonic bacteria. Towards gaining a better understanding of the biology of phages interacting with biofilms, in this monograph I provide an overview of the subject, divided into five areas: (i) The many facets of phage-biofilm interactive biology including consideration of virus trapping, phage hydrolytic enzymes such as EPS depolymerases, infection of biofilm bacteria, and phage prevalence within natural biofilms. (ii) Prophage-biofilm interactions including in terms of prophage modification of biofilm structure or function along with the potential for biofilms to resist phage attack. (iii) A critical review of the literature concerning phage use as biofilm prevention or eradication agents (phage therapy or phage-mediated biocontrol). (iv) Discussion of phage-biofilm interactions from the perspective of phage-plaque development, since plaque formation might inform phage-biofilm interactions plus may be better understood at microscales than phage interactions with biofilm bacteria. And (v), exploration of issues pertaining to phage penetration into the bacterial microcolonies. I also provide discussions related to phage-biofilm interactions including of Poisson distributions, multiplicity of infection, calculating killing titers (phage numbers killing bacteria), calculating decimal reduction times of bacteria following phage application, and the concept of pseudolysogeny. I stress that key to understanding the dynamics of phage-bacterial interactions within biofilms is a combination of addressing how phages move towards and move away from target bacteria, including in terms of the phage potential to burrow into bacterial microcolonies. I also suggest that that it may not be necessary for phages, even if they specialize on biofilm bacteria, to extensively destroy naturally occurring biofilms in order to prosper.
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(A) Free phage diffusion above biofilm (B) Free-phage encounter with biofilm EPS resulting in local EPS digestion (C) Phage encounter with a susceptible bacterium that is followed by a phage burst and phage progeny release (D) The path of a single phage is traced which encounters a bacterium associated with a nearby microcolony (E) Burst proceeds as previously indicated except that here the path of released phages towards the biofilm surface is indicated (F) Free-phage dissemination into bulk water towards subsequent biofilm acquisition (ii) Enzymes that degrade bacterial capsular material should facilitate phage movement towards individual and so coated bacteria (ii) Multiplicity of infection which is the key variable underlying Poisson distributions as used to predict levels of bacterial survival following phage exposure but which (unfortunately) is erroneously defined in a large number of phage publications (ii) Prophage-biofilm interactions including in terms of prophage modification of biofilm structure or function along with the potential for biofilms to resist phage attack (iii) A critical review of the literature concerning phage use as biofilm prevention or eradication agents (phage therapy or phage-mediated biocontrol) (iii) Enzymes that degrade the bacterial cell wall i (iii) Killing titers which is an old term with new relevance as we seek to kill bacteria using phages (and which is what we achieve upon successful combination of the concepts of Poisson distributions and if correctly defined phage multiplicity of infection) (iii) Lastly phage-encoded enzymes exist that contribute to the lysis of already phage-infected bacteria (iv) Decimal reduction time for bacteria following phage exposure which at a minimum can supply an intuitive understanding of why it is that phage titers kill bacteria (giving rise to what now commonly is called MOIactual) rather than ratios of phages added to bacteria (describing instead an ?MOIinput?) (iv) Discussion of phage-biofilm interactions from the perspective of phage-plaque development since plaque formation might inform phage-biofilm interactions plus may be better understood at microscales than phage interactions with biofilm bacteria (More completely stated this would be NPkt but where N and t are both equal to (though the latter meaning is often implied) ) impact determination Animal testing EPS depolymerases ; see also Catheters treatment of (implanted devises) Effort has been made to engineer phages so that they encode hydrolytic enzymes ; for a review of phage modifications in general for phage therapy application see ) In words: the rate at which a given bacterium becomes phage infected is independent of bacterial density though with two caveats 85% after min) determined as functions of bacterial viability release of intracellular ATP and biofilm dry weight a ?dense latticeworks of Pf4 filaments surrounding small-colony variant cells? p a lysogenic or pseudolysogenic infection Secondary adsorption Attachment (adsorption) of a phage to an already phage-infected bacterium (especially primary adsorbed); secondary adsorption may or may not lead to superinfection Superinfection Phage infection of an already phage-infected bacterium; the original infection may be productive reductive or even destructive Superinfection immunity Prevention of temperate phage infection of a lysogen as mediated by a prophage of the same (or similar) type; immunity is an intracellular process that follows phage adsorption Superinfection exclusion Prevention of infection of a phage-infected bacterium (productively or lysogenically infected) in which adsorption occurs but phage genome uptake is blocked Tailed phage A phage such as coliphage T4 that possesses both a DNA-containing head and bacterium-adsorbing tail; tailed phages display lytic productive infections; contrast tailless Temperate phage A phage that is capable of displaying either productive or lysogenic infections; sometimes the phrase ?lysogenic phage? is incorrectly used as a synonym (bacteria are lysogenic not phages) In the two chapters that follow this one I explicitly consider the spectrum of possible phage-biofilm interactions first from the phage perspective (chapter 3) and then from the bacteriums perspective (chapter 4) active penetration active penetration; Figure 1) active treatment adult form are either too small to be seen with the naked eye or are nearly so algae an adsorption constant for a given diffusion rate or bacterial density that maximizes plaque wavefront velocity and the variable M is equal to MOIactual Appendix 5) application of drops of bacteria-killing but replication-incompetent phages to immature bacterial lawns are active against a target that is present randomly in a diversity of organisms as a prophage; prophages often but not always are integrated into the bacterial chromosome Lysogenic infection A reductive infection that results in ongoing phage genome replication (as a prophage) without virion progeny production; contrast with productive infection Lytic infection A phage infection that destroys i as an extension of expedient versus economical musings indicated for scout versus settler phages (Chapter 8) as biofilms as by settler phages a process that I take up in the following chapter as can be addressed via the use of phage cocktails rather than monophage formulations; Figure 1) as discussed by as discussed in as ending in phage production and in many instances bacterial lysis as envisioned under certain circumstances for biofilms (previous paragraph) as equivalent to preservation (above) is crucial since otherwise food spoilage will have commenced prior to phage amplification as found in other biofilms as in single-step growth experiments as just described as noted perhaps resulting in less of a boost in wavefront velocity from increased infection numbers than otherwise could be the case as occurs during the interval spanning from about min to about min and given a phage density of 1010/ml (see their Figure 7) as one expects also among planktonic bacteria as plaques as well as from the study of phage-biofilm interactions themselves as plaques traditionally have been both studied and modeled as via diffusion to within the vicinity of a bacterium; (ii) the likelihood of collision with a bacterium once the phage is found in its vicinity (a function mostly of bacterium target size); and (iii) the phage affinity for a bacterium given collision which itself is a multi-component process that ends with and therefore is defined by irreversible adsorption as via greater overall productivity) bacterial doubling versus phage burst size) bacterial killing that is dependent on phage population growth; (ii) cloudy plaques as literally a potential failure of active penetration by phages into bacterial microcolonies; and (iii) solely passive treatment as what may or may not be going on when failing to detect phage activity as a function of plaque formation based on phage numbers that have actually adsorbed both give rise to low EOP along with death of the infected host) I am also of the opinion that the infections displaying reduced infection vigor should not always be categorized as abortive infections by using higher phage densities) or one or more additional phages should be employed chronically) or upon cell lysis and the latter type of reproduction as mediated by phages is what I focus on here cloudiness) nonetheless remains corresponding to unlysed bacteria turbid lysis debris and/or partially intact bacterial microcolonies coliphage T7 ) collision followed by irreversible adsorption) cuticles and stomach acid) mechanisms by which parasite components are recognized as foreign (e days rather than months or years) and techniques are employed that serve to assure that phage densities are maintained at high levels near surfaces then prevention of biofilm formation on implantable devices in principle should be achievable densities approaching those associated with stationary phase) diffusion) and an attachment component (i efficacy (a emphasizing rapidity of peripheral movement even at the expense of focus fecundity unless multi-focus phage exploitation of a single biofilm is especially rampant end-point adsorption determinations EPS depolymerase that is virion associated) excessive reliance on active treatment) poor infecting properties by phage ?S1 under the conditions employed (perhaps especially in terms of adsorption rate) poor phage penetration into the interior of biofilms insufficient overall experiment duration (only far more than just one-half and therefore consistent with active treatment faster population growth) and greater competitive ability between environments (e Fe2+ to Fe3+) and biomass production by free-living groundwater bacteria fluid flow) along with bacterial diffusion simply does not occur food processing) from drug-like application of phages as antibacterials how closely packed those microcolonies are within bacterial lawns) along with the affinity that individual phages have for the microcolonies lining those corridors (phage adsorption constant) in such a way that upon revival the bacteria are able to support productive infections by the same infecting phage in terms of phage dissemination between bacteria should not also occur in more mature lawns in terms of the expense of treatment that follows wound infection as well as patient morbidity and mortality then longer-term treatment of open wounds likely would be economically permissible in the following chapter in which bacteria are increasingly being lost to phage-induced lysis over time (and therefore as one moves towards the center of plaque) in which biofilm presence was quantified using a crystal violet staining protocol infection burst) including out of the biofilm itself and into the overlying fluid initially × or 1010/ml and then recirculated past biofilms just as one can speculate that ?a dense biofilm structure would render phage penetration difficult? p just as we can envisage biofilms as virus- or particle-filtering gels just as we would predict they would appear less distinctly as foci of infection within genetically heterogeneous biofilms just sufficient to achieve passive treatment but with repetition may make more sense ?Kill the winner?) killing titer; Appendix 3) kinetic rather than much less precise single data point i larger phage burst sizes likelihood of phage dissemination to bacteria not yet infected by that phage or instead in terms of lysogen survival and reproduction maximum added-phage-to-bacterium ratio was in the range of MOIactual (see Appendix 2) more cells infected my considerations of why phage plaques grow at a constant rate despite the maturation of the harboring bacterial lawn non-photosynthetic bacteria as well as photosynthetic organisms i of bacterial lysogens and eventual dissemination Stephen T one decimal reduction) then the D-value is Stephen T one distinctly separate from the parental focus p passive treatment was not attempted though this might have allowed a determination of whether failure to remove remaining bacteria was a problem of insufficient in situ phage generation versus physical or phenotypic resistance by bacteria to phage-mediated removal penetration that involves lysis of overlying bacteria before infection of deeper or microcolony-interior bacteria could occur perhaps phages/ml final in situ density or more) or if active treatment can be counted upon to provide sufficient phages to eradicate bacteria perhaps particularly for shorter generation times even at the expense of phage burst sizes so that foci expansion is more rapid phage amplification = active treatment; Figure 1) can be presumed to have taken place phage endolysins normally operate by allowing phages to move out of infected bacteria during normal bacterial lysis phage T7 seems to have at best only a limited ability to enter into the T4 zone of either reduced turbidity or scout-phage infection) PhageBioderm ) phagelysed bacteria) and secondary adsorption (attachment of a phage to an already phage-infected bacterium) are not shown explicitly phages which are Earths most prevalent category of viruses while viruses are Earths most abundant category of ?organisms phages/ml units with the goal that in situ phage densities at least at the start of experiments are unambiguously known preferential phage adsorption to nearer permissive bacteria or instead blocks on phage movement through EPS prevention of bacterial adherence to surfaces or more precisely killing of those bacteria that achieve such adherence primary pharmacodynamic effects) prior to reaching a fully infection-refractory stationary phase producing more explorer phages which then disseminate out of biofilms to found new foci of infection (Figure 14) rapid flow of overlying fluid) can lead to even more dramatic changes such as biofilm sloughing (see ?Release upon sloughing? Table 3) reduced burst size) and which consequently display low efficiencies of plating (EOP) see Appendix 2) or must be experimentally verified (though alternatively it can be possible to determine rates of phage adsorption based on increases in measured killing titers ) see previous paragraph) and with an absence of regrowth (phages alone reduced densities by about three log) ?settlement? ) so called depressor effect ) solid-phase phage propagation on or in agar-containing media) submerged vegetationassociated) was less than 10-4 (= 0/4 970) such as in the course of the treatment of wounds such as into biofilms covering the benthic surfaces lining bodies of water such as one would see with a multiplicity of such as operating rooms susceptible bacteria versus ?non-host material? that presumably coincides with lawn entrance into stationary phase the focus lineage plus indicate phage movement through space as well as time the phage is not itself genetically modified the potential for improvements in one aspect of an organisms biology to give rise to declines in other aspects the scenario seemingly envisaged by Doolittle et al their means of attachment to bacteria thousands or more) through biofilm ECM versus through channels and pores or instead through cracks and other defects tissue debridement ) and/or as noted improvement in phage delivery approaches such as to better assure phage retention at biofilm surfaces to bacterial debris which might be relevant to phage population dynamics to practice preadsorption) rather than as free phages to S toll-like receptors or complement) and phagocytosis towards eventual formation of new lysogens on new hosts) towards passive treatment visible plaque equivalents) wasps ants and bees) or the eusocial naked mole rats different individuals that have developed under different circumstances can take on different functions towards the collective good (in this case as measured ultimately in terms of phage dissemination and subsequent founding of one or more new foci of infection i weeks months or perhaps even years (see ?Biofilms as non-specific virus reservoirs? Table 3) when calculating the fraction of bacteria that have not been phage adsorbed where fraction of cells that survive following phage addition is equal to e-MOI (Appendix 1) where phage generation and loss from biofilms maintains a phage density sufficient to balance biofilmbacteria reproduction with bacterial loss or at least insufficient phage production to effect further bacterial decline which are Appendices adsorbed) with the delay until adsorption approaching or given phage decay even equaling infinity woodlice) congregating under rocks or when fallen leaves accumulate either in or on uncovered swimming pools ?S1) on P explored the impact of phages on up to twenty-day-old P also employed their ?bred? phages to remove preexisting biofilms formed by L-form L are suggestive that phage performance was slowed by poor adsorption resulting in turn in active treatment rather than the expected given the phage densities employed passive treatment evolved an ATCC-obtained phage to be specific for a cell-wall deficient L study are suggestive that substantial killing of bacteria did not occur until phages reproduced to densities higher than the >109/ml initially applied or alternatively that bacteria became more sensitive to phages after the first few hours of the experiment a two-log reduction in biofilm viable counts was observed over a short time frame (three hours) when phages were applied to biofilms consisting of phage-resistant bacteria that is mutants of the above-treated strain that (presumably) retained the same EPS but nonetheless did not support phage infections speculate that a single phage could potentially destroy an entire biofilm following multiple rounds of bacterial infection studied the impact of phages on Enterobacter agglomerans biofilm now Pantoea agglomerans that was formed on polyvinylchloride with disruption reportedly occurring due to a combination of EPS degradation and bacterial lysis refer to as a ?traditional? description of MOI also reported a positive relationship between phage production and cell death within P describe released phages as ?lytic? plus seem to equate ?bacterial death? with ?lysis? and ?autolysis? with ?phage activity? though no actual phage characterization was performed and as the authors note ?isolates may contain multiple phages? (p found no association between that phenotype and filamentous phage production suggesting that Webb et al explored the impact of phage T7 on E found reduced biofilm-forming ability by P echoing the sentiment of a number of earlier publications (see ?Biofilm resistance…? Table 5) claim that biofilms are ?resistant against attacks by bacteriophages? (p found that prophage integration could disrupt a gene involved in biofilm formation similarly speculated that prophage-induced lysis within S for their application to a similar system upon prophage deletion in P treated L thus found that efficiency of plating (EOP) was low for phages encoding a soluble EPS depolymerase when infecting a K1 capsule-producing strain and that an isogenic phage not encoding the depolymerase is ?unable to form plaques on lawns of this strain? (p employed a phage as well as various cleanser and disinfectant solutions to kill E contributed to biofilm eradication versus those phages that were both replicating and staying within the biofilm over the course of treatment employed a phage cocktail to treat two-species biofilms formed on stainless steel surfaces where the second species was S follows up a 2004 paper which also considered the impact of phage ?S1 (a may be viewed as a hybrid passive-active action where most bacterial depletion was observed during the passive phase due to the high phage densities supplied while relatively little bacterial depletion was achieved during the active phase treated broth-suspended P used a different phage phiIBB-PF7A (also indicated as ?IBBPF7) to eradicate P describes specific parameters that should affect the ability of biofilms to serve as virus reservoirs I distinguish between ?natural and ?artificial biofilms where ?natural biofilms arise outside of the laboratory on natural surfaces while ?artificial biofilms arise inside of laboratories studied the impact of both a newly isolated phage and its endolysin on S is a follow up to a previous E with the phage that displayed both lytic and EPS depolymerase activity used both a phage and the antibiotic ciprofloxacin against K explored the impact that ?cryptic prophage genes? (p following up on the 2003 study characterized phage-associated small-colony variants of P reported an association between release of filamentous phages subsequently isolated from biofilm effluent and death of host bacteria found in the interiors of microcolonies and the formation of these phages into an ECM-like state (i in a short note describe the use of a phage against S observed a decline in biofilm-forming ability in a strain of P treated 24-hour S explored phage T4 impact on E obtained some eradication of a S explored the impact of phage T4 on E considered treatment of biofilms consisting of either E employed fluorescently tagged phages also against a P and as quoted in Chapter it is possible that with biofilms an alternative route for phage movement between microcolonies is over rather than through microcolonies or associated ECM rather than first diffusing laterally into adjacent biofilm (Figure following page) found that phage infection of benthic bacteria Stephen T present a list of factors that could give rise to low likelihoods of phage infection of bacteria in benthic environments is not necessarily so rare that it is unable to support an ongoing phage presence within biofilms that it is those phages that by chance ?jump sooner? by displaying stochastically shorter latent periods that will contribute more to the speed of plaque spread is a follow up on the Curtin and Donlan study described above though employing phages targeted against P than by Curtin and Dolan with one- to twolog recovery in phage-treated biofilm density observed in the Fu et al also in terms of the total number of bacteria infected data it is possible that there could exist a peak positive impact of attachment likelihood on plaque wavefront velocity i provide a discussion of phage life-history evolution within plaques as a model of phage-biofilm interaction (for further consideration of phage life-history evolution in plaques see my own work in this area ) ) and therefore the total number of bacteria that become phage infected per focus as settlement however provide experimental evidence in support of the original negative-impact hypothesis and therefore contrary at least to the idea that changes to higher adsorption rates will not impact plaque growth rates which is limited in its breadth to describing what I call explorer phages describe prophage-induced bacterial lysis that in part or in whole has the effect of reducing an E Wang et al is from the same group as Goldman et al studied the application of phages to combat biofouling of ultrafiltration membranes used in water treatment of various effluent sources ? Alternatively quote Adams p ? Biofilm removal was assayed via safranin staining as well as electron microscopy ? However at least in terms of mass action the rate of bacterial adsorption by phages is a function of phage density as has been shown also with phage adsorption to biofilms but not of bacterial density (see Appendix 4) ? In this section I attempt to integrate these concepts of settlement production and emigration with those of settler scout and explorer phages ? It should be noted that in experiments consisting of biofilm growth on stainless-steel coupons bacteria were removed into ml of buffer prior to enumeration but coupons were approximately cm2 in total surface area (assuming that the ?size of microscopy slides? is × inches and mm thick) suggesting that the presented per ml densities for bacterial densities in biofilms are similar numerically to per cm2 densities ? Knezevic and Petrovic describe a simple in vitro method for exploring the impact of phages on biofilm formation ? Multiple enzymes too might be applied in tandem to biofilms with or without co-application with phages ? Note though that these observations may be difficult to interpret ? See Abedon and Thomas-Abedon along with papers cited there for further discussion of these concerns ? These activities similarly appear to have been associated with ?seeding dispersal of intact bacteria from biofilms ? These depictions can be viewed as a description of active penetration of phages into the biofilm i ? These environments may be especially suitable to a phage-mediated biocontrol because of a relatively low diversity of potentially biofouling bacteria: ?In comparison with effluent the diversity of well bacterial species is smaller (attributed to groundwater lack of organics presence of metal ions such as Fe2+ and Mn2+ and variable oxic/anoxic conditions); therefore only specific bacterial strains such as iron and manganese oxidizers/reducer and sulfate-reducing bacteria will be present if external pollution is not present ? What I assume is a ratio of added phages to bacteria of ? The Bacteriophages 2/e 2006 Oxford University Press) one as sole editor (Bacteriophage Ecology 2008 Cambridge University Press) and one as editor of a special topics issue of the journal Current Pharmaceutical Biotechnology (The Nuts and Bolts of Phage Therapy 2010 Bentham Scientific) ?Efficiency can be achieved by impregnating phages into surfaces or alternatively by retaining high phage densities locally by using for example phage-impregnated wound coverings or gels ?Efficient strategies should be viewed as preferable especially to the extent that phage formulations are costly to produce ?Efficient strategies as I define them here are ones that retain phages (or other antibacterial agents) near to the surface that is being treated thereby avoiding excessive phage wastage ?Inefficient application would be phage addition to large volumes of bulk water to reach a relatively small biofilm presence such as via the drinking of phage formulations to fill the gastrointestinal lumen with phages in order to reach gastrointestinal biofilms the addition of phages to industrial vats to block biofilm formation on surfaces or even the systemic delivery of phages to treat otherwise local infections s results do not extend at least to all P in the lower range versus one of 0 in the upper range of phage densities 0 0 0 0 0 0 0 1 1 1 1 13 While the above calculations have been performed using an Excel® function note that the traditional means of calculating Poisson distributions is via the following formula: POISSON(x M False) = Mxe-M/x! where x! is x factorial that is 0! = 1! = 2! = 3! = = × 4! = = × × etc 0002 was used implying a reliance on active treatment 0 imply that active treatment was used phages to bacteria implying that active treatment was relied upon though there is no exploration of phage population dynamics other than that phages were incubated with biofilms for hours 0016 0 0 0067 which is also equal to e-5 (and not coincidentally equal to that value; see EXP(-5) using Excel® along with further discussion below); note that this value rounded up is the approximately 1% bacterial survival rate noted above given MOI = 0 0 initial phage-to-bacterium ratio) though it is uncertain whether this result was a consequence of employing multiple phages or instead of including at least one phage that was especially effective and 0 0 0 0 0 0 0 05 the number instead is about 98% to 30% of the remaining cells were found to be phage T7 resistant True) Is there for example a 100-fold difference Hardly! Instead with MOI approximately 3% of phage-infected bacteria will be expected to have been singly adsorbed whereas with MOI It is equal to POISSON(1 True) = 96% versus 0 to 0 0 0 0 (i = e-Pkt = e-MOI = e-M where t in this case is the Dvalue) 0 of and as well as Figure of ; see also the last figure presented in this book found on p 1% = POISSON(1 1) 1) 1)/Pk (this is a rearrangement of 1 Log reduction in bacterial density for a given phage multiplicity (MOIactual); this is calculated as log(POISSON(0 MOI False)) 10) or in conjunction with an associated repression of bacteria fimbriae aid in the generation of planktonic dispersal cells 108) 108) 108) for further illustration including a microscopic view of what I interpret to be microcolonies of lawn bacteria Fraction of bacteria that are not infected = POISSON(0 MOI False) emphasis mine) 1164) have on E 1171) Fraction of bacteria that are adsorbed by only a single phage = POISSON(1 MOI False) inches = Fraction of bacteria that are adsorbed by two phages and only two phages = POISSON(2 MOI False) 135) of Fraction of bacteria that have been adsorbed by at least one phage = POISSON(0 MOI False) or about 15% 1446) ?In another experiment it was confirmed that bacteriophage SAP-2 is able to remove the SA1 biofilm (data not shown) 0 Fraction of bacteria that have been multiply adsorbed = POISSON(1 MOI True) = POISSON(1 MOI False) POISSON(0 MOI False) 15) calculated by the authors was only 15) ?that some biofilm cells perhaps those closest to the medium flow may be infected by multiple phage particles? (or alternatively phages might have been trapped in EPS) 150) ?that addition of phages at later stages of biofilm formation… is not effective due to increased barrier formation through EPS formation and its limited penetration by the specific phages 151) Fraction of bacteria that have been adsorbed by more than one phage (not or 1) as a fraction of those bacteria that have been adsorbed by at least one phage (not 0) = (1 POISSON(1 MOI True)) / (1- POISSON(0 MOI False)) 0 0 186) were turbid due to slow adsorption Lysis inhibition Multiply adsorbed T-even phage-infected bacteria show a delay in lysis that could hamper active phage penetration into microcolonies; greater cloudiness is seen towards plaque exteriors perhaps in combination with stationary phase microcolony interiors Debris Retention of light-scattering ability by phage-lysed bacteria; greater turbidity towards plaque exterior due to greater bacterial numbers and therefore of lysed bacteria as lawn matures Mixed indicator Lawns intentionally supplied with bacteria that are at least potentially phage insensitive; cloudiness is found throughout plaques Chapter CONCLUSION The movement of bacteriophages occurs primarily within fluids 2 Detection of loss of viruses or virus-like particles from fluids that have been exposed to biofilms implies a dependency on active treatment 0 1 23) for a scenario for phage-biofilm interactions one involving lytic infections along with phage display of a virion-linked EPS-degrading depolymerase enzyme 24) is also known as gel filtration or Stephen T 263) though latex beads alone have been shown to penetrate fairly deeply into biofilms 27) I distinguish studies into categories of eradication prevention and ?control 0 ) Reductive infection A phage infection that is neither productive nor destructive i × etc 0 30): ?…the student of phage should be familiar with the Poisson distribution…? This point continues to be true even if one is working with complex bacterial cultures such as biofilms since it is useful to estimate how many bacteria would be expected to be killed upon phage addition if only to compare this value with the fraction that are actually killed were approximately as much killing as one would expect given a Poisson distribution of phages adsorbed to bacteria 3264) as observed microscopically; see also Webb et al 3264) ?Bacteriophage activity was detected in the maturing biofilms of all CF strains examined and the amount of phage produced paralleled the degree of cell death seen in the biofilm 3271) 337): ?Infected cells on the biofilm surface (closest to the bulk flow) increased in size and burst prior to those cells lying deeper in the biofilm… Patches of infected cells spread radially and downward into the biofilm to include cells attached to the glass surface 337-338): The spread of infection more closely resembled that of plaque formation… than that of plume formation ?gradients (eg nutrients oxygen) present within the biofilm may affect the rate of phage replication within infected cells 0 = e-1 which when using Microsoft Excel® is equal to Exp(-1)) that do not 4 = e-0 Biofilm-forming ability reduces sensitivity to phage 4% removal as determined by crystal violet staining) following addition of to phages/ml ?final concentration? which suggests that active treatment was occurring ?Most of the biofilm bacteria had been removed from the substratum by the action of the depolymerase long before the phage-infected bacteria had lysed 0 43 emphasis his: ?Multiplicity of infection: Ratio of adsorbed phage particles to bacteria in a culture 4587) since filamentous phages historically are known to be able to form plaques without necessarily lysing hosts 4588) when added to biofilms ? 4872) 4893) 10-4 visibly productive phage infections per benthic bacterium observed would appear to be a low prevalence 4893) can be rare 4896) = 5 = Exp(-0 = Exp(-0 × 5 × 5 × 1010 phages in ml (>109 phages/ml) was then allowed to pass over the biofilm for min suggesting that passive treatment was at least initiated (indeed an over two-log reduction in bacterial viable counts was recorded in the first halfhour of treatment though it is not certain that these cells were actually killed versus released from biofilms) × bacteria in many instances would be much less than one square centimeter of biofilm × benthic bacteria examined including no phage-infected bacteria found among cells isolated from submerged vegetation-associated biofilm one infected bacterium found in association with sediments and three others associated with ?decaying plant litter? 0 Biofilm-forming ability declines upon acquisition of phage resistance cm2 total surface area) without phage treatment but with phage treatment for six hours still a fairly substantial nearly bacteria remained (~104 bacteria/cm2) describes actual infections then theoretically 40% of bacteria should have been phage infected (0 hours) and/or the existence of perhaps temporarily phage-resistant bacteria within biofilms is MOI and the one-minus construct is used to determine the fraction of bacteria that are expected to have been phage infected; the fraction of bacteria expected to remain uninfected prior to phage reproduction is e-0 logs to 5 to 5) = 5) = 5) = 5 that is the ratio of the number of phages adsorbed to the number of bacteria present is one-half then e-0 too that is ?about half? (Appendix 1) where 0 Can also be passive if sufficient phage dose are employed 6% of biofilm density via vancomycin treatment or days respectively 61 = 61; see Appendix 1) versus the 0 1 7 / (1010 × 10-11)) = 1010 × 10-11 × (see Appendix for discussion of similar calculations) = MOI Includes lysis from without as well as infection by various lysis-negative bactericidal phage constructs 74 = 0 ? only about half of the bacteria would be expected to have been initially phage infected implying a reliance on active instead of passive treatment emphasis his: ?…multiplicity of infection which is defined as the ratio of the number of phages (strictly speaking the number of adsorbed phages) in the inoculum to the number of bacteria in the culture ?This idea…?) Bactericidal-type chemical antibiotics 8068 ) 1 85) 869) but neither reference this claim nor provide evidence for it (see also ) 0 5 0 0 0 0 a a a a a a A common measure of antimicrobial impact is the decimal reduction time (D-value) which is the time it takes to reduce the viability of a culture by one log A consequence of these immediately productive lytic infections (step 3a Figure 6) could be phage-mediated disruption of biofilms that otherwise might contain newly formed lysogens potentially reducing the ability of these lysogens to sustain their initial biofilm association A consequence of this is that complex phage-bacterial dynamics likely are at play in these experiments as reflected by the high efficacy seen even when initial phage titers are low such that bacterial growth and Phages as Anti-Biofilm Agents subsequent active treatment (i A diffusion-driven ?extracellular search? is followed by phage attachment to a susceptible bacterium (adsorption) A failure to clear cultures macroscopically thus should be more likely especially for biofilms consisting of bacteria of heterogeneous phage susceptibility types A faster adsorbing phage (arguably) might be less discriminating in terms of the bacteria it adsorbs to e A fifth notion is that to the degree that biofilms are formed from abundant Phage-Biofilm Interactions Phage Perspective planktonic bacteria then phage decimation of these planktonic ?winners? could lead also to substantial loss of the biofilm bacteria A focus of infection (Figure next page) is a plaque-like unit of biofilm exploitation that is initiated by a single phage where prior to that infection this phage was either diffusing external to the biofilm environment or was generated within the biofilm by phages released from an induced lysogen A focus of infection is initiated with phage infection of single bacterium A followup animal study found no phage impact A forth consideration also included is pharmacological A fourth consideration is that when sampling biofilms for productive phage infections prevalence may be higher if sampling occurs by chance within a focus of phage infection versus within a region in which phage exposure has not recently occurred A given focus of lytic infection by a temperate phage might also give rise to multiple lysogens as the focus spreads A glossary of relevant phage terms can be found in Table (next page) A good rule of Stephen T A great number of possible phage-bacterial interactions exist though for our purposes the most important are those associated with phage productive infections i A handful of reviews on phage Stephen T A hybrid between these two cases involves the submersion of materials such as a ?coupon or a membrane within a non-laboratory environment such as drinking water distribution pipes or sewage effluent A number of recent general reviews of phage ecology exist A phage that is optimized for highly effective infection of healthier surface cells thus might pay a cost in total phage progeny produced if such optimization leads to a reduced potential to infect less-healthy cells to the degree that those cells are exposed upon phage-induced bacterial lysis A Poisson distribution is the default description of phage adsorption to Phage Therapy bacteria given that a single bacterium can be adsorbed by more than one phage A population that is holding steady can be said to exist at a steady-state equilibrium A pseudolysogenic infection contrasting these other phage infection types can be best described by what it is not: It is not productive (no virions are produced) it is not lysogenic (no prophage or phage-genome intracellular replication occur) and it is not destructive (the phage survives at least in the near term) A reasonable conclusion therefore is that phage infection of biofilm bacteria though seemingly relatively rare as consistent with the observations of Filippini et al A second consideration is that even phages that cannot productively infect stationary phase bacteria may still be able to infect those bacteria reductively i A second consideration is that if these infections are abundant then ?Kill the winner? might operate meaning again that it will be only over relatively short intervals that lytic infections by common phage types will be prominent within naturally occurring biofilms A second consideration is that killing titers are only as applicable as the Poisson distribution is applicable which will not be the case if all bacteria are not equally susceptible to phage adsorption (Appendix 1) for example if bacteria exist within biofilms A second consideration is that particle movement may slow upon entrance into the biofilm matrix as also experienced with size-exclusion chromatography (Figure 4) A second impediment is that bacteria making up even otherwise phage-susceptible bacterial clones may not necessarily serve as physiologically permissive hosts such as bacteria within biofilms that are found further from nutrient sources A second spatial constraint occurs with regard to bacterial clonal association where surfaceassociated bacteria can readily form into arrangements microcolonies and even ?macrocolonies? A six-hour treatment resulted in three-log reduction in bacterial densities A somewhat consistent observation is that plaques do not decline in their wavefront velocity (rate of size increase) as bacterial lawns reproduce to higher densities at least until an abrupt cessation in plaque growth that presumably coincides with lawn entrance into stationary phase A third implication is that phages specializing on biofilm bacteria may not contribute substantially to the total number of phages observed planktonically within a given environment unless of course biofilms make up a substantial volume of a particular environment (such as in soils) A total of bacteria are shown on each side A value of ?False? yields the fraction corresponding to the x term A working assumption therefore might be that phages adapted for exploiting biofilms should be more settler like emphasizing fecundity over expediency to at least some extent rather than more scout like i A yet additional consideration is the ?efficiency of phage use during biofilm biocontrol Abedon their infection process Abedon only an approximation with deviations from this calculation occurring as a consequence of differences in bacterial sizes within a population or equivalently if bacteria display varying degrees of availability to phages Abedon expression: (number of free phages) ? (adsorption constant) = Pk (where P is phage density) Abedon ln(0 Abedon considerations are relevant to the study of phage-biofilm interactions since in most cases the initiation of a focus of infection in a biofilm will not involve preadsorption Abedon interactions as they may occur within biofilms Abedon interactions Abedon BIOFILMS Many or indeed most bacteria have a propensity to adhere to surfaces creating oftenslimy layers known as biofilms Abedon aspects: their genes and everything else Abedon might be more profitable to display productive infections so as to advance virus-like population growth so too it might be safer to do so in comparison with remaining bacteriaassociated for too long Abedon though I consider phage-biofilm interactions both as described in the literature and as may be inferred especially from a more phage-centered perspective on phage biology Abedon gel permeation chromatography i Abedon Figure Abedon otherwise inhibit a phages search for bacteria Abedon Figure Abedon host lysogeny-establishing infections Abedon sampled from ?sediment decomposing plant litter and biofilms on aquatic vegetation? (p Abedon numerous to halt this bacterial advance (i Abedon of the phage as a prophage within a disseminating bacterium (step 5b Figure plus Figure next page) Abedon Table Abedon one case of gene deletion of an integrase the result was a decrease in biofilm formation however Abedon Physiological impediments I consider in Chapter in terms of phage penetration into bacterial microcolonies Abedon discussion of dosing considerations see the concept of killing titers ( and Appendix 3) Abedon to chemical antibacterials Abedon biofilms sufficient to adequately amplify phage numbers at the stage of development where treatment is envisaged Will bacteria in real-world biofilms which might consist of more than one bacterial species have the same potential to amplify phages to sufficient densities as model biofilms in the laboratory Chapter PHAGES AS ANTI-BIOFILM AGENTS In Table (p Abedon least to slow infection disease and even colonization such as may be accomplished by coating submerged surfaces with anti-fouling agents or by pressure treating lumber Abedon evolution of bacterial resistance Abedon subsequent air drying Abedon mediated removal from coupons or alternatively via ?crushing? of biofilm-harboring coverslip) Abedon provide an overview of phage-bacterial interactions exploration of how phage population growth may be shaped by biofilm properties and critical review of the literature concerning phage-mediated control of biofilm bacteria Abedon cleanser) Abedon that of phage T4 adsorbing to planktonic E Abedon The study by Gino et al Abedon Given these disadvantages prevention therefore may be most applicable under circumstances in which ? The window during which phage treatment is necessary is short (or if phages can be retrained in place while remaining active for long periods); ? The bacteria to be treated are anticipatable; ? It is economically feasible to employ large numbers of phages Abedon thumb is that at least phages per ml must be present to effect such passive treatment with this number increasing given the use of phages that are poor absorbers or which are inefficient in their penetration into biofilms (in this case penetration through EPS just to reach biofilm-surface-located bacteria) Abedon Related to the question of what phage titers per dosage may be required to achieve sufficient biofilm eradiation is the question of dosing repetition Abedon plaques and their formation by myself and collaborators have recently been published Abedon but in biofilms instead may give rise to the bulk of those phages that disseminate out of the biofilm matrix to if they are lucky eventually initiate new foci of infection (Figures and 8; Chapter 3) Abedon There should be additional consequences to phage population dynamics resulting from microcolony formation Abedon Table Abedon FORMATION OF PHAGE PLAQUES LATE STAGES The outward progression of phages from a central focus within a biofilm is perhaps best modeled by later stages of plaque formation Abedon secondary adsorption should impact plaque wavefront velocity by reducing the total number of virions that survive to reproduce (above) Abedon Figure Abedon with regard to the infecting phage Abedon metabolic wastes will more readily diffuse away Abedon (?efficient treatment; Chapter 5) Abedon phages to bacteria Abedon viii portion of phage ecology Abortive infection systems as a phage resistance mechanism especially can be viewed as a phagocytosis analog and one which is effective particularly given bacterial existence as multi-celled and clonal microcolonies ABOUT THE AUTHOR Steve Abedon is an Associate Professor of Microbiology at The Ohio State University where he has served on the faculty primarily as an instructor of undergraduates since 1995 ABSTRACT Bacteriophages (phages) are the viruses of bacteria and biofilms represent a frequent niche for bacteria where they are embedded in extensive extracellular polymeric substances (EPS) and can be structured into complex microcolonies Achieving a steady state is not necessarily facile however since it implies that the rate of reproduction of an organism is closely tied to its exploitation of resources such that the population despite growing fails to overshoot its environmental carrying capacity Achieving such potentially conflicting goals can require greater sophistication than one may imagine might be available to a simple virus though to a degree I will argue otherwise Active penetration implies a role for phage-induced bacterial lysis in exposing underlying layers of bacteria found within biofilms Active release often involves bacterial production of EPS- or even surface-degrading extracellular enzymes Active treatment by contrast requires in situ phage amplification placing a premium on phage population-growth performance Actual phage therapy treatment can take on a number of forms as illustrated in Figure (next page) Additional consideration of phage biology include the following: The first phage to adsorb a bacterium can be described as a primary phage Additional experimental observation by contrast has been inconsistent with declines in plaque wavefront velocity as bacterial lawns reproduce to higher densities at least until an abrupt cessation in plaque growth i Additional explanation for this relative failure in biofilm removal is that phage application was maintained only until nearly this degree of removal had been attained plus not all bacteria may have been immediately available to phage penetration such as biofilm-interior cells Additional Phage-Biofilm Studies Locations Attributes References Laboratory Prophage contribution3 to biofilm development see also Lysis or reduction in biofilm (also Table 4) see also ? Phage infectivity with ? biofilmforming ability4 see also ? Phage infectivity with ? biofilmforming ability5 Phage gene expression in biofilms Biofilm resistance to phages (speculation) Environment Archaeal virus references Metagenomics study Electron microscopy study CRISPR study see also Acidophilic communities Archaeal virus references Metagenomics study Electron microscopy study Webb et al Adsorption allows phage infection or simply the killing of bacteria Adsorption rates should by contrast be under weaker selection at Viewing Phages from a Phage Perspective higher bacterial densities aeruginosa aeruginosa aeruginosa aeruginosa biofilm but found that only upper layers of the biofilm were reached aeruginosa biofilm eradication in addition to the prevention experiments discussed above i aeruginosa biofilms aeruginosa biofilms here derived from cystic fibrosis-patient clinical isolates aeruginosa on agar plates? (p aeruginosa rather than S aeruginosa strain Mooij et al aeruginosa strains or prophages aeruginosa upon lysogenic infection with a specific temperate phage aeruginosa virulence in a mouse acute pneumonia model aeruginosa both with fluorescently labeled phages and followed microscopically After hours biofilms consisted of ~106 bacteria/cm2 (viable count as determined following scalpelStephen T After two or three hours an increase in phage density was recorded implying infection burst and potential for subsequent active treatment Against planktonic bacteria I am assuming that this treatment is not purely passive but instead that in situ phage reproduction first occurs agglomerans biofilm-eradication study agglomerans single-species biofilm to approximately zero (0 All animals plants and fungi together make up only a fraction of an even larger taxon called domain Eukarya All viruses are obligate intracellular parasites and pass through a life cycle that consists minimally of attachment to cells (adsorption) infection of cells during which virus particles are produced and release of virus particles into the extracellular environment or some alternative means of infectious passage between cells Also consistent with active treatment up to about 100-fold more phages were found in bulk media than were initially applied Also due to bacterial shielding within microcolonies the number of bacteria that may be infected and therefore contribute to plaque growth rates might not increase as fast as overall bacterial densities increase during lawn maturation Also of interest isolated phages were found to be able to form plaques on lawns of the parental lysogen (as well as forming clearings known as spots) plus CsCl-gradient purified phages contributed to ?early cell death? (p Also of interest though not addressable using plaques is the dynamics of phage movement out of and then away from biofilms towards new bacteria to infect Also biofilms often contain voids and channels that are presumably lined with bacteria to which phages can adsorb plus there is ample evidence that bacterial lysogens can form biofilms and even modify biofilm structure (above) Also such calculations are so crucial to performing phage-based experiments that they are a prerequisite to experimental competence Alternatively a drug for which this ratio is very small would need to be supplied approximately as rapidly as the drug is inactivated or eliminated Alternatively and as noted it can be difficult to ascertain without proper experimental controls just what the contribution of individual ingredients might be towards biofilm removal or for that matter whether synergistic or antagonistic interactions between formulation elements might be occurring Alternatively bacteria can be detached in smaller numbers (erosion) either by predators actively on the part of the biofilm or more passively by shear forces Alternatively broader utility may be more difficult to engineer given biofilms that are more difficult to disrupt or that can be reached only given systemic application a process which is inherently diluting Alternatively depolymerizing enzymes (ii) exist whose function is the removal of EPS serving more effectively as barriers protecting individual bacteria from phage attack; see for example and also for references and discussion Alternatively disregarding this practice can force readers to invest time in deriving actual dosing titers which under all circumstances should have been explicitly provided by authors Alternatively for more localized movement then soluble depolymerases may suffice such as for phage dissemination out of biofilms Alternatively how many more bacteria would you expect to be singly adsorbed of those bacteria that are phage infected if one started with an MOI of versus an MOI of Alternatively if bacteria are increasing in number then foci too may increase in number without terminating this bacterial increase though at a certain point of bacterial density phages will become sufficiently Stephen T Alternatively if new locations are not easily reached then it may pay for phages to emphasize overall productivity thereby better assuring that at least one phage goes on to found a new population in a new environment Alternatively in still-growing plaques there should exist bacteria-free ?corridors between microcolonies that is regions within which bacterial growth has yet to invade Alternatively infection prevalence explicitly in biofilms (i Alternatively it is possible that diffusion is more constrained if a phage is surrounded by bacteria that are not adsorbable so long as phage movement is slowed through EPS that a phage is unable to depolymerize Alternatively it may be that interior less metabolically active and/or multiply adsorbed bacteria could be both better anchored into the biofilm and more likely to Stephen T Alternatively it requires that bacterial biofilms form prior to their eradication which strictly is not a biofilm prevention strategy but rather an eradication strategy (or ambiguously we might describe such strategies as efforts in biofilm ?control rather than prevention) Alternatively larger phages with relatively long tails may be less able to enter into or migrate about within biofilms Alternatively lysogenic microcolonies might produce phages via induction over much of the life of a microcolony albeit with phage release from individual induced productive infections not necessarily occurring at biofilm surfaces Alternatively lysogens formed within biofilms might subsequently release phages following induction with those phages then going on to infect other bacteria perhaps establishing additional lysogens (step 5c Figure 6) Alternatively phage addition in sufficient numbers to kill planktonic bacteria without relying on additional phage reproduction may not supply sufficient phage densities to clear bacterial biofilms Alternatively phage T7 which is able to productively infect even bacteria making up stationary-phase lawns would appear to be ideally suited to penetrate into bacterial microcolonies Alternatively phage-resistant microcolonies can be present due to the use of a mixed-indicator technique employed to elucidate phage host range Alternatively phages can display strategies that involve retention of individual bacterial cells over longer periods including as lysogens or to produce larger burst sizes Alternatively phages that are released from below a biofilms surface may be more likely to encounter and then pass into ECM associated with adjacent microcolonies envisage phage release within a hole that had been bored into a multi-species biofilm Alternatively setting ?cumulative? to True yields all probabilities up to and including the x value provided Alternatively the approach might be employed to explore the potential of non-host bacteria to impede phage plaque formation Alternatively the enzyme may enhance phage movement away from infected bacteria following phage release (i Alternatively this is because most drugs are dosed so that densities remain as constant as is safe or convenient rather than intentionally allowed to decline towards zero Alternatively with productive infections phages can display three general strategies: penetration of biofilms perhaps layer by layer into associated microcolonies (active penetration; Figure and Chapter 1) lateral movement to infect new microcolonies (Chapter 8) and movement out of the biofilm to establish new daughter foci of infection (Chapters and 8) Alternatively within biofilms the association of lytic infections with reductive ones might reduce a microcolony to just newly formed lysogens perhaps releasing those lysogens from the biofilm as a consequence (above) Although the prophages seem to be exerting a negative impact on biofilm formation that result should not necessarily be presumed to indicate that these prophages also bestow a negative impact on long-term biofilm fitness under natural conditions An ability to recognize specific EPS enzymatically also should allow phages to more readily target biofilms that potentially contain susceptible bacteria that is bind to and then enter these biofilms An actual MOI of should result in 37% bacteria survival (= e-1) which is similar to the extent of survival observed suggesting that phage K was either not able to reproduce or not able to reproduce quickly on biofilm or stationary phase bacteria under the conditions employed An additional category of only partially destructive infections is often grouped among abortive infections (sensu here rather than Lwoff ) An additional concept is active penetration where phage access especially to interior-located biofilm bacteria may be aided by lysis of overlying bacteria (as indicated with the statement ?Bacterial lysis required here? An additional consideration is that ongoing phage production should be possible only given sufficient bacterial densities within biofilms particularly to the extent that phages from foci of infection can be lost from biofilms to bulk water An additional consideration is that under circumstances where virus-like growth is more productive so too may be the likelihood of lytic infection of a bacterial lysogen such as by a different phage type inhabiting the same environment An additional consideration is the duration of phage application where shorter intervals presumably will be less able to effect passive treatment than longer applications particularly if either phage adsorption or penetration into biofilms is poor An additional consideration is the potential for phages to infectiously migrate within biofilms An additional general concern is that to understand the consequence of phage impact on bacterial populations it is crucial to understand Poisson distributions An additional mechanism that can lead to plaque cloudiness which also might be a consequence of infections failing to lyse as lawns mature into stationary phase is lysis inhibition An additional parallel can be made between primary prophylactic vaccinations versus the use of antimicrobials as preservatives such as of food An alternative perspective to outright killing of bacteria such as via passive treatment is that mechanisms which can allow bacterial contamination from environments also would allow downstream introduction of disinfecting phages thereby giving rise to subsequent control of biofilm formation following active proliferation of phages in situ An alternative perspective however is that phagesensitive bacteria hiding within biofilms could serve to replenish planktonic bacteria given phage-mediated decimation of the latter An alternative way of viewing these processes is that virus availability within biofilms is lowered owing to reductions in rates of virus movement An approximately two-log reduction in cell count was obtained from an initial ~109/cm2 to a resulting ~107/cm2 An ideal organism may display both greater competitive ability within environments (e An indicated An obligately lytic phage particularly as viewed through a dissecting scope can display gradations of turbidity with greatest clearing seen in the center and increasing turbidity going outward And (v) the biology of phage plaques in a bit more detail than as provided in the main text And (v) exploration of issues pertaining to phage penetration into the bacterial microcolonies Another consideration is phage inactivation as a consequence of adsorption to already lysed bacteria i Another important distinction is that by biofilm ?interiors I am implying distance from not only the fluid overlying biofilms (bulk water) but also from any channels within biofilms through which nutrients dissolved oxygen metabolic wastes and even phages might readily move Another interesting consideration ecologically is that an ability to quickly clear a biofilm enzymatically wont necessarily also represent an effective strategy for phage predatory exploitation of a biofilm Another mechanism that could lead to cloudy plaques is the formation of microcolonies that are phage resistant for reasons other than lysogeny such as due to mutation to phage resistance Another perspective is that particles might enter into ?cracks and fissures? of biofilms which quickly close as a consequence of bacterial growth Another way of making this same point is that there is no reason to handicap phages by providing them simultaneously at low and infrequent doses relative to how antibiotics are dosed unless there are particularly compelling reasons to do so Another way of saying this is that Another way of stating this idea is that environments typically contain spatial structure which is a description again of the ability of an entity to move from one location to another: Absence of spatial structure is equivalent to a well-mixed broth environment one in which location changes rapidly over time and reaching all subsequent locations is essentially equally probable Another way of stating this is that the more bacteria that are present in a culture then the faster free phages will be lost to adsorption Antagonistic interactions can be between different antibiotics between antibiotics and phages or even between different phages anthracis as also did Rice et al Antibiotics if effective however can be antagonistic in some cases to phage action particularly if phage-induced bacterial lysis is sought Appendices As a consequence phage workers employing high bacterial densities (and phages that adsorb reasonably fast) can ?get away? with defining multiplicities of infection as equivalent to multiplicities of adsorption in terms of initial free-phage-to-bacterium ratios Appendices The species heterogeneity of bacterial biofilms has an analog in phage plaques APPENDICES For the sake of clarifying various concepts that are relevant to phage-biofilm interactions as well as phage biology in general as follows I provide five appendices APPENDIX 1: POISSON DISTRIBUTION In working with phages there are two key mathematical concepts that are critical APPENDIX 2: MULTIPLICITY OF INFECTION To many phage workers the concept of multiplicity of infection has a standard meaning: It is the ratio of phages added to bacteria which is MOIinput using the terminology of Kasman et al APPENDIX 3: KILLING TITER Though multiplicity of infection (Appendix 2) as well as Poisson distributions (Appendix 1) are relevant to phage biology in general they are especially relevant to the pharmacodynamics of phage therapy APPENDIX 4: BACTERIAL REDUCTION TIMES The reason that MOIinput does not necessarily equal or even approximate MOIactual is that the length of time over which adsorption is allowed to occur is not infinite APPENDIX 5: PHAGE PLAQUES In this section I provide an overview of phage plaques working under the assumption that properties known or thought to be associated with phage plaques may be applicable to phage-biofilm interaction Applied phage densities were 107/ml implying reliance on active treatment (Appendix 4) and resulting in reduction in cell numbers ranging from one to five logs Arrows indicate the initial stages of virion diffusion following lysis of the infected bacteria Arrows indicate the spread of phage virions released from these cells Arrows on top indicate adsorptions whereas arrows on bottom indicate production of a phage burst where ?One Burst? refers to one burst size Arrows pointing to the left act to slow (decrease ?) plaque wavefront velocities Arrows pointing to the right indicate mechanisms that either increase (?) or mitigate the slowing of plaque wavefront velocity Arrows represent immediately local (solid) or moredistant (dotted) pathways of virion diffusion As a consequence of the resulting spatial structure and heterogeneity phage-bacterial interactions within biofilms can be more complicated than those between phages and planktonic bacteria As a consequence of this reduced movement it is relatively easy to conceptualize the process of phage acquisition infection and then exit from a bacterium As a consequence as bacterial lawns mature we should have an expectation that wavefront velocities will decline due to an increased likelihood of secondary adsorption As a consequence microbiology can be viewed more as a collection of techniques than as the study of phylogenetically distinct types of organisms As a means of distinguishing phages with greater versus lesser potential to control biofilms the approach has merit particularly given that biofilm-density measurements are easy to perform and can be carried out with high throughput As a result active treatment places a higher demand on the performance of the phages involved: With active treatment phage population growth is required versus simply phagemediated bacterial adsorption killing and lysis As a result the closer to their parental infections that phages adsorb during their diffusion away from the center of a plaque then the less time they spend diffusing and therefore the slower the plaques growth As a result there is no direct benefit to an individual bacterium to the display of this mechanism As a rule of thumb one should assume that phages/ml or more may be required to achieve passive treatment over reasonable time frames As a second example lets assume an actual multiplicity of As above such bacterial dissemination also should be viewed in terms of a steady state As above though the number of pathogens that could be simultaneously targeted would be limited As Adams noted over fifty years ago (p As both bacterial and phage densities were low no more than about bacteria/ml at first while subsequently concentrated on membranes no more than about 106/ml it is unexpected that observed efficacy 3-log drops in bacterial densities and 5-log lower densities versus the no-phage control was achieved As discussed by Doolittle et al As evidence for this claimed failure of phage biology to coalesce as a field that is centered upon itself I note that nearly a century after their discovery a phagecentered journal has as of 2010 yet to be published As follows I consider and critique a number of studies in which phages have been employed to remove bacterial biofilms As further evidence for the possibility that phage ?S1s adsorption rates were poor to this P As noted above I speculate that the phages used in these experiments poorly adsorbed to their target bacteria resulting in substantial lags between phage addition and bacterial eradication As noted in Chapter various biofilm- EPS attacking enzymes might be used in conjunction with phages (Figure 1) serving either to enhance phage penetration or alternatively to extend the spectrum of bacteria or biofilms against which a specific formulation might be effective As phage dosing was indicated as MOI = this level of bacterial removal is highly suggestive of active treatment As planktonic bacteria were otherwise present and at ~107 colony-forming units/ml (which eventually were reduced in density by six logs using phages alone) it is difficult to tell to what extent phages were reproducing using planktonic versus biofilm bacteria as hosts As such the initiation of plaques is not identical to the initiation of infection foci in moderately mature biofilms and therefore does not necessarily serve as a reasonable model for the initiation of phage exploitation of biofilms As the alternative to passive treatment the term ?active? in ?active treatment? is used to imply that phage reproduction is involved in generating phage titers of sufficient levels to result in substantial killing of bacteria; see for review As the authors note however this observation may instead have been artifactual reflecting limitations of observation rather than failures of phages to fully penetrate into biofilms As they note ?The subsurface part of a typical well is made of a stainless steel (SS) screening device that prevents soil particles from being collected during pumping As with biofilm prevention my standards for success tend to be relatively high As with comparisons to size-exclusion chromatography one can speculate that similar processes might occur during phage exploitation of biofilms As with immature lawns lawns that do not yet contain microcolonies (above) it is possible to speculate that it will be those phages that diffuse some optimal distance before encountering and then adsorbing to a microcolony a distance that best balances phage generation time and extent travelled which will best define the leading edge of phage-plaque formation As with my analysis of biofilm-prevention studies (above) I consider these publications in approximate ascending-date order As with phage-encoded virulence factors however the interesting questions will be whether these contributions can be shown to be distinct from more general contributions to bacterial fitness as may be measured in terms of rates of planktonic bacterial growth and to what degree the contributions otherwise make ecological sense As with prevention protocols can be differentiated into those that are more ?efficient versus less so in their application of phages as well as in terms of whether or not active treatment is relied upon that is in situ phage amplification As with previous figures phages are not drawn to scale Association of enzyme with virions thus is less important for infection of bacteria that are nearer to a phages parental infection but more important for infection of bacteria that are more distant Association of phages with implantable medical devices is more problematic Assume that on average five phages are adsorbed per bacterium Assuming that their ?average number of phage added per bacterium? (p Assuming that there is a steady-state equilibrium for foci across ecosystems meaning that total numbers of foci are neither increasing nor decreasing then on average only a single additional focus needs to be generated per existing focus to sustain the population of a given phage/focus type (Figure next page) At a minimum however passive treatment is defined as the addition of as many phages as are required to achieve desired levels of killing of bacteria or biofilm removal without relying on supplementation of phage numbers generated via in situ phage reproduction At an extreme a biofilm consisting of clonally related lysogens will have few or no bacteria that can serve as subsequent hosts to the phage progeny of lysogen induction At an extreme a phage may become trapped within a biofilm that either does not or does not locally contain susceptible bacteria At an extreme biofilms can be formed by bacteria that are already phage infected that is by bacteria that exist as lysogens At an extreme cells can individually die though when they do so this death may not interfere with phage adsorption At least five plaques as taken through the ocular lens of a dissecting microscope At least some EPS thus likely evolved as a means of protecting EPSproducing bacteria from phage attack (below) At some intermediate distance a balance between diffusion duration diffusion distance and phage latent period should occur defining a maximal outward rate of phage progression At steady state replacement is assumed meaning that on average each focus of infection establishes only a single additional focus over the course of its existence At the same time microcolonies may become large enough that parental microcolonies themselves sop up more phages At the same time phage densities found in catheter lumens increased suggesting ongoing phage production Attempts were made to increase phage doses but never to the point where passive treatment would have been expected (i aureus biofilm in vitro (nearly one log or aureus biofilms could supply nutrients to other cells contribute to biofilm differentiation or serve as an apoptotic mechanism aureus biofilms emphasizing the latter in their analysis aureus strains versus only five of those strains by the intact phage aureus) blockage of biofilm formation is necessary for only short periods (e Azerdo and Sutherland suggest that phages using EPS depolymerases ?must destroy enough of the matrix if they are to release the embedded bacteria and make them vulnerable to the phage themselves? (p Bacteria are indicated as darker ovals except those depicted as releasing phages upon lysis (lighter ovals) Bacteria associated with biofilms are constrained spatially in various ways and to various extents Bacteria reached a density of >108/cm2 while phages were added to a final densities in the range of 109/ml suggesting a potential for passive treatment (Appendix 4) Bacteria that are found on the surface of microcolonies or biofilms in contact with bulk water thus are definitely not found in biofilm or microcolony ?interiors Bacteria within aging lawns in addition to inhabiting larger microcolonies may enter into physiological states that less vigorously support phage infections i Bacterial densities in these biofilms were approximately 106/cm2 which they describe as ?early stage? Bacterial densities subsequently were reduced by six logs in five hours or less to approximately colony-forming units/cm2 i Bacterial densities tended to be reduced to about 103/cm2 (given that cm2 of biofilm was removed into six-ml buffer) Bacterial densities then recovered relatively rapidly from their low points Bacterial densities then remained more or less constant for the next ~20 hours Bacterial infection by phages can be broadly differentiated into three basic types: productive infections reductive infections and what I describe as destructive infections Bacteriophages and bacteria are not drawn to scale BACTERIOPHAGES For historical reasons the viruses that infect members of domain Bacteria are called bacteriophages or phages for short Based on a figure found in Abedon and used with permission Based upon considerations of Poisson distributions killing titers and phage multiplicities of infection (MOIactual) it is fairly simple to calculate decimal reduction times particularly when phage densities are assumed to hold constant Because in most circumstances a given bacterium can support only a single infection but can adsorb multiple virions Stephen T Beginning with cell densities ranging from to 108/cm2 log reductions ranged from one to approaching four for singlespecies biofilms with reductions generally greater for older (72-hour) versus younger (24- hour) biofilms Biofilm Bacteria in Cited Studies Bacterial species References Acinetobacter johnsonii Bacillus anthracis Bacillus subtilis Escherichia coli Enterobacter cloacae Enterococcus faecalis Klebsiella pneumoniae Lactococcus lactis Leptospirillum group II bacteria Listeria monocytogenes Mycobacterium smegmatis Pantoea (Enterobacter) agglomerans Pseudomonas aeruginosa Pseudomonas fluorescens Sphaerotilus natans Staphylococcus aureus Staphylococcus epidermidis Staphylococcus lentus Table Biofilm bacteria often reproduce by producing progeny that do not remain surface associated Biofilm bacterial densities were reduced to between and 106/cm2 after two Phages as Anti-Biofilm Agents to four hours of phage exposure during which time phage densities above biofilms increased in most cases to between in excess of to 1010 phages/ml an increase in the range 100-fold or more over phage densities as initially supplied BIOFILM ERADICATION STUDIES Roy et al BIOFILM ERADICATION USING PHAGES Studies involving the application of phages to remove existing biofilms are far more common than those designed to prevent biofilm formation in the first place (see ?Eradication…? versus ?Prevention…? Table 4) Biofilm exploitation by bacteriophages thus can be both extensive and ongoing while at the same time barely perceptible macroscopically either visually or in terms of bacterial infection or loss rates Biofilm growth also was followed for about twice as many hours by Fu et al Biofilm presence was determined via a bioluminescence assay which indicated biofilm growth in the presence of phages even two or three hours after initial phage exposure though at a slightly lower rate than that seen without phages BIOFILM PREVENTION STUDIES In this section I consider studies in which phages are added either prior to or during bacterial challenge and which therefore at least potentially result in biofilm prevention Biofilm slimes can have DNA-based matrices for example and at least one phage is known that encodes an infection-secreted DNase whose function may be to clear this DNA presumably for the sake of boosting rates of phage movement through this matrix Biofilm treatment following two-day growth involved application of glycolic acid (2% v/v) for one hour to disrupt ECM Biofilms also often consist of more than one type of bacterium thereby complicating issues of availability of bacteria to phages even locally BIOFILMS AND RESISTANCE TO PHAGE ATTACK The susceptibility of biofilms to phages is oddly uncertain Biofilms as non-specific virus reservoirs Release upon sloughing (observed speculated) Biofilm removal2 of viruses from water Membrane bioreactors as cited by Drinking water (distribution treatment) as cited by Wastewater treatment as cited by Use of wetlands (non-natural mesocosms) Figure Biofilms can foul surfaces as well as contribute to bacterial pathogenicity Biofilms initiated with fewer bacteria were more susceptible to eradication after hours of growth than biofilms initiated with more bacteria Biofilms often also have numerous voids through which bulk water can flow supplying nutrients as well as removing wastes plus biofilms can contain cell-free voids within microcolonies Biofilms thus are complex ubiquitous and in many cases nuisances threats to health or both Biofilms thus can be viewed as virus ?sponges? in terms of either non-specific virus sequestration lysogen formation and persistence or with resistant but nonetheless phagereceptive bacteria serving as phage killing machines Biofilms thus may contribute to the ecology of phages solely by storing them until their subsequent release occurring either through occasional diffusion into bulk water or as a consequence of biofilm bulk deterioration (see ?Release upon sloughing? Table 3) Biofilms for example also can non-specifically trap viruses or modify the activity of prophages found within constituent bacterial lysogens Biofilms however may possess multiple mechanisms that result in their displaying resistance to phage infection or resistance to biofilm eradication even when infection is still possible (as discussed both immediately above and below) Biofilms Phage-Biofilm Interactions Phage Perspective by slowing particle movement relative to that observed in bulk water thus can nonspecifically accumulate viruses Biotic components can be either conspecifics or different species while abiotic components can be further differentiated into chemical and physical aspects Birge for example in the 2006 edition of his text Bacterial and Bacteriophage Genetics defines multiplicity of infection as ?the ratio of virus particles to potential host cells? Both experiments imply a substantial impact on biofilm structure from EPS depolymerase alone Both of these results I would view as consistent with poor performance by phage ?S1 as a biofilm-removing agent which I speculate could be a consequence of insufficiently aggressive treatment (e Both phage adsorption to bacterial debris (i Both researchers and reviewers should be conscious of this problem as it can result in a substantial waste of time and resources plus can potentially give rise to dangerous dosing errors in clinical practice ?Bursts? are represented as stars with the points on these stars indicating the extent of the burst that at least initially will be expected to diffuse in an outward direction (total number of points are equal to or and higher numbers remaining mean greater phage diffusion away from the parental microcolony) By applying these phages just prior to packaging the opportunity for further contamination is limited By contrast ?control as I define the term here represents an unspecified combination of eradication and prevention By contrast I spend little time considering molecular genomic or phylogenetic aspects of phage biology By contrast movement is faster in bulk water since there are no gels interfering with either particle diffusion or fluid flow increasing particle encounter rates with surfaces By contrast with abortive infections as I define the term here both phage and bacterium die By contrast with active treatment phages are also required to generate sufficient progeny per infection (high burst size) to make up for initial inadequacies in phage dosing By definition only temperate phages are able to display lysogenic infections Cerca et al Chapter INTRODUCTION There are limits to the densities at which bacteria can sustain active metabolisms while macroscopically filling environments Chapter provides an overview and conclusion Chapter VIEWING PHAGES FROM A PHAGE PERSPECTIVE Phage biology often exists as a field seemingly apart from itself Chapter PHAGE-BIOFILM INTERACTIONS PHAGE PERSPECTIVE Phage interactions with biofilms can be differentiated by category Chapter PHAGE-BIOFILM INTERACTIONS BACTERIUM PERSPECTIVE From the perspective of a bacterium the impact of phages can range from a profound contribution from especially lysogenic conversion to interference with reproduction to of course death Chapter PHAGE THERAPY The deliberate application of phages to remove bacterial biofilms can be described as phage-mediated biocontrol or as a form of phage therapy where the difference in terminology can be used to distinguish respectively non-clinical (e Chapter PHAGE THERAPY LESSONS GLEANED In this chapter I provide a summary of the material presented to this point as viewed from the perspective of anti-biofilm therapy or biocontrol Chapter PHAGE PLAQUES AS MODELS OF BIOFILM EXPLOITATION When attempting to understand complex systems it can be helpful to simplify Chapter PHAGE PENETRATION INTO BACTERIAL MICROCOLONIES Microcolonies along with biofilms can be relatively large masses of clonally related bacteria Chapters and reflect on and review the use of phages as anti-bacterial and anti-biofilm agents Chemical exchange particularly at biofilm surfaces thereby remains possible Chronically infecting phages which are unable to lyse bacteria might not be able to penetrate into microcolonies in this manner Circularly arrayed arrows indicate initial directions of phage diffusion away from the lysing bacterium Circumstances that Can Lead to Increased Plaque Cloudiness Circumstance Comment Stationary phase microcolony interior Failure to infect interior bacteria in more-mature microcolonies; creates bulls eye as larger microcolonies are found towards plaque exteriors Bacteria mutation to resistance Phage-resistant bacteria may be found in greater numbers towards the exterior of plaques due to higher bacterial densities and therefore more mutations to phage resistance as lawns mature Lysogeny Lysogens are resistant to the same phage types initiating these reductive infections; lysogenic microcolonies are larger towards a plaque interior because these lysogens have incubated longer Pseudolysogeny Similar to stationary phase microcolony interiors but implying that phage infection of these cells is reductive rather than destructive Slow adsorption Stent suggested that so-called ?ht?-type plaque mutants (p Clear plaque centers thus we denoted as ?zones of clearing? cloacae is explored Cocktails as the term is typically employed are the use of more than one phage type simultaneously though the term also could imply the use of phages with other products (or various phage products with other products) coli and digest the associated EPS reduced biofilm densities by another two logs coli B coli biofilm development coli biofilm treated with phage T4 coli biofilms biomass under conditions where lysis is IPTG inducible coli biofilms coli biofilms coli biofilms formed on polyvinylchloride coli K12 coli O157:H7 found in biofilms adhering to stainless steel surfaces coli or P coli parent culture however so there is no means of determining whether the resistance arose prior to or following biofilm formation coli biofilm densities were reduced by a doubly engineered T7 by up to Coliphage T7 in particular is able to continue its outward progression even following the entrance of a bacterial lawn into stationary phase com/ journals/bacteriophage) Combination therapy was notable instead for its ability to inhibit the appearance of phage-resistant bacteria Combined treatment of purified EPS depolymerase and various chemical disinfectants were also studied Combining prophages reduced biofilm-forming ability and insertional mutagenesis Ojha et al Combining soluble depolymerase with various disinfectants (depolymerase added first) was also more effective in biofilm removal than any one agent alone Combining the two hypotheses from Yin and colleagues with the Gallet et al Consequently the effective burst size is smaller than the actual burst size Consequently it is possible to succeed in doing many aspects of phage biology with little understanding of this important calculation Consequently it is reasonable to describe multiplicity of infection as a multiplicity of adsorption rather than strictly of infection Consequently phages may or may not be able to infectiously penetrate deeply into biofilms Consequently protocol development can require extensive trial and error Consequently though abortive infections and reduced infection vigor could resemble one another mechanistically and perhaps also operationally (i Consequently though promising we nonetheless remain at only early proof-of-principle stages of exploring the phage potential for total eradication of biofilm bacteria from surfaces consider Considered is a lytic phage that is physically associated with an EPS depolymerase and which encounters from the overlying bulk water an EPSdisplaying biofilm Consistent especially with this latter mechanism a slowing of plaque radial growth rates with higher initial bacterial lawn densities has been observed Consistent with conclusions of ?strikingly low incidence of impact of phages in the benthos? (p Consistent with the idea that phage infection of biofilm bacteria may be rare and/or will rapidly fade from view Filippini et al Contrasting the eukaryotes (members of domain Eukarya) these so-called prokaryotic organisms lack cell nuclei but nonetheless are both the most numerous and most genetically and physiological diverse organisms on Earth CONTRIBUTION OF PHAGES TO BIOFILM STRUCTURE One can view biofilm structure as representing an ongoing balance between bacterial growth microcolony differentiation bacterial release into the planktonic state (Figure 9) and various sources of bacteria as well as ECM degradation CONTROL OF BIOFILM FORMATION USING PHAGES Biofilms can be controlled by preventing them from forming in the first place Control of biofilms before excessive colonization occurs actually represents a fairly common use of phages as antibacterials CONTROL REQUIRES EFFECTIVE FORMULATION Whether towards prevention or post-formation eradication key to successful biofilm control is proper choice of ingredients in anti-biofilm formulations CONVENTIONS Per Skraber et al Corbin et al Cost of secondary adsorption Countering these advantages however are a number of disadvantages: ? Phages must be present for as long as surfaces may become contaminated; ? It may be more difficult to anticipate what bacteria to target rather than to identify bacteria after they become present; ? Active treatment is inherently unable to block bacterial cultures that have not yet reached relatively high densities so therefore active treatment cannot be employed as a biofilm prevention method Cross section of biofilm showing the lysis of bacteria found on its surface Curtin and Donlan filled catheters with an anti-S Curtin and Donlans protocol seemingly would represent an approach albeit unoptimized towards biofilm prevention D Dark ovals represent successfully reproducing foci while white ovals are dead ends Darker ovals are uninfected bacteria while lighter ovals (three in total) are phage-infected bacteria Darker ovals are uninfected bacteria gathered into microcolonies (rendered here in two dimensions as consisting of seven bacteria each) Definite conclusions regarding the relative merits of this follow-up active treatment are difficult to reach as experiments were terminated before substantial active treatment could have occurred Degradation can be due to biofilm-consuming eukaryotic organisms such as protists or may occur as a consequence of lysogen induction and EPS depolymerase production deep within biofilms Del Pozzo et al Deletion of one of these prophages in its entirety increased biofilm early-stage formation but in later stages biofilms decreased in bulk which the authors describe as representing an ?increased dispersal? (p Depolymerases can also enable phage adsorption following phage encounter with bacteria that display phage-adsorption-inhibiting EPS Despite their smaller size wild-type plaques contain substantially more phages than do r-type plaques Despite this plethora of possible dissemination scenarios (Figure 6) unless a phage population is growing in number then we would expect that on average only one of these strategies will succeed at a time for a given phage Destructive infections thus can be differentiated into two categories depending on whether in fact the host bacterium also dies: what I will call restriction or instead what I will call abortive infections Different pathways followed in the course of focus of infection development Different phage reproductive strategies optimizing either phage population growth rates or phage survival can vary greatly in their utility as a function of bacterial density Different phages therefore can be envisaged as taking on different ?behaviors? as a function of their location within an expanding plaque DIFFERENT PHAGES WITH DIFFERENT JOBS Phages found near a plaques periphery call them scouts are responsible for moving a plaques boundary outward Different roles for different phages within biofilms DIFFICULTIES OF PENETRATION (CAUSING CLOUDINESS IN PHAGE PLAQUES) Microcolonies can be difficult to penetrate into or otherwise eradicate Discussion of suitability of different phage infection types to phage therapy treatments Display of lysis inhibition by phage T4 is associated with the wild-type genotype whereas r mutants are defective in this latent-period extending and burst-size enhancing phenotype Disruption of bacteria or biofilms using phages or phage products can be accomplished via the application of EPS depolymerases single phage types (monophage) multiple phage types (cocktails; shown as multiple arrows top of figure) or bacteriolytic enzymes (endolysins) Distant from the periphery are phages called settlers whose role is to acquire uninfected bacteria within the already phageoccupied volume such as via penetration into bacterial microcolonies Donivan Sphar then an undergraduate and Cameron Thomas-Abedon my wife performed the experiments to test this hypothesis Doolittle et al Doolittle et al Doses were made in phages-per-well units but volumes were ?l meaning that maximum phage densities applied were 108/ml Dosing considerations are impacted by whether active or passive treatment is employed (Figure 1) if only because active treatment all else held constant should require an addition of fewer phages than passive treatment DOSING CONSIDERATIONS As with any pharmaceutical proper dosing can be crucial to efficacy DOSING LEVELS AND REPETITION Dosing in phage therapy can be distinguished into levels that achieve passive treatment versus those that achieve only active treatment During lysogeny the prophage can express genes that modify the phenotype of the bacterial host so-called lysogenic conversion During plaque development this lowmultiplicity phage exposure can be assumed to occur on the very periphery of plaque formation During plaque development therefore lawn maturation in combination with pseudolysogeny may result in microcolony retention and therefore cloudy plaques During these early stages of plaque formation note that target bacteria are well separated and it is relatively easy to envisage some phages colliding with close-by bacteria while other phages diffuse further before colliding with more-distant bacteria e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e Each of these categories of enzymes may be employed towards deliberate eradication of biofilms (for example see ?EPS depolymerases? Table 4) Each of these components of innate immunity has a bacterial analog some of which are illustrated in Figure (next page) ECM degrading enzymes also may be used independent of phages to effect biofilm disruption (below and Chapter 6) ECOI determination thus separates determination of the ability of a phage to productively infect a given bacterium from any need for the phage to form visible plaques on a lawn of the same bacterium Ecological utility can be either in terms of phage virus-like population growth i ECOLOGICAL UTILITY OF SUCCESSFUL PENETRATION Why penetrate into microcolonies The consequence for phages of more or less fully exploiting bacterial microcolonies is the production of additional phage progeny Ecologically EPS depolymerases improve phage movement that occurs either adjacent to or distant from a phages parental infection Economical phage strategies are of greater utility when bacteria are less available and are geared more towards producing higher phage numbers overall Either all or a majority of cellular organisms are thought to be parasitized by one or more virus types Elucidating ecological utility and doing so within an experimental biofilm context however is not necessarily a trivial process Environmental disinfection presumably serves only to reduce the likelihood of contamination from environments along with subsequent biofilm formation unless of course disinfection is achieved with 100% efficacy Enzymatic removal of substantially diverse EPS presumably could be similarly complicated (e epidermidis epidermidis biofilms with × 108/ml of phage K suggesting at least an approximation of passive treatment (Appendix 4) epidermidis in ?an in vitro catheter model? (p epidermidis phage (~1010 phages/ml) incubated these phage-filled catheters for one-hour at 37°C (followed by ?removal?) and then exposed catheter lumens to a flow of bacteria (108 to 109/ml) for two hours also at 37°C (which was then followed by sterile media for the rest of the experiment) EPS degradation also is not always necessary for phage infection even of biofilm bacteria EPS depolymerases tend to produce halos around plaques where EPS has been cleared but does not remove the actual EPS-producing bacteria a plaque morphology that is the result of faster diffusion of the smaller soluble enzymes relative to the much larger virions where only the latter can effect actual bacterial lysis EPS is indicated as a barrier surrounding an internal coccus with wedge-shaped loss of EPS a consequence of depolymerase activity whereas intact EPS is illustrative of an absence of EPS depolymerase in the vicinity of a phages bacterial target Equivalent to Figure except that bacterial reproduction occurs via binary fission following the acquisition of abiotic nutrients rather than via productive infection following bacterial acquisition Eradication is the removal of an existing biofilm Eradication of biofilms using the traditional means of chemical or physical disinfectants or chemical antibiotics is hampered however by the tendency of biofilm bacteria to resist such measures e Especially if too much sample is applied then the matrix can be overloaded such that concentration gradients no longer favor particle movement from carrying buffer into matrix Even for active phage therapy to be reasonably effective then so too must phage densities reach titers of approximately 108/ml Even given that information it can be difficult to develop meaningful insights into multifactorial phenomena Even if not serving as absolute barriers EPS within biofilms may slow virus movement Even with the higher multiplicity Exactly how that movement occurs and how it is limited by biofilm structure clearly is not well understood e Exactly what the distance is to these bacteria is less important than the idea that plaques can be differentiated into two phage populations based solely on the location of the bacteria that by chance they happen to infect Exceptions include ssDNA-containing icosahedral phages (family Microviridae) ssDNA-containing filamentous phages (family Inoviridae) ssRNA-containing icosahedral phages (family Leviviridae) and dsRNA-containing enveloped phages (family Cystoviridae) Expedient strategies by contrast emphasize more rapid increases in phage numbers usually at the expense of the numbers of phages eventually produced Extent of diffusion away from microcolonies should also affect the likelihood simply of re-infection of the parental microcolony Far better in fact is to experimentally determine MOIactual or at least to calculate this value and then to check experimental results against this estimation (e Faster phage adsorption should result in greater likelihoods of secondary adsorption where adsorption likelihood and therefore adsorption rapidity both increase with bacterial density Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Finally phages released near a biofilms surface may have the greatest likelihood of diffusing along that surface thereby reaching the tops of adjacent microcolonies i Finally the utility of settler phages is to increase the productivity of a focus of infection the other aspect of production sensu Gallet et al Finally while viruses themselves are capable of aggregating and bacteria can become surface associated for reasons other than intentionally attaching my focus here is on biofilms that consist predominantly of bacteria that have deliberately established a surface-associated life style First is that bacteria can be multiply adsorbed by phages and the second is that this adsorption can be modeled as a function of multiplicities of infection (MOIactual = M) and assumptions of adsorption distributed over bacteria Poissonally First I describe research that has indicated roles especially for prophages in biofilm development First in terms of plaque spreading it will be phages that are moving away from microcolonies rather than simply away from individual bacteria that will drive the outward diffusion process First Stephen T First the argument assumes that free phage densities remain constant (which actually is a good approximation given low bacterial densities; see Appendix 4) this is another way of saying that phages in the phage-adsorption reaction are not limiting First there may be no need for a substantial number of bacteria within biofilms to be productively phage infected at any given time in order for biofilm-exploiting phage populations to persist First they assume that all phages have adsorbed to bacteria which either is not necessarily the case (e fluorescens biofilms Sillankorva et al fluorescens biofilms formed on stainless steel surfaces fluorescens biofilms with a phage after determining an optimal temperature for phage treatment fluorescens biofilms here as formed adhering to glass fluorescens eradication remained efficacious despite the presence of the second biofilm species though this was not the case under all of the culturing conditions explored fluorescens following transposon mutagenesis of a potentially phage-associated gene fluorescens host note that the time it took bacteria to decline in viability by one-half was close to min in the presented experiments e fluorescens the sole target was an only minority constituent (see for similar efforts towards two-species biofilm eradication using different phages and bacterial species; see also ) Foci in non-clonal biofilms in other words can be highly ?cloudy? or ?turbid? to borrow terms from the plaque-morphology literature (Chapter 9) FOCI OF INFECTION AND STEADY STATES One can view these rare but arguably not non-existent biofilm-infecting phages as inhabiting foci of infection Focus-of-infection steady state Follow up with additional depolymerase did not result in additional biofilm eradication however perhaps consistent with the failure of whole phages to completely eliminate biofilm (previous paragraph) Follow up with additional phages i Following settlement and ensuing productive infection these explorer phages can give rise to settler and scout as well as explorer phages Following up the work of García-Contreras et al For 1010 phages/ml and an adsorption constant of 10-11 ml/min it would take about min to reach this MOI (= For a list of bacterial species employed in cited phage-biofilm studies see Table (next page) For additional discussion of Poisson distributions see Appendix For additional discussion of these various concepts see For additional perspectives on phage-biofilm interactions see the following: For additional Stephen T For an elaboration on this figure see Figure For different trials between For example chronic infections can persist despite a combination of antibiotic sensitivity and treatment using high antibiotic doses For example one could be interested in the number of bacteria that are not adsorbed by any phages (in which case this number x would equal 0) the number adsorbed by only a single phage (x = 1) the number adsorbed by two phages (x = 2) etc For example phages along with other viruses can become trapped non-specifically within biofilm extracellular polymeric substances (EPS) can degrade these polymers can infect bacteria making up the biofilm and can infect planktonic bacteria lysogenically that could go on to make up biofilms For example substantially more phages are likely to secondarily adsorb and superinfect already phage-infected bacteria if those bacteria are part of microcolonies than if bacteria instead are not physically associated with other clonally related bacteria (Figure 15) For example the application of just EPS depolymerases in certain instances may be sufficient to achieve control (see also ) For example we can consider the number of bacteria that have been adsorbed three times (x = 3) given an MOI of (average across the population) = POISSON(3 False) = For further consideration of conflicts within phage populations between longer-term and shorter-term fitness see For further information I point the reader to various reviews and overviews: For MOI = the fraction of bacteria also which do become phage infected is For most phages productive infections of bacteria found in stationary phase do not readily occur (phage T7 as noted seems to be an exception in this regard i For phages these would be conditions where either bacterial densities are low phage decay rates are particularly high or some combination of both For plaques and the net-outward flow of phages away from plaque centers what may be most relevant similarly is the ratio of the volume of the corridors between microcolonies (i For references and further discussion see: For review see Skraber et al For simplicity we can distinguish these into a movement component (i For summary of this model of phage plaque formation in light of microcolony formation see Figure (p For the sake of long-term utility the goal of this disinfection would be either complete bacterial eradiation such that resistance to applied phages does not evolve or phage formulations are modified over time so as to keep up with such evolution For the same reason phage adsorption rates or adsorption likelihood should not increase as fast as bacterial densities increase within lawns (Figure 18) FORMATION OF PHAGE PLAQUES EARLY STAGES Plaque formation begins with phage placement into a solid-phase or semi-solid environment FORMATION OF PHAGE PLAQUES MIDDLE STAGES There is no reason that the same plaque-formation processes outlined above i found in the crude lysates would be expected to compete with phages for binding sites on the catheter material Four hours post phage exposure a decline in bioluminescence was apparent however Frequency of Multiplicity of Infection Classes (Poisson Distribution) log not multiply not reduction infected infected infected dividing MOI\x9 011 213 not not or not or From a Darwinian perspective this is phage survival followed by phage reproduction From epidemiology we would describe the reproductive number of this successful focus as being equal to one where values greater than one imply an expanding phage population or at least expanding focus ?population whereas values of less than one imply a contracting phage population From this logic it can be predicted that immediately productive phage infections may be relatively rare in natural biofilms Fu et al Further a number of these bacterial mechanisms can be viewed more fruitfully in terms of microcolony protection rather than protection of individual bacteria Furthermore a substantial literature exists indicating a potential for phages to infect biofilms (Tables and 5) Furthermore as a consequence of that spatial heterogeneity during plaque formation phages interacting with bacteria might act a lot more like phages interacting with biofilms which also are made up of bacterial microcolonies than I would have previously thought Furthermore as reviewers consider correcting authors who use MOI incorrectly as the ratio of phages added to bacteria by quoting Stent p Furthermore even if phages are successful in mostly eradicating phage-susceptible bacteria this success could result in overgrowth of resistant bacteria Furthermore exploitation of even clonally related bacteria may not be identical in its efficiency across biofilms because different bacteria within biofilms or microcolonies may not display identical physiologies Furthermore M can be estimated from measures of bacterial survival following phage exposure Furthermore successful or subsequent bacterial acquisition by a phage virion that is produced within a biofilm is by no means assured Furthermore the bacteria targeted can be limited for example to specific pathogens such as Listeria Furthermore to effect passive doses densities of added phages should both reach and ideally be sustained in situ over the course of treatment at levels of 108/ml or more Furthermore we can view (i) plaque formation as analogous to active treatment i g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g Gaining regulatory approval too may be simplified so long as the replicative medicinal i Gallet et al García-Contreras et al GENERAL PRINCIPLES Phage application as antibacterial agents sometimes simply works Generally adsorption rates and latent periods are fairly simple in terms of their genetics and perhaps even in terms of bacteria- or environmentmediated variation whereas effective burst size is extremely complex particularly as viewed in terms of why the phage eclipse period is the length that it is what the phage maturation rate is during the post-eclipse period of a latent period how phages survive to acquire bacteria and even how well phages survive once they have successfully adsorbed Generally if new locations are easily obtained then selection should favor more rapid population growth over overall population productivity Generally we can distinguish methods in a number of ways which I outline in this chapter prior to considering individual studies (next chapter) Generally if efficacy is inadequate then approaches towards enhancement can include improvement in phage choice (such as one or more phages with demonstrably better ability to lyse and kill bacteria ) increase in phage numbers applied (switch from relying on more active to more passive treatment ) increase in the number of doses applied (thereby providing phages with multiple opportunities to encounter and infect bacteria ) improving upon site preparation strategies (e Generally then phage adaptations can be viewed as contributing to phage survival (and therefore increasing the phage effective burst size) shortening the phage generation time (by increasing adsorption rates or decreasing the time until phage-progeny production) or by increasing phage per-infection fecundity (either by extending the phage latent period Viewing Phages from a Phage Perspective increasing the likelihood of phage survival or increasing rates of phage-progeny maturation within infected bacteria) Generally then we can view these various hydrolytic enzymes as positively impacting phage movement vis-à-vis bacteria displaying susceptible polymers either towards or away Given observations of reduced efficacy with greater phage application then it would be helpful from a pharmacological perspective to make some effort towards investigating why such treatment is less efficacious than expected Given successful penetration into bacterial microcolonies however these settlers may ultimately produce more explorer phages (wider vertical arrow) than will directly descend from scouts (narrower vertical arrow) Given the high phage densities used (>109/ml) these results of Hibma et al Given the latters larger size but lower productivity I reasoned that r mutants may succeed in out-competing wild type phages only if plaque numbers were extremely high on plates e Given this microcolony physiological differentiation it is not necessarily simple for phages to effectively penetrate into microcolonies that is to more or less fully exploit the bacteria making up a microcolony or at least to successfully kill all of those bacteria Going yet further from the plaques center we enter into the realm of scout phages Goldman et al Greater biofilm removal was seen by Tait et al Greater versatility generally may be formulated into biofilm eradication protocols when there are fewer constraints on either delivery to susceptible bacteria or efficacy once delivered Hanlon et al He sees the core of his scientific training as being in biochemistry having received his BS in that subject but overlain with a strong tendency to explore issues of organism-level phenotype and ultimate causation Hence cells deeper in the biofilm would most likely become infected with greater frequency upon release of progeny phage from the initially infected cells Here a fraction of infections display very long productive rather than reductive infections along with a failure to lyse under stationary phase conditions Here I consider possible impediments to biofilm eradication by phages A major impediment to phage-mediated biofilm eradication is biofilm genetic diversity Here I consider three aspects of the phage impact on biofilms Here I provide a descriptive model of these processes one that attempts to address phage-bacterial interactions at organismal levels such as between individual phages and individual bacteria as well as between phages and microcolonies Here I Stephen T Here I suggest that phage plaques may represent a reasonable model of phage propagation as it occurs from a central focus within biofilms Here I will argue that it is within this zone of reduced turbidity that settler phages reside especially penetrating into persisting microcolonies which if present presumably would be the dominant contributors to the turbidity of these zones Here since e-MOI is the fraction of bacteria expected to remain not-infected assuming a Poisson distribution then - e-MOI is the fraction expected to be infected Here the longer that infections produce phages then the more phages that are produced all else held constant Here treatment of a dual-species biofilm with E Hibma et al Higher planktonic phage densities appear to have been present at the end of experiments when starting with 72-hour versus 24-hour biofilms possibly explaining the greater susceptibility of 72-hour biofilms to eradiation by phages versus 24-hour biofilms His principal research interest is in the evolutionary ecology of bacteriophages a Historically phage characterization has been done mostly under conditions where bacterial densities are relatively high Horizontal arrows indicate the presence of fluid flow above the biofilm with greater arrow length and width indicating faster flow However an alternative interpretation is that with the coarseness of time points taken no more than once every two hours the Fu et al However a counter argument could be that though infection numbers may increase as lawns mature infection fecundities (burst sizes) instead may decrease i However a number of studies reviewed in the previous chapter seem to indicate limitations on the degree to which active treatment can reduce bacterial densities However a shortcoming to the employment of these enzymes both ecologically to the phage and in terms of their application as biofilm-disrupting agents is the diversity of polymers found in biofilms in combination with the typically high specificity of phage depolymerases specific enzymes thus will not be effective against most biofilm EPS However as a means of protection of clone mates from phage attack there would be obvious utility However at lower bacterial densities latent periods make up an increasing smaller fraction of the overall phage generation time so are under weaker selection than at higher bacterial densities However at steady state where phage densities in environments are neither increasing nor decreasing then we have the expectation that any given focus of phage infection within a biofilm will produce only one new focus on average just as no matter how many acorns an oak tree produces over its lifespan at steady state only one acorn on average will give rise to a successfully reproductive new oak tree (Figure 23) However because phage densities were not determined it is difficult to tell whether titers were sustained at high levels either at surfaces or in bulk water over the course of the study which in any case did not involve ?efficient phage application However come 2011 this sorry state is scheduled to change (see www However for a number of reasons this observation actually is difficult to explain for plaques even though bacterial lawns during plaque formation in fact are somewhat spatially homogenous at least at larger scales However for lytic phages the longer phages are produced the longer the phage generation time since with these phages the release of intracellular phage progeny coincides with a loss of the ability of an infection to produce additional phages However given that phages are selfish products of Darwinian selection it is not unreasonable to view phage molecular aspects as existing solely for the sake of their contribution to phage organismal aspects However if planktonic bacteria are allowed to replicate to the point where they can support phage population growth then presumably biofilm formation will have at least been initiated and therefore control would be a more reasonable descriptor than outright prevention of biofilm formation However it is also possible that the recombinant phage T4 used did not display lysis inhibition since the LacZ gene was cloned into the lysis-inhibition-associated phage rIIB gene However it is at the surface of microcolonies that the physiologically youngest bacteria should reside and also the ones which phages are most likely to encounter However it seems reasonable in terms of biofilm prevention to expect that the blocking of biofilm formation will occur quite early in the course of development However just as our understanding of the Conclusion pharmacology of phage-mediated biocontrol of biofilms could benefit greatly from a better appreciation of phage-bacterial interactions in biofilms on microscales so to too would our understanding of plaque development benefit from such fine-scale analysis However key considerations are ? Use of ingredients that are not antagonistic to each others actions; ? Use of ingredients that display demonstrable biofilm-disrupting ability; ? Use of ingredients with sufficient breadth of action at least when used in combination; ? Use of sufficient dosing that ingredients are maintained at effective levels throughout the course of treatment However little consistent difference is seen between the different doses with all providing three- to four-log reductions in target bacterial densities whether in single or dual-species biofilms (though removal of non-target bacteria in dualspecies biofilms was not as consistently efficacious) However MOIinput = MOI is true only under a narrow set of conditions and ones which are routinely violated in modern phage biology However note that there also is an expectation that mechanisms which serve to reduce the duration of a phages infection period will also reduce a phages burst size and it is not well understood which of these phenomena larger bursts versus shorter latent periods will have the larger impact on rates of plaque-size increase However phage movement away from a microcolony will be less efficient than away from individual bacteria since part of a given phage burst will tend to encounter and then infect the parental microcolony However phages carry nucleic-acid based genomes and are subject to Darwinian evolution However phages may not be as competent at this infection as we necessarily ?want? them to be However such enhancement should be readily confirmed using properly controlled experimentation However taking on this life style can dramatically reduce the per-infection fecundity of these phages (i However the authors point out that phages should concentrate on membrane surfaces which could result in both a more ?efficient strategy of phage use along with a concentration of phages to sufficient densities at membranes to eliminate biofouling bacteria: ?a portion of seeded phages will be left attached to membrane surface and available for continous sic infection of the oncoming bacteria without interfeering sic with filtration process? (p However the details likely become more complicated due to a tighter packing of bacteria first into microcolonies (Figure 13) and then into collections of adjacent microcolonies However the eventual bacteriophage production was insufficient to achieve complete eradication of the biofilm formed over the course of treatment However the rate at which free phages are lost to infection is equal to only the product of bacterial density and the phage adsorption constant However there is a profound difference between knowing that something exists and actually seeing it with ones own eyes However there still is relatively little understanding of the extent to which complete bacterial eradication of surface-associated bacteria may be achieved and this in part is because biofilm-eradication positive controls consisting of the repeated application of very high densities of phages are rarely attempted However to the extent that the alternative is costly i However together they would seem to have a constraining impact on rates of phage plaque enlargement However while infections found towards the periphery of a focus of infection might produce all three phage ?types scouts settlers and explorers infections that are found far from that periphery will produce only two types settlers and explorers However while sitting in a car as my daughter visited and cared for her horse I realized that I might be able to recycle some older as yet unpublished material into that chapter Hughes et al Hughes et al I additionally present five appendices I also provide discussions related to phagebiofilm interactions including of Poisson distributions multiplicity of infection calculating killing titers (phage numbers killing bacteria) calculating decimal reduction times of bacteria following phage application and the concept of pseudolysogeny I also suggest that that it may not be necessary for phages even if they specialize on biofilm bacteria to extensively destroy naturally occurring biofilms in order to prosper I am also grateful to the feedback provided by Dwayne Roach Catherine Loc-Carrillo Philip Serwer Susan Lehman Hans Ackermann Ariane Toussaint Ing-Nang Wang Sarah Kuhl Ben Chan and Cameron Thomas-Abedon on various portions of the manuscript I can envisage three general scenarios by which such control may be accomplished: ? Use of phages to disinfect environments such that bacterial contamination is limited; ? Treatment of planktonic bacteria so that bacterial adherence to surfaces again is limited; ? Passive treatment of biofilm bacteria during early stages of biofilm development I consider these and other phage impacts on biofilm structure etc I employ this model in part because plaquing is a ubiquitously used technique in phage laboratories and because the mechanics of phage plaque formation have been explored theoretically as well as experimentally to at least a modest degree I follow this with review of studies where phage-mediated biofilm-eradication is explicitly attempted I provide additional discussion of the biology of phage plaques in Appendix I realized however that it would be helpful to expand the chapter to provide additional material including more background an expanded conclusion various appendices and some more-speculative phage-centered prose e I show this zone in two parts to provide an impression of its dynamic nature i I stress that key to understanding the dynamics of phage-bacterial interactions within biofilms is a combination of addressing how phages move towards and move away from target bacteria including in terms of the phage potential to burrow into bacterial microcolonies I suggest especially that microcolony formation should slow changes in bacterial lawns that otherwise could reduce wavefront velocities I then provide a more ecological discursion on phage-biofilm and phage-microcolony interactions (Chapter and 9) particularly as informed by theory pertaining to phage propagation in the laboratory within plaques (i I view this as a prevention study because phages were added only ten to twenty minutes after bacteria implying a lack of biofilm formation prior to phage application (this the authors describe as ?simultaneous?) I will go further and say that if a given phage is unable to supply sufficient biofilm-disrupting utility then either an effort should be made to increase associated efficacy (e I would also like to thank the Battelle National Biodefense Institute for support which provided me with the time and motivation to develop my organizational skills self-confidence and critical eye towards phage-therapy experimentation all of which were essential to the writing of this monograph Ill define pseudolysogeny subsequently after considering destructive infections Ill start with examples Ideally in either case bacterial colonization is brought under control before damage occurs which with the clinical application of phages is before actual disease symptoms commence Ideally in fact it will be bacterial colonization that is blocked i If a newly formed lysogen remains biofilm associated then ideally from the lysogens perspective the dissemination potential of the original microcolony (Figure 9) will be transferred to it If a Poisson distribution can be assumed then the expected phage-free bacterial frequency following phage adsorption is e-MOI where MOI as noted above is defined strictly i If a population is not growing in size then each individual on average produces a single new individual If at all possible one should employ in vitro experimentation prior to in vivo or in situ testing if only for the sake of saving time and money If at any given time only 10-4 biofilm bacteria are productively infected perhaps implying that still tens hundreds or even thousands are productively infected per cm2 (Chapter 3) then it may be that at least some phages are competent at infecting biofilm bacteria If bacteria are sufficiently susceptible and numerous then phage propagation through a biofilm may result in substantial bacterial loss If distant then movement towards bacteria will be enhanced by physical linkage between virions and depolymerases If foci are increasing in number then susceptible bacteria should decrease in number resulting in reductions in focus productivity or focus numbers If movement to this adjacent microcolony isnt too far then secondary adsorption potentially may occur there as well again leading to loss of phage infections If relatively low phage densities are employed then one can view these approaches to biofilm control as more equivalent to vaccination albeit of an atrisk population If so then it would be in the vicinity of multiply phage-infected and then lysed microcolonies that this debris should be most prevalent and therefore of greatest importance If this interpretation is correct then it would imply an adsorption rate for this phage that is more than one-hundred-fold lower than Stephen T If those microcolonies consist of phagesusceptible bacteria then this moves the periphery of a plaque a small distance outward from the central focus here defining that plaque in terms of phage infection rather than simply phage presence If we assume that k = 10-9 ml/min then for P = and the corresponding D-values are Illustration of impact of EPS depolymerase depending on position and association Illustration of impact of microcolony local curvature on phage outward diffusion Illustration of propagation of infection foci assuming steady state Illustration of reduction in virion mean free path due to a lack of shielding of bacteria given random bacterial dispersion (right) immunity? (superinfection immunity) lysogenic cycles (upper-left) lytic cycles (bottom-right) destructive infections (bottom-left plus lysis from without) and chronic infections (upper-right) Impediments to the ability of phages to diffuse through a bacterial lawn could similarly reduce plaque wavefront velocities Implication is that corridors between bacteria within which virions can diffuse relatively unobstructed will be larger given bacterial formation of microcolonies versus existence as unassociated bacteria Important non-mathematical biological models include that of the double helix or the use of fruit flies to better understand mammalian developmental biology Importantly my ambition had become excessive as suddenly I realized that I wanted to take on the entire English-language literature considering phage-biofilm interactions Importantly the contribution at least to bacterial death has been rigorously shown in a subsequent study to be dependent on phage presence In a biofilm these are found on a focus of infections edge In a continuation of earlier studies involving phage-mediated eradication of P In a dual-species biofilm this phage displayed a greater negative impact even on the non-susceptible bacterium (particularly so given higher phage dosing) implying a role for EPS in the stability of both susceptible and not-susceptible bacteria In a similar study of small-colony variants of a different P In addition to cellular organisms microbiology also considers entities that may or may not be described as organisms but instead which may be referred to as infectious agents In addition to seeking to prevent biofilm formation (above) Hibma et al In addition all of the members of domain Bacteria and of domain Archaea are microorganisms which along with Domain Eukarya describe all known cellular organisms In addition and also relevant to that first argument to the extent that concentrating phages to higher densities is not feasible such as for economic reasons then phage application at lower doses e In addition and related I consider how phage-biofilm interactions may be informed by interactions as they can commonly occur in the laboratory especially given phage population growth within plaques In addition at any one time these foci may not contain substantial numbers of bacterial infections In addition biofilm aging along with abiotic factors (e In addition biofilms can consist of heterotrophic organisms e In addition consideration of phage interaction with bacterial microcolonies as components of bacterial biofilms rather than simply phage interaction with individual planktonic bacteria may be key to understanding a substantial Stephen T In addition he founded in 1996 the Bacteriophage Ecology Group (www In addition indicated ratios are In addition it is difficult to ascertain what phage densities were employed since doses are given in multiplicities of infection by which I presume the authors mean ratios of added phages to bacteria (see Appendix 2) In addition mixing (i In addition non-specific diffusion of viruses into biofilms may slow or Stephen T In addition note that the Poisson distribution is The value x across the top of the table is the number of adsorptions under consideration e In addition phage ?IBB-PF7A alone was used to treat dual-species biofilms where P In addition phages as well as phage products can be employed to artificially disrupt bacterial biofilms as a form of phage-mediated biocontrol or therapy of nuisance or pathogenic bacteria In addition phages can display differences in their durability in the face of environmental degradants In addition phages were pumped through catheters for two hours rather than statically filled for one In addition potentially more infections will occur near the surface of microcolonies assuming movement over time of the location of phage infections from the outside of microcolonies towards their interiors such that diffusion of resulting phage progeny out of biofilms may be more likely In addition to the extent that active treatment ?works? then concentration of phages to sufficient densities to achieve passive treatment may not be necessary In addition with biofilms there may be utility to active penetration the lytic movement of phages towards biofilm interiors In any case at steady state we can expect by definition that bacteria die no faster than they are born and similarly that phages are born no faster than they die In any case from a phages perspective the larger the effective burst size or shorter the generation time all else held constant the better In any case reductive infection of a planktonic bacterium seems less likely to result in subsequent lysogen establishment within a biofilm relative to reductive infection of bacteria already making up a biofilm In any case the Excel®-based calculation for the fraction of phageadsorbed bacteria that are multiply adsorbed is (1 POISSON(1 MOI True)) / (1- POISSON(0 MOI False)) In at least Stephen T In circumstances where enzymes may not be directly supplied it should thus be advantageous for those enzymes to be carried by virion particles if only to increase the efficiency of initial infection In comparison with another experiment in which both bioluminescence and viable counts were performed it is possible to estimate that a biofilm of approximately bacteria/cm2 remained after six hours of phage exposure though with a trend towards ongoing removal In comparison with wild-type T7 as well as a phage-untreated control of biofilm consisting of otherwise phage-T7-resistant E In each case it should be obvious that treatment of the biofilm proper either is not occurring in an ?efficient manner (that is something other than just the biofilm is being treated) or alternatively biofilm formation still occurs but just not to the same extent that it otherwise might In either case densities of susceptible bacteria were reduced only to the range of to bacteria/cm2 In either case these are potentially economical means of avoiding much more costly wound-infection treatment that would be required after visible infection has commenced In either case what is being described is a clonal population of phages where both earlier- and later-adsorbing phages are both sourced from the same parental infection and otherwise are genetically identical In fact selection should be towards greater effective burst sizes faster adsorption and shorter latent periods no matter the bacterial density In fact the actual number of infected bacteria was no more than 1% after this phage pulse prompting the authors to speculate (p In fact to achieve more uniform plaques it can be helpful to pre-adsorb phages to bacteria before mixing either with agar In general only once substantial efficacy has been observed should researchers back away from more aggressive bacterial clearance protocols to ascertain for example whether application of fewer phages per dose or fewer doses will also achieve sufficient efficacy In his spare time he cooks (Schezuan Chinese-inspired) tends to his family (two children and loving wife) takes care of his ?farm (when there isnt any snow about) and otherwise skis as much as possible In host range determination this approach provides a facile means of visualizing the potential of phages to lyse multiple bacterial types where phages will produce clear plaques only if all indicator bacteria used to initiate a lawn are susceptible to phage-induced lysis In indicating the potential utility of passive treatment I dont want to imply that active treatment is lacking in usefulness In many instances phage treatment is of bacteria that are present either fully or partially in direct physical association with other bacteria as well as non-bacterial surfaces i In medicine the constant irrigation of not-yet visibly infected wounds or periodically soaking bandages with much smaller volumes of phages represents ?inefficient and more ?efficient strategies of biofilm control (respectively) in Microbiology with a minor in Ecology and Evolutionary Biology (University of Arizona 1990) he has published approximately articles chapters and other ?citable units In most of these studies prevention of biofilm formation does not actually occur however so in the majority of cases ?control in fact may be a more accurate descriptor of outcomes In order: (i) Enzymes that degrade the structural matrix of biofilms may allow phages to more effectively move towards bacteria to infect In other experiments by Hughes et al In other words an overnight incubation of phages with bacteria in LB broth in no way separates phage impact on biofilm formation from phage impact on planktonic bacterial growth In other words for a given adsorption interval MOIactual will be larger given greater bacterial densities while MOIinput will stay the same In other words how might biofilm eradication in these experiments have fared were the planktonic phage pool not continuously in contact with biofilms throughout experiments but instead allowed to flow or diffuse away as might be the case under conditions better resembling those existing within the dairy plants where the two species might form dual-species biofilms Alternatively could more ?efficient treatment strategies have retained similar phage densities in biofilm contact It also would be of interest to determine whether the failure to effect further bacterial removal than that observed in this as well as the other studies discussed above could be improved given prolongation of phage exposure higher exogenously provided phage densities application of EPS depolymerases use of multiple phages targeting the same bacterial species or some other means of improving the ability of phages to infect what I speculate are biofilm-interior bacteria and/or bacteria that otherwise are resistant to the treatment protocol employed In other words multiplicity of infection defined literally or simply in terms of phage adsorption (Appendix 2) should be high in the vicinity of a burst In other words no matter how many phages a given focus produces nor its duration of phage production a focus should be considered successful if it goes on to form a single new focus of infection i In other words the phage-bacterial dynamics seen in the Hibma et al In other words there is no guarantee that temperate-phage interaction with biofilm bacteria will result in the retention by biofilms of newly formed lysogens both because not all such infections are reductive and because productive infections could interfere with the biofilm retention of reductive ones In other words there is no reason to expect that phages over long periods will do any better within environments other than to simply persist and the same can be said for biofilm bacteria themselves In particular (p In particular (i) secondary adsorption may not increase as fast as bacterial density increases due to the geometry of microcolony size increase (Figure next page) (ii) the ability of bacteria to support robust phage infections may decline less rapidly if the healthiest bacteria persisting on the surface of microcolonies are what phages are most Phage Plaques as Models of Biofilm Exploitation likely to encounter or (iii) the average path length along which free virions are able to diffuse before colliding with bacteria may decrease less rapidly to the extent that microcolonies shield interior bacteria from phages (Figure next page) In particular adherent viable bacteria were recorded per ?square? (0 In particular and contrasting the potential for phages to infect individual bacteria that inhabit biofilms what many phage researchers are concerned with is the potential for phages to effectively remove biofilms from surfaces In particular experimental protocols that are reliant on such amplification by planktonic bacteria will be successful outside of the laboratory only if these planktonic bacteria are likely to also be present under real-world treatment conditions and at sufficiently high densities that phage amplification to therapeutic densities is guaranteed In particular I discuss lessons that may be garnered from that material which I provide towards development of phage therapy (or biocontrol) best practices In particular I do not accept that statistically significant differences in end-point bacterial densities represent a sufficient aspiration for phage therapy protocol development but instead look for biologically or medically relevant levels of removal In particular plaques can be initiated using multiple strains of lawn bacteria the so-called mixed-indicator technique In particular they noted (quoted from their abstract) that (p In particular while lawns tend to lack macroscopic spatial heterogeneity they in fact are temporally heterogeneous In passive treatment sufficient phage numbers are supplied via exogenous dosing (application of phages) to acquire and kill most or all target bacteria over some reasonable span of time (see Appendices and 4) In phage therapy (or biocontrol) a fundamental measure of efficacy is in terms of reductions in bacterial density and the simplest predictor of bacterial killing in situ involves determination of killing titers in vitro that is what degree of bacterial killing should be expected for a given phage dose In Phage-Biofilm Interactions Phage Perspective comparison with an induced productive infection however more phages should be generated by immediately productive infections given exploitation of an entire microcolony because greater numbers of productive infections should be involved i In plaques newly formed lysogens can also be seen as microcolonies though ones that form in plaque centers presumably reflecting the longer incubation of earlier lysogenized and now phage-resistant bacteria in the course of plaque development In principle biofilm treatments should display sufficient breadth of activity to achieve presumptive treatment with some reasonably high frequency or instead rapid methods of testing biofilm susceptibility should be developed so that phages and other ingredients can be chosen over short time frames that have demonstrable utility In principle this approach to biofilm prevention should be labeled ?efficient since treatment of the bulk culture was not attempted In recent work using published models of phage growth I and others have explored the impact of modifying various parameters burst size (per-infection phage fecundity) latent period (per-cell phage-infection duration) adsorption constant (a measure of the rate of virion attachment to bacteria) and bacterial density all on rates of radial plaque formation (plaque wavefront velocity ) as well as total phage numbers produced per plaque (plaque fecundity ; see Appendix for additional discussion) In short all phage molecular adaptations can be viewed in terms of their impact on (i) phage latent period including as extended in the course of reductive infections (ii) phage burst size or (iii) phage adsorption plus avoidance of virion inactivation or destructive infections (a component of the phage effective burst size) In some cases passive treatment also may be achieved by using so many phages that bacteria are literally lysed from without In Table I compare different phage infection types along with bactericidal antibiotics in terms of their potential to effect active versus passive treatment of bacterial infections In terms of log reductions in bacterial densities however there is an exponential decline in bacterial viability as a function of MOIactual (bottom graph) In terms of phage dosing during biocontrol or therapy an additional consideration is that most antibacterials are not dosed as ratios of drug to bacteria In terms of population ecology and particularly if predation of the focus species is not an issue then such a population can be said to be existing at an environments carrying capacity for that species In terms of productive infections if free phages encounter susceptible hosts within biofilms ones that are amassed for example into microcolonies then multiple bacteria will be present locally to infect In the ~20 years since receiving his Ph In the center zone most or all bacteria have been lysed and no microcolonies remain except those associated with phage-resistant bacteria In the course of this research I got into the habit of peering through dissection scopes at plaques and it was this latter tendency that led to a deeper interest in the interaction of phages with spatially constrained bacteria In the laboratory this is typically one that is agar containing and for plaque formation to occur the environment also must contain phage-permissive bacteria In the middle is a single bacterium that has been co-adsorbed by two (or more) phages In the zone of reduced turbidity bacterial lysis has occurred but turbidity (i In their second phage-biofilm study Doolittle et al In this case the number of bacteria that are expected to not become phage infected prior to any phage reproduction is e-0 In this chapter I first review the handful of existing biofilm-prevention phage studies In this chapter I provide overviews of these various phage-biofilm interactions In this chapter I take a standpoint that is in contrast to the existence of phage biology for every possible reason other than to better understand phages: Consideration of phages from a phage perspective In this latter case × phages/ml was added to × bacteria/ml for what given reasonable rates of phage adsorption would give rise to an MOI of (as reported) In this model different phages are indicated as playing different roles In this role EPS barriers thus can serve in some capacity as receptors for phages equipped with these enzymes In this section I consider various perspectives on phage exploitation of microcolonies In this section I provide an outline of phage therapy general principles various concepts that together reflect decades of tinkering by practitioners towards improving the usefulness of phages as antibacterials In this way restriction-modification systems are reminiscent in their action to that of the alternative Phage-Biofilm Interactions Bacterium Perspective complement pathway which is active against both self tissues and non-self invaders except that self tissues thwart complement-mediated recognition via modification In this way in plaques and perhaps also with biofilms we can distinguish between outward phage migration which may occur mostly on microcolony surfaces and more complete exploitation of the bulk of a microcolony i In words this is the fraction of bacteria that are neither singly nor not adsorbed divided by the fraction of bacteria that are not not adsorbed (i Increased infection number in particular should increase wavefront velocity while the rest of the mechanisms pointing rightward should serve to mitigate declines Increases in plaque size by contrast are indicated by the larger dashed lines which basically consist of scout phages producing more scout phages (involving an alternative form of emigration to that concerning explorer phages) Increases in the efficacy of phage treatment potentially could have been possible had higher phage densities been used or phages better able to adsorb and lyse bacteria under the treatment conditions Indeed all phage molecular functions ultimately can be viewed simply in terms of their impact on both the size and the rapidity of formation of the phage effective burst size Indeed as stated in the latter review (of results found in ) p Indeed biofilms need not be limited to members of domain Bacteria but also can include members of domain Archaea as well as microscopic members of domain Eukarya Indeed even when experimental biofilms are exclusively responsible for this phage amplification two important questions should still be asked: Are bacterial densities in these Stephen T Indeed for a time I wasnt sure whether I would ever even write the chapter Indeed given sufficient host rarity a focus might even involve the exploitation of only a single bacterial microcolony or even a single bacterium Indeed it can be difficult to ascertain whether phage infections within biofilms occur naturally at all (below) Indeed it is only after one has established consistent bacteria-infecting efficacy for a given phageaddition protocol (see killing titer Appendix 3) under well controlled conditions that MOIinput may be legitimately employed as a description of phage dosing Indeed it is possible that many aspects of phage molecular biology are difficult to optimize in ways such that improvements in adsorption rate latent period or effective burst size are not in conflict Indeed on a per infection basis there should be no net gain in effective burst size (Chapters and 2) nor distance jumped by any one phage (this chapter) but instead decreases in both of these quantities Indeed particles may randomly diffuse throughout the biofilm matrix and/or display weak and non-specific chemical binding with biofilm materials just as phages can non-specifically bind with various materials found in soils such as clays and humus (see for further if brief discussion) Indeed phages as a phenomenon was discovered in terms of the phage impact on bacteria particularly the lysing of bacterial cultures Indeed taxonomically we can speak of a kingdom Animalia a kingdom Plantae and a kingdom Fungi Indeed these are strategies of biofilm eradication rather than prevention (above) since phage densities sufficient to combat either bacterial colonization or infection will not be present until colonizing or infecting bacteria are there in the relatively large numbers Indeed they observed only four productive phage infections in Indeed under circumstances in which phage delivery is inherently inefficient such as following systemic delivery then active treatment may be the only form of phage therapy that is possible Indicated are that scouts give rise to either settlers or explorers as well as to new scouts whereas settlers give rise to other settlers or explorers but not to scouts Individual planktonic bacteria by contrast may be more regularly encountered by predators though with fewer bacteria obtained per encounter Induction is the conversion of lysogenic infections into productive infections and the majority of temperate phages display lytic productive infections Infection numbers increase because of the additional numbers of bacteria found as lawns mature but by growing as microcolonies this increase should be slower than expected as bacteria become shielded by bacteria found on microcolony surfaces (though this shielding also should slow declines in phage mean free path Figure plus will bias phage infection towards physiologically younger bacteria found on microcolony surfaces) Infection spans the phage latent period which ends in bacterial lysis Infections by both scouts and settlers produce phages explorers which can diffuse out of biofilms (top of figure) to potentially found new foci of infection Infections can be either productive reductive or destructive Stephen T Infections displaying reduced infection vigor should display a substantially larger ECOI than EOP whereas for abortive infections sensu stricto ECOI and EOP should be similar Infections for phages can be destructive productive or reductive Infections provide new phages to this ?reaction-diffusion? process but also temporarily halt phage movement Inhibition of biofilm formation in this study likely is attributable to infection and Phages as Anti-Biofilm Agents lysis of a combination of both planktonic and stainless-steel-adherent bacteria Initial density of each phage type in experiments was × 106/ml indicating active treatment Innate immunity as seen among vertebrate animals as well as both invertebrate animals and plants by contrast is fixed such that it adapts only over evolutionary time Instead bacteria become phage adsorbed on a per-bacterium basis at a rate that is equal to this Stephen T Instead such infections are characterized by a stalling of the phage infection such as may occur following phage infection of sufficiently starved bacteria Instead the answer is that nearly 1% of the bacteria would be expected to not have been phage adsorbed and therefore would be expected to survive phage exposure at least in the near term Instead what appears to have occurred is inefficient active treatment Interestingly by spot testing the isolated endolysin displayed ?activity? against S Interestingly the amount of bacteria removed from these early stage biofilms was nearly the same as that seen with phage ?S1 application to more mature biofilm Interestingly the decline involved an interaction with the bacteriums CRISPR systems Introduction Figure Introduction Table Introduction MICROBIOLOGY Microbiology is the study of organisms that in their mature i Introduction of fresh media however increased pre-phage bacterial densities phage titers produced and biofilm-eradication efficacy It also rendered those resistant bacteria that did appear less robust in a number of ways including in terms of their ability to form biofilms It also requires sufficiently productive phage infections of biofilm bacteria that phage or depolymerase levels are boosted in situ to densities that are sufficient to remove all unwanted bacteria It also results in some degree of physical degradation of the bacterial cell It can be concluded that there are multiple paths by which phages can infect bacteria inhabiting biofilms (Figure 6) It consists of simply growing bacteria in microtiter plates and then staining washed wells using crystal violet It especially can be less important to show the number of bacteria that are removed from a given surface than the number that remain It goes without saying that phages should not be included in anti-biofilm formulations unless they can be shown to display at least some ability to disrupt biofilms It is also feasible to attempt to augment phage treatment with chemical antibacterials (see ?Treatment…? Table 4) It is also possible however that destruction has positive effects in its sculpting of biofilm morphology supplying nutrients to remaining bacteria or even easing the process of bacterial translocation from within the biofilm matrix to a dissemination stage outside of it (Figure 9) It is an open question by the way the extent to which demonstration of anti-bacterial activity in broth or on plates will necessarily translate into anti-bacterial activity in biofilms It is conceivable that as phages penetrate lytically into microcolonies the physiology of the exposed cells therefore may change in a manner that is favorable to productive phage infection It is difficult definitively to say just how these two consequences should affect plaque formation without either detailed modeling or experimentation designed explicitly to explore these possibilities It is difficult to establish from the study just what the remaining biofilm density was since density was reported in units of bacteria per ?peg? It is difficult to say to what extent the high densities of planktonic phages observed by Sillankorva et al It is especially the organismal perspective of phages that has been somewhat missing particularly in modern phage biology It is important to realize though that the failure of a phage to plaque is not necessarily equivalent to the failure of a phage to productively infect but can result instead from reduced infection vigor such as reduced burst size It is my opinion however that ecologically there is a qualitative difference Introduction between out-right phage inactivation (as with a fully phage-destructive infection) and simply a reduced phage productivity resulting in reduced plating efficiency It is not always necessary to achieve both enzymatic digestion of EPS and bacterial lysis to clear biofilms but instead just one of these can accomplish ?a substantial degree of biofilm degradation? p It is not obvious how inoculation differences translate into resulting biofilm properties though perhaps the higher starting density cultures were either further into stationary phase at the point of phage addition or further into biofilm formation It is phages that by chance diffuse down these corridors that may come to infect more distant bacteria It is possible also that phages may be limited in their ability to even reach bacteria found deep within biofilms if efficient bacterial lysis is necessary to expose those deeper bacteria to phages (see the concept of ?active penetration? in ) It is possible also to return to the analogy of gel filtration/size exclusion/permeation chromatography (Chapter and Figure 4) but with the added property of affinity thus affinity chromatography It is possible that more-complete eradication of these bacteria might have occurred following the lysis of these lysis-inhibited bacteria though this possibility was not explored It is possible that this phage production might be a normal part of biofilm differentiation that occurs (I speculate) in conjunction with phage-virion dissemination (i It is possible though that some phages are better at infecting more refractory especially stationary phase bacteria than are others (e It is these x values that are Poissonally distributed It may be less crucial to be more ?efficient during biofilm eradication versus prevention however since phage application for eradication need not be as ongoing; nevertheless fewer phages may be necessary or higher phage densities may be achieved if phage application can be targeted to the biofilms rather than to the environment in general It seems that prophages may contribute to biofilm structure in many ways just as they contribute to bacterial pathogenicity through a diversity of mechanisms via their encoding of bacterial virulence factors It therefore can be necessary not just to have the right phage in the right place to infect a single type of biofilm bacteria but instead to have multiple phage types to remove biofilms consisting of multiple bacterial types (i It would be helpful to employ facile models towards improving our understanding of phage-biofilm ecological or pharmacological interactions johnsonii B Just why phage-associated bacterial death tends to be Positive contribution that is other than solely bacterial loss due to phage-induced lysis k k k k k k Keep in mind in such explorations that adequate doses for passive treatment only start at a realized density of about phages/ml and in many cases may require even higher doses (see also Appendix 4) Keep in mind that penetration into microcolonies should not be considered to be equivalent to phage acquisition of microcolonies: Whereas acquisition involves simply infection of surface bacteria penetration presumably entails infection of bacteria found in a microcolonys interior Keep in mind however that the goal is to develop a framework a model that can be used for understanding phagebiofilm interactions and that these interactions obviously and ultimately are going to be more complex in biofilms than they are in plaques Key: free phages (? black) phage genomes (? gray) prophages (? white) bacteria (?) lysed bacteria (? dashed) binary fission products (? overlapping) inactivated phages (smaller ?) inactivated phageinfected bacteria (larger ?) ?co- super-infection? (coinfection and/or superinfection) ?1? phage? (initial adsorbing phage) ?2? phage? (additional adsorbing phage) ?superinf Killing of bacteria as a function of multiplicity of infection Kinesis is a description of the tendency for some organisms to be more kinetically active in certain environments while less active in others Kinesis is seen for example with pillbugs (a Kirov et al Kirov et al Knezevic and Petrovic explored P landesbioscience Larger particles (dashed line) are unable to enter the bead matrix so follow a path that is much less complex shorter and faster than that of smaller particles (larger particles hence can exit columns sooner than smaller particles) Lastly I reflect on how biofilm multicellularity might impact our perception of this bacterial resistance as analogous to aspects of the immune systems of animals Lastly I would like to dedicate this book to the memory of Professor John Spizizen a mentor and friend recently deceased Lastly the burst on right is more likely to enter the fluid flowing above the biofilm and perhaps is less likely to encounter adjacent surface-located bacteria found below its perch Lawns too can be spatially heterogeneous at smaller scales from bacterium to bacterium and particularly in terms of microcolony formation lentus lentus in these experiments into the planktonic phase Less literally the concept of multiplicity of infection was invented prior to an understanding of many of the limitations to phage infection such as superinfection exclusion where not all adsorbing phages succeed in infecting Less rigorously we can speak of ?control? of biofilm formation where some biofilm development occurs but that biofilm is either then removed or is prevented from progressing beyond some low density Less speculative suggestion of ecological relevance is supplied by experiments showing that deletion of the prophage from bacteria results in a decline in P LIFE CYCLE OF A FOCUS OF INFECTION Gallet et al Like viruses in general phages are diverse in terms of their virion structure the types of nucleic acid that make up their genomes and the details of Stephen T Likelihood of phage acquisition of microcolonies found in different biofilms however presumably increases the greater the number of explorer phages that are produced per focus of infection Little additional detail is provided though the ?preliminary results? presented appear to indicate some success in biofilm eradication with complete eradication seemingly achieved in at least one trial though interpretation is difficult since the x axis on the one graph presented is labeled only with ?level of bacteria? Location of intersections of lines have no meaning Logic though would suggest that it must since how else would depletion of lawn turbidity occur as plaques increase in size To differentiate these various morphological ?zones? found within such plaques in Krone and Abedon we proposed various terms that might be employed if there is a need for strict descriptors Low phage densities as well as reported ratios of added phages to bacteria in the range of Lower multiplicities in turn could be a consequence of lower rates of phage adsorption than one would expect given such high starting phage densities (see also Appendix 4) Lu and Collins engineered phage T7 to express apparently as a soluble enzyme an EPS depolymerase not normally associated with this phage lyses its host cell at the end of its latent period in order to release intracellular phage progeny; contrast with lysogenic or chronic infection Lytic phage A phage with a productive infection that is lytic; most phages including most temperate phages are lytic phages Multiplicity of infection (MOI) Literally the ratio of adsorbed or infecting phages to total bacteria but instead is often mistakenly used to describe the ratio of added phages to total bacteria Obligately lytic phage A lytic phage that upon infecting is unable to display a lysogenic infection; often the term ?virulent? is used as a synonym Permissive host A bacterium that can support a productive phage infection; failure to support an infection can include due to either a bacteriums genotype or its current physiological state; permissive hosts (and conditions) for example are those used to propagate phage stocks; contrast with restrictive hosts (or conditions) Plaque Phage growth within a spatially constrained environment typically involving sufficient local depletion of bacteria that a visible clearing is observed in an otherwise turbid background Productive infection A phage infection that directly leads to the production of virion progeny with virion release either by lysis or chronically Stephen T Lysis from without is phageinduced bacterial lysis that is effected by phage adsorption alone; contrast lysis from within Lysis kills the infected bacterium and terminates the phage infection Lysis of overlying bacteria by phages though might also result in improvement in the physiology of these otherwise buried bacteria and therefore their more effective removal Lysis results in outward phage diffusion establishing new infections and producing additional phage progeny Lysogeny thus can be viewed as costly under circumstances where virus-like growth is more productive than bacteria-like growth Lytic productive infections produce phages that either diffuse laterally within the biofilm (or bacterial lawn) diffuse vertically out of the biofilm or stay approximately in place Many biofilms thus can be viewed from the perspective of predators as consisting of bacteria that are densely available locally but more sparsely available across environments Mathematically the absolute rate at which bacteria become infected in other words the rate at which infected bacteria are created is the product of phage densities bacterial densities and the phage adsorption constant Mayr-Harting expressed a similar idea while addressing the question of why plaque growth does not slow as lawn bacteria reproduce (below) Mean free path decreases less rapidly as overall bacterial densities increase if bacteria instead are found in microcolonies (left) so long as at least some of these additional bacteria are located within the interiors of these microcolonies Microbiology in this way is unlike the study of animals (zoology) plants (botany) or even fungi (mycology) each of which considers at their core groups of organisms that in fact are fairly closely related from genetic developmental and physiological perspectives that is animals are fairly similar among themselves as are also plants and fungi also among themselves Microbiology thus is the study of all members of domain Bacteria all members of domain Archaea and various larger coenocytic members aside all unicellular members of domain Eukarya plus various simple but nonetheless more-multicellular organisms Microcolonies are assumed to have developed to the stages indicated prior to phage exposure (thus going from left to right does not represent a progression in phage propagation or bacterial division in the presence of phages but instead bacterial propagation as it occurred prior to phage contact) Microcolonies during different stages of plaque formation Microcolony steady state (ignoring phage impact) Microorganisms by contrast are not limited to a single kingdom or even a single domain min or about Minimally however keep in mind that biofilm eradication with passive treatment is a function of phage adsorption rates and potentially also of resulting lysis rates (such as to clear overlying bacteria within biofilms for further phage penetration i Minimally these mechanisms imply that plaques will grow in size at Appendices greater rates the faster phages diffuse or the less time they spend infecting Mixing of phages with a quaternary ammonium compound (QUAT) increased bacterial removal over QUAT alone though only slightly Model of phage exploitation of microcolonies and by extension biofilms along with phage movement between and penetration into microcolonies Models need not be mathematical and in biology typically are not MOI which here is equal to -ln(fraction of cells that survive following phage addition) i monocytogenes monocytogenes (L-form) monocytogenes bound to surfaces with over phages/ml (implying an approximation of passive treatment; see Appendix 4) Moons et al More complex phages may display superior adsorption capabilities or survival potential but potentially at the expense of the rate at which their virions can be produced More distantly adsorbing phages however will better define the leading edge of plaque development though at the expense of a longer generation time More familiarly and as noted at least potentially requiring EPS penetration phage infections can be ?immediately productive beginning with phage adsorption and ending with release of phage progeny after a single normal-length latent period (step 3a Figure 6) More formally Skraber et al More generally I consider the ability of prophages to impact biofilm structure (see ?Prophage contribution…? Table 5) More minor processes especially as measured in terms of phage numbers involved are indicated using dotted lines More often however phage-mediated biocontrol or therapy requires forethought and strategy to either achieve or improve upon efficacy More recently they have been identified as powerful agents affecting bacterial ecology and evolution plus are thought to be important players in the global carbon cycle More specifically these represent phage-productive phage-reductive and phage-destructive processes though I often will drop the ?phage-? suffix as a given More-rapid population growth makes it easy to overshoot environmental carrying capacities however ultimately resulting in phage population declines MOVEMENT TOWARDS NEW ENVIRONMENTS Phage population growth can be considered not just within a given environment but also as occurring between environments MULTIPLE DISSEMINATION OPTIONS FOR TEMPERATE PHAGES A given focus of phage infection within a biofilm if initiated by a temperate phage in many cases should produce free phages as well as phage infections that are lysogenic rather than productive (Figure 6) Multiplicity of infection thus can be legitimately described as the ratio of adsorbed phages to bacteria My argument is that historically it was understood that at best MOIinput was an approximation of MOIactual = MOI whereas with time that institutional knowledge was lost resulting in a mistaken tradition that MOIinput = MOI My assumption is that phage-biofilm interactions are more similar to those depicted as a ?Late? stage than they are to those depicted as an ?Early? stage of plaque/lawn development My interpretation is that biofilms at least in their beginning stages of development were not blocked from forming due to insufficient densities of adherent phages but that subsequently formed biofilm was able to support sufficient phage production to result in substantial biofilm eradication i My perspective especially is a view of phages as whole organisms rather than as assemblages of otherwise disparate molecular parts and to focus on how phages might behave ecologically rather than how phages as model systems instead can serve to inform our understanding of other non-phage aspects of biology Namely phages that instead (or additionally) stochastically ?jump? further into bacterial lawns through bacteria-free corridors between microcolonies should similarly contribute more to plaque spread than phages that diffuse relatively short distances before adsorbing natans) that was isolated from a deep well one containing high ferrous ion densities and which they grew as biofilms upon stainless steel coupons along with stainless-steel wire baskets Nearly English-language phage therapy or biocontrol studies in vivo in situ or in vitro have been published in the past years a number of which have explored specifically the phage potential to serve as an anti-biofilm technology (Table 4) Net flow of particles into beads is concentration dependent as also is exit Nevertheless after two hours of exposure to bacteria bacterial densities on catheters were little different with versus without phages (~105 bacteria/cm2) Nevertheless if possible these deviations should be characterized by comparing levels of killing following phage exposure with expected levels of killing given what phage titers are applied the fraction of these added phages that have succeeded in adsorbing and what MOIactual that degree of adsorption translates to Nevertheless it should be clear that if one wants to achieve rapid killing of bacteria both without delay and at a high rate once initiated then employment of passive doses doses which are maintained at high levels and doses which themselves are quite high (such as 108/ml or more) should be assumed to represent a reasonable default protocol unless such efforts are either demonstrably less efficacious or otherwise demonstrably unnecessary Nevertheless it should serve as a reasonable starting point towards further advancement or understanding of both basic and applied aspects of phagebiofilm interaction such as the development of more efficacious approaches to phagemediated biofilm eradiation Newly formed lysogens are unlikely to become established either within biofilms or planktonically unless the newly acquired prophage provides a selective advantage to its bacterium host Nian et al No effort appears to have been made to improve levels of killing of bacteria or otherwise to experimentally explore the infection dynamics leading to the results obtained No matter the route required to reach sufficient phage numbers or sufficient phage penetration passive treatment active treatment or active penetration it nevertheless is crucial in combating biofilms that both adequate phage densities and adequate phage penetration be achieved Nonetheless development of simplified models arguably represents the primary means by which all sciences progress if only because it can be rare to possess all relevant information associated with a given phenomenon Nonetheless if a cm2 area of biofilm were to contain just bacteria a conservative estimation then productive infections per cm2 would still be of bacteria (= × 10-4) Nonetheless if technically true then it is still uncertain what may have caused this failure e Nonetheless innate immunity is quite diverse consisting of physical and chemical barriers to parasite penetration into hosts (e Nonetheless some degree of infectious phage mobility has been observed within experimental biofilms as well as descriptions of phage propagation within biofilms as being plaque-like Nonetheless subsequent stages of plaque formation once bacterial microcolonies have formed likely do better represent phage exploitation of biofilms Nonetheless the study is notable in showing that the addition of ten-fold higher phage densities (producing a 000:1 phage-to-bacterium ratio) gave rise to approximately ten-fold greater killing of bacteria after Phages as Anti-Biofilm Agents 24 hours Nonetheless to the extent that pathogens targeted can be limited (e Not all bacteria necessarily remain perfectly stationary however while nonetheless remaining surface associated Not all phages are successful in this search as they are lost due to decay Not apparent is whether this improved outcome associated with the cocktail was due to the use of multiple phages or instead whether one or more of the phages found in the cocktail was simply superior to the single phage M4 treatment Not indicated is formation of lysogenic microcolonies starting from planktonic lysogenic bacteria or the potential for lytic clearance of host bacteria prior to initiation of a new lysogenic lineage within a biofilm (see text) Not shown is phage adsorption to lysed bacteria that is to bacterial debris Not shown is the extent of possible EPS degradation further phage infection of microcolony bacteria nor potential free phage movement out of the biofilm Notably the publication does do not supply direct evidence for the occurrence of these consequences Note also the collision between plaques especially the lower two T4 plaques (? and ?) with the T7 plaque (?) where the latter is limited in its ability to enter the T4 plaques due presumably to previously existing infections in the latter (i Note especially the delineation of the lower T4 plaques to their left and right which involve temporally later collision than that seen towards the bottom of those plaques Note that before making the latter calculations it will be necessary to determine phage adsorption constants these adsorptions constant determinations are preferably accomplished using multiple data point i Note that different components of the illustration are not drawn to scale Note that focus success can be measured at different stages (as too can also oak tree success) such as in terms of released explorer phage at the point of initiation of a new focus or instead at the point of successful initiation of a subsequent focus though in any case at steady state each existing individual focus produces on average only a single other individual focus Note that for MOI = M = approximately 1% of bacteria survive (actually a bit more than a two-log reduction) whereas with MOI = M = the reduction is over four-log Note that the more heterogeneous a bacterial lawn is in terms of phage susceptibility types then the less clearly any of these zones will appear particularly macroscopically i Note that this formula reduces to POISSON(x M False) = e-M when x = i Note that this scenario to some degree contradicts You and Yin who argue that virus and infected host densities are too low at the leading edge of plaque formation for secondary adsorption to greatly impact rates of plaque development Note that though these examples are interesting I concentrate in the rest of this chapter on studies in which biofilms are explicitly controlled rather than delving into numerous studies where bacterial growth presumably but not explicitly as biofilms has been prevented via phage application such as phage prophylaxis of burns or other wounds Note that with most phages only a single burst is produced under these circumstances (T4 phages with their secondary adsorption-induced lysis-inhibition phenotype are exceptional) Note the turbidity surrounding the central zone of clearing in all four T4 plaques but that it is much more prominent in the T4 wild-type plaques (? and ?) Notwithstanding that interpretation some cell lysis was reportedly observed among dead bacteria in biofilm microcolonies Notwithstanding the above-noted exceptions the implications of this description of phage-biofilm interactions as steady states are many Notwithstanding these caveats the default assumption for understanding phage-bacterial interactions particularly prior to phage-mediated amplification of phage numbers is that Poisson distributions of phages adsorbing bacteria will be an accurate descriptor that results in both an ability to predict phage titers based on bacterial survival (killing titer) and an ability to predict bacterial survival based on phage multiplicity of infection Notwithstanding these concerns the rate at which bacteria become phage adsorbed is a function of phage density and not of bacterial density meaning that the more phages one supplies to bacteria then the faster bacteria will become phage adsorbed Notwithstanding this criticism certainly multi-log reductions in bacterial densities have been repeatedly achieved (Chapter 6) though not in association with development of substantial pharmacological understanding Notwithstanding this evidence Oh et al Now I knew very well that bacterial lawns consist of bacterial microcolonies which are found jammed together to varying degrees Oddly though crude phage lysates were used rather than highly purified phages where the bacterial debris etc Of particular interest is development of a better understanding of phage penetration into bacterial microcolonies of movement between microcolonies and of overall phage productivity during biofilm infection in situ all things that may be addressable in terms of phage plaques Of particular interest was the release of S Often this impact occurs as a consequence of lysogen induction and is a function either of phage production or bacterial lysis On a number of occasions I use this formula e-MOI to gain better understanding of study results (Chapter 6) such as in one occasion where reported ?low rates of killing? p On the one hand it is conceivable that the infection abilities of some phages may be biased towards planktonic bacteria rather than bacteria that are encased in biofilm-specific EPS or vice versa On the other hand (iv) successful spotting of a bacterial lawn can be achieved even given purely passive treatment i On the other hand colorimetric approaches typically have relatively low sensitivity which could allow substantial bacterial presence to go undetected On the other hand non-specific phage diffusion into biofilm ECM is not necessarily blocked (Table and Chapter 3) On the other hand the use of a three-phage cocktail reduced bacterial densities in an E On the other hand there exist numerous clues as to how these phage-biofilm interactions might occur Once completed the effort was so long (about words in main-text alone) that it made sense to market it also as stand-alone book one targeted to those individuals with as much of an interest in phages as in biofilms One also might argue simply that prudence would dictate that phages should not be applied in too-excessive densities for fear for example of anaphylactic responses to either phages or concentrated impurities One barrier to both microcolony penetration and eventual eradication via phage infection alone is bacterial physiology One can assume that non-specific phage capture by the catheter material was the goal which ideally would have resulted in the infection of any bacteria that came into contact with the catheter surface One can speculate that at least some of the genes of unknown function especially found in tailed phages might be involved in optimizing the robustness of phage infections under different host physiological states One category of bacterial predator is their viruses known as bacteriophages or phages One consideration that has not been addressed is the impact on plaque wavefront velocity of phage adsorption to already infected bacteria so-called secondary adsorption One even might differentiate phage strategies into more expedient versus more economical in terms of the degree to which microcolonies are more completely exploited i One is lysogenic infection which in biofilms might develop into microcolonies (Figure 6) One obvious approach as noted involves the mechanical cleansing of contaminated surfaces including the removal of necrotized tissue in the phage therapy of wounds One therefore should be asking whenever viewing a phage molecular adaptation which of these three aspects the adaptation contributes to adsorption latent period and/or effective burst size One way to tie together population growth rates and resource consumption is for resources to be sufficiently low in density that growth rates are inherently low One way to view this slowness is that particle contact with the interface between the bead matrix and the carrying buffer is less likely coming from within beads due to inhibition on particle movement imposed by that matrix than it is from outside of beads Or at least rare unless conditions are biased towards such observation as can be attained through deliberate exposure of biofilms to high densities of phages to which they are susceptible (Table and Chapter 6) org) an ensemble of all things phage ecological Other antibacterial agents such as bacteriocins including the food-additive nisin can be employed in combination with phages Other tradeoffs exist Our model of what happens during biofilm infection by phage assumes that during initial exposure to phage present in the bulk liquid the probability of being infected is greater for cells at the biofilm-liquid interface Outside of this zone of reduced turbidity is where the plaque wavefront is located (at least in terms of phage densities) consisting of a zone of scout-phage infection that in turn is surrounded by the true periphery of the plaque where virion invasion occurs (lateral movement into the bacterial lawn by scout phages) Over the rest of the current chapter I provide an overview of the properties of bacteriophages as well as of biofilms beginning with consideration of the place of phages within the grander scheme that is microbiology Over time foci increase in size laterally while phages that move out of foci have some potential to establish new foci of infection (settlement) Overall as I will suggest not only are the constraints on rates of plaque growth ones that should develop as lawns mature more complex than one might expect but so too are possible mitigating factors ones that go beyond simply that infection numbers should increase as bacterial numbers increase Overall biofilm production was reduced three logs given phage presence but still consisted of bacteria/cm2 at the end of experiments Overview of phage exploitation of a biofilm Particularly with biofilms initiated with lower bacterial numbers low phage doses were capable of providing substantial biofilm clearing implying active treatment Particularly depolymerase can be important for penetrating through capsular material associated also with planktonic bacteria; see for references and review Particularly if there are numerous bacterial hosts within a given environment then a phage may be better off instead displaying a productive infection Particularly the world can be differentiated in terms of the potential for an organism to reach a given location starting at a different location Particularly when greater efficacy is seen with cocktails versus individual Phage Therapy Lessons Gleaned phages it is crucial to explore the efficacy associated with all phages found within a cocktail individually before claims may be made that cocktails provide greater efficacy Passive treatment can be mediated by phages that have been modified so that they are unable to carry out productive infections because of defects in producing progeny phages in releasing those phages once produced or by using non-lytic phages carrying genes that serve as antibacterial poisons Passive treatment in this case i Passive treatment was used (1010 phages/ml) Perhaps a better comparison is that phage T7 engineered to infect this particular bacterial strain was able to reduce biofilms by about two logs while the doubly engineered strain able to both infect the targeted E Perhaps not surprisingly if phage adsorption rates really were that low this level of phage dosing was not sufficient to provide any more than a one-log reduction in bacterial densities phage Phage adsorption and genome translocation thus occur but subsequent replication of the phage genome does not at least not immediately Phage densities at membrane surfaces however were not determined Phage densities were relatively high i PHAGE ECOLOGY AND PHAGE THERAPY Ecology is the study of the interaction of organisms with their environments Phage encounter with these biofilms can result either in phage sequestration away from susceptible bacteria or alternatively and preferable from the phages perspective infection Phage exposure however was short-lived and ?the apparent multiplicity of infection (p PHAGE HYDROLYTIC ENZYMES Many viruses seem to readily enter into biofilms (above) and migrate within them once they have entered (see ?Diffusion within biofilms? Table 3) Phage impact also was relatively small where bacterial densities after three hours were reduced an average of about one and a half log in comparison with a phage-free culture Phage impact was studied under both static and dynamic conditions (without or with media shaking) and though phage association with surfaces was greater under static conditions no difference in bacterial eradication was observed between the two conditions Phage infection characteristics Phage Infection Types and Active versus Passive Phage Therapy Infection type Phages live Phage replication Virions released Bacteria replication Therapy type Chronic Yes Yes Yes Yes Poor Lytic Yes Yes Yes No Active Lysogenic Yes Yes No Yes Poor Pseudolysogenic Yes No No Yes Poor Restrictive No No No Yes Poor Abortive No No No No Passive Antibiotics NA NA NA No Passive It is possible to debate the relative merits of active versus passive phage therapy Phage movement respectively with regard to these three phage types roughly can be vertical that is out of the biofilm (as well as the initial step into biofilms) can be lateral within the biofilm or can occur only minimally (See Figure for summary) Phage penetration into biofilms was monitored as well and ?Putative infected cells became enlarged and spheroplast-like with white dots encircling their periphery? suggesting lysis and possibly subsequently lysis from without PHAGE PLAQUES A plaque is a macroscopic focus of infection that occurs within a bacterial lawn Phage Plaques as Models of Biofilm Exploitation Figure Phage Plaques as Models of Biofilm Exploitation Those phages that by chance diffuse away from their parental microcolony likely will come into contact with adjacent microcolonies Phage Plaques as Models of Biofilm Exploitation Figure Phage Plaques as Models of Biofilm Exploitation Figure Phage plaques can be initiated either as free phages or with phage-infected bacteria with either qualifying as a plaque-forming unit (PFU) Phage plaques grow through a combination of phage infection and virion diffusion Phage progeny are released in this burst to begin their extracellular search Phage propagation within a biofilm as plaque like PHAGE RESISTANCE AS IMMUNE-SYSTEM ANALOG The physical association of bacteria that are clonally related as can be seen in microcolonies or biofilms can result in a ?spatial vulnerability? to phages Phage strategies that emphasize rapid acquisition of bacteria such as shorter latent periods can be favored for example when bacteria are more available Phage T7 was supplied for one hour at a concentration of 1010/ml Phage Terms and Their Definitions Terms Definitions Burst (size) Lysis of a phage-infected bacterium releasing free phages; burst size is the number of phages released in a burst (usually measured as a population average) Chronic infection Productive phage infections in which phage progeny are released in a manner that does not terminate the phage infection; chronic infections are not lysogenic infections though the productive cycle for a small number of temperate phages is chronic rather than lytic Destructive infection A phage infection during which the phage is inactivated such that neither a productive nor reductive infection is completed Filamentous phage A phage such as coliphage M13 that is long and thin in morphology and displays a chronic productive infection Free phage A bacteriophage virion that has been released from its parental infected bacterium but has not yet adsorbed to another bacterium Immediately lytic infection A phage infection that immediately upon infection enters a lytic cycle; contrast with both lysogenic infection and induced lytic infection Induced lytic infection A lytic infection that follows the induction of a lysogenic infection; contrast immediately lytic infection as well as induced chronic infection Induced productive infection A productive infection that follows the induction of a lysogenic infection; for most phages these infections are lytic though for temperate filamentous phages they instead are chronic Induction The conversion of a lysogenic infection into a productive infection Lysis inhibition Associated with T4-like phages lysis inhibition is an extension of the normal phage latent period and is induced via secondary adsorption by a similar phage Lysogen A bacterium that contains a phage displaying a lysogenic infection i Phage therapy Phage Therapy The impact of different treatment approaches often cannot be predicted before hand and particularly so without substantial understanding of both biofilm properties and basic principles of phage biology and ecology Phage therapy doses in most instances can be and therefore should be administered such that phage densities do not substantially decline over the course of treatment even if or perhaps especially if one is effecting active treatment and this is rather than towards establishment of some hypothetical added-phage-to-bacterium ratio Phage therapy in addition can be viewed as a form of applied phage ecology specifically community ecology where phage-bacterial interactions are encouraged to result in declines in bacterial densities Phage Therapy Lessons Gleaned CONTROL IS MORE COMMON THAN PREVENTION Just when during biofilm formation a ?biofilm? truly has become a biofilm will differ depending on how one defines the term Phage therapy also known as phage-mediated biocontrol is the application of phages to either control or eliminate nuisance or pathogenic bacteria Phage therapy/biocontrol of biofilms can be viewed as employing this natural focal exploitation by phages but with sufficient foci and/or phage types applied that a majority of bacteria are infected and cleared Phage treatment (up to phages/ml) at cold temperatures (4°C) reduced bacterial densities by a little more than one log within a single day (compared to five or more logs for alkaline Stephen T Phage treatment alone reduced biofilms to the same extent as combination therapy about six logs though this was insufficient to completely eradicate the biofilm Phage treatment in this study seems to have reduced initial measurements of biofilm density by about one log compared to not at all for Curtin and Donlan Phage-bacterial steady states with planktonic bacteria are also possible but only given sufficiently low bacterial and controlling nutrient densities in combination with sufficiently high phage loss rates Phage-Biofilm Interactions Bacterium Perspective confined to the interiors of microcolonies is an important question as Webb et al Phage-Biofilm Interactions Bacterium Perspective Filippini et al Phage-Biofilm Interactions Phage Perspective Table Phage-Biofilm Interactions Phage Perspective Table Phage-Biofilm Interactions Phage Perspective INFECTION OF BIOFILM BACTERIA BY PHAGES LYTIC AND TEMPERATE Degradation of EPS can be relevant to phage infection of many bacteria not just those found in the biofilm state Phage-Biofilm Interactions Phage Perspective Figure Phage-biofilm interactions may be a great deal more complicated than can be captured even in terms of tradeoffs in phage-biofilm exploitation strategies Phage-destructive infections are ones in which the phage dies that is they are destructive to infecting phages but not necessarily to the infected bacterium (for Lwoff these would be abortive infections) Phage-infected clusters of bacteria even if consisting of multiple infected bacteria too can serve as individual plaque-forming units Phages and various products can be supplied in combination as illustrated via the application of a cocktail in Figure Phages are distinguished by their position rather than genotype or necessarily phenotype and arrows connect phages with roles Phages as Anti-Biofilm Agents Given the mid-course cessation of passive treatment the eradication approach employed by Sillankorva et al Phages as Anti-Biofilm Agents Attributes References Reviews (marked with in subsequent rows) Eradication of existing biofilms (full partial) Prevention of biofilm formation ?Control of biofilms Use of cocktails Mixed biofilm treatment Treatment along with chemical antibacterials Electron micrographic impact documentation Crystal violet (etc Phages can even become non-specifically trapped within biofilms that lack bacteria susceptible to those phages Phages can increase their mobility given encounter with EPS by deploying EPSdegrading depolymerases (see for reviews as well as ?EPS depolymerases? Table next page and Figure 3) Phages containing a LacZ (?-galactosidase-encoding) gene were also employed resulting in chromogenic detection of T4-infected bacteria (which otherwise were unable to metabolize lactose) Phages found further from a plaques or focus of infections periphery call them settlers are responsible for acquiring plaque- or focus-interior bacteria ones that have not yet become phage infected Phages seem to be capable of diffusing within biofilms (see ?Diffusion within biofilms? Table as well as discussion of phage EPS depolymerases above) Phages that are released especially high above a biofilms surface such as from biofilm surface-projecting microcolonies may be less likely to reach adjacent microcolonies due to a combination of vertical distance and potential to enter flow that carries phages away from their parental microcolony Phages were isolated from sewage and grown to stocks of only to phages/ml Phages for example can be supplied in sufficiently high numbers that subsequent phage reproduction in situ is unnecessary to achieve complete infection and killing of target bacteria Phages for example can be tailed or tailless Pharmacodynamics can also be subdivided into considerations of the negative impact of drugs that is side effects (also known as secondary pharmacodynamic effects) along with the positive impact i Pharmacodynamics refers to the impact of drugs on the body where body can be defined broadly to include not just the bodys own tissues but also symbiotic organisms such as pathogenic bacteria PLAQUE FORMATION AS A MACROSCOPIC PHENOMENON To the extent that plaques may serve as a tractable model of phage interaction with surface-associated bacteria then it may be profitable to consider plaque development on more macro scales i Plaque formation can be considered to occur in a number of stages such as initiation progression outward from that initiation and in many cases an eventual termination of the formation process Plaques are visible in this media either because bacteria are prevented from growing extensively because bacteria are outright killed or because bacteria are killed and lysed Plaques can be clear or display varying degrees of cloudiness Plaques can be formed by carnivorous bacteria (such as Bdellovibrio ) or as a consequence of virus propagation Plaquing is a vital component of phage host-range determination pneumoniae biofilms Points that are external to the circular microcolonies (numbers indicate how many) thus represent those virions that have at least started their journey away from rather than towards the parental microcolony Poor adsorption is also suggested by the many hours (four) required by these phages to start to reduce viable counts in separate broth-culture experiments given a starting phage density of 109/ml versus a starting bacterial density of 107/ml (see Appendix for relevant calculations) POPULATION GROWTH A population consists of a group of similar individuals that either are clonally related or instead which together make up a single inter-mating gene pool Populations generally can be considered to be increasing decreasing or holding steady in terms of their size or density Possible explanations can include unexpectedly low adsorption constants (which in any case should be determined) or antagonistic interactions if multiple phage types are simultaneously applied (i Preadsorption can play roles in host-range determination as well where one can distinguish between an EOP (efficiency of plating) on a given host versus an ECOI (efficiency of center of infection) Preservatives are used by contrast to prevent or at Stephen T Presumably they do this by allowing particles to diffuse into the gel-like biofilm extracellular matrix (ECM; see ?Trapping by biofilms? Table 3) PREVALENCE OF PRODUCTIVE PHAGE INFECTIONS IN NATURAL BIOFILMS The consequence of lytic phage infection of biofilm bacteria will be eventual erasure of evidence of that infection Prevention in medicine can be described as prophylaxis such as treatment without recognition that any bacterial pathogen is present whereas eradication would be the goal of treatment of an already apparent infection PREVENTION IS OF LIMITED UTILITY The assumption when striving towards prevention of biofilm formation are either that bacteria are more susceptible to phages prior to biofilm formation or that not giving bacteria a chance to form biofilms is otherwise desirable Prevention is the blocking of the initiation of biofilm formation Prevention of biofilm formation literally must entail a failure on the part of target bacteria to either adhere to surfaces or given initiation of colonization then a failure to replicate to the point where EPS and other biofilm aspects come to dominate cultures Principally if levels of phage adsorption are assumed to be equal to the product of phage density (P) phage adsorption constant (k) and time and bacterial survival is given as equal to Prior to that point however the continued maturation of the lawn gives rise to various relevant changes Probably as a consequence learning the Poisson distribution seemingly has fallen out of favor among phage biologists much to the detriment I would argue of our understanding of efforts to use phages as antibacterial biocontrol agents (phage therapy) Problems associated with such inter-phage interactions however I would argue are ones associated with insufficient repetition of dosing Productive infections can be either chronic or lytic and I focus on the latter Productive infections produce phage progeny that are then released from infected bacteria either via lysis (the majority of phages) or alternatively some phages instead are released chronically (especially filamentous ones) Protection of individual bacteria from phage infection is reviewed elsewhere Protocol augmentations include multiple dosing as well as the use of a phage cocktail to prevent Stephen T Pseudolysogeny (middle-left) is shown being resolved as either a productive infection or as a lysogenic infection (though the arrows indicating this resolution are themselves not labeled) Pseudolysogeny thus may be viewed as a phage means of avoiding destructive infections that could result from infection of starved bacteria or alternatively as a phage means of delaying productive infections until a time when phage per-cell productivity (burst size) may be greater Rate of movement from carrying buffer into bead matrix thus initially is higher than rate of movement out of bead matrix Rates of secondary adsorption shouldnt increase as fast as bacteria increase in number due to a dependence by the former on the immediately local geometry of bacterial associations which should not change as fast as bacterial numbers given bacterial growth as microcolonies (see Figure 17) Recently the journal Current Pharmaceutical Biotechnology published a special issue on phage therapy including articles on phage choice isolation and preparation phage modification phage therapy pharmacology phage treatment of plants phage treatment of foods and phage treatment of infections in people ; see also Reconciling these contrasting views that biofilm bacteria can both be susceptible to phages and not and as discussed in Chapter a perhaps reasonable working hypothesis is that some phages are better at infecting biofilm bacteria than others that such infections do not necessarily give rise to macroscopically observable damage to biofilms (hence are difficult to appreciate) and that phage infection of natural biofilms may be common but nonetheless not abundant Reduction in cell density by one-half requires an MOI of Reductions in plaque wavefront velocities at higher bacterial densities could also result from declines in phage burst sizes or increases in phage latent periods that can occur due to bacterial nutrient limitation or due to aging towards stationary phase Reductions were at most about one log as determined via crystal violet staining (though note that crystal violet staining can potentially greatly underestimate cell killing ) Reductive infections are either lysogenic or pseudolysogenic Regarding biofilm-forming bacteria it is uncertain whether these newly reductive infections predominantly are of planktonic pre-biofilm bacteria or instead involve bacteria already making up biofilms Regarding impact of phages alone they report (p Relative radii (r) of microcolonies are as indicated (1 and infinity) Release also can occur with dispersing bacteria remaining instead as biofilm-like clumps of cells (sloughing or clumping dispersal) Relevant questions therefore include: Are lysogens of biofilm-producing bacteria generally first formed within biofilms If yes then for how long following that establishment do these infections tend to remain biofilm associated Figure Removing biofilms via phage action or even just preventing biofilm formation consequently can be a challenge Resch et al Restriction-modification systems nonetheless are generally acting i Restriction-modification systems of bacteria by contrast are a means of distinguishing self from non-self DNA but are fixed in their specificity except over evolutionary time Reviews of numerous aspects of phage biology can be found in a number of recently published phage books ; see also Hyman and Abedon for a general overview of this biology S57) Scenarios for temperate phage interaction with and dissemination from biofilms Schematic of lytic phage life cycle Scholl et al Schuch and Fischetti noted a positive relationship between lysogeny biofilm formation and EPS production in B SCOPE AND OVERVIEW OF BOOK As indicated by the title my emphasis here is in the exploration of phage-biofilm interactions from ecological as well as applied (that is phage therapy) perspectives Scouts settlers and explorers in phage exploitation of solid-phase bacteria Second I consider biofilms from the standpoint of their resistance to phage infections considering for example whether biofilms in fact are more resistant to phage infection than are planktonic bacteria Second while biofilms may display an ongoing susceptibility to phages it may be relatively rare for phages to damage biofilms in a manner that is macroscopically visible and particularly so to the extent that biofilms are highly heterogeneous in terms of the types of constituting microorganisms Second while this calculation estimates the rate of phage adsorption to bacteria it does not necessarily describe the rate of bacterial loss to phage adsorption particularly given high multiplicities of infection (actual) since phages have the potential to multiply adsorb individual bacteria (see Appendix 3) See Abedon for earlier formulation of these latter ideas that phages found in different locations within plaques may play selectively different roles See Adams and Stent for application of Poisson calculations to single-burst experiments determinations of phage lysis by turbidimetric means the probability of recombination of genetic markers among phages phage survival following UV irradiation etc See also See also Appendix See also Chapter See also Figure (next page) See also Figure See Appendix for further discussion of these calculations See Chapter for further discussion of these concepts See discussions of killing titers (Appendix 3) and bacterial reduction times (Appendix 4) for consideration of just what phage densities in broth cultures may be necessary to achieve passive treatment See Figure (next page) for illustration of the logic involved See Figure for illustration (next page) See Figure for illustration See Figure for overview See Figure (previous page) for summary of these ideas See Figure for summary of the various pathways described in this section See Figure for summary (following page) See Figure (p See Figure (p See Figure for definition of focus of infection (shown here against a biofilm background top-middle) See similarly the bottom panel of Figure See Table (next page) for summary of factors that can lead to increased plaque cloudiness See Table for a comparison of various Poisson-based predictions given different values of x or MOI See Table for estimations of NM/N0 for various values of M Selection acting on scouts can be for greater expedience i Semantically ?infection? implies phage adsorption to a bacterium followed minimally by phage-genome translocation into the bacterial cytoplasm Settlement is the initiation of a focus of infection emigration is movement relative to the parental microcolony and penetration is movement further into the parental microcolony Settler phages produce progeny that contribute to the fecundity of the plaque Stephen T Sharma et al She postulated that plaque wavefront velocity is controlled by distances between bacteria with greater distances resulting in faster plaque growth: ?…as the individual bacteria develop into microcolonies the distance between them is only slowly reduced at first and the advance of phages between the individual infection-centers goes approximately as slowly and this results in the arithmetic growth curve of the plaque diameters? (as translated from the original German see ) Shown is a hypothetical plaque within a bacterial lawn or focus of infection within a biofilm Shown is a vertical cross section of a column containing filtering beads Shown is an elaboration of the figure presented as Figure Sillankorva et al Sillankorva et al Sillankorva et al Similar results were seen with application of phage-free depolymerase alone to phage-sensitive bacteria Similarly analogous is the potential for bacteria to sequester and then inactivate phage invaders just as phagocytosis serves animals as a means of sequestration of foreign materials Similarly important it is absolutely necessary that dosing information be provided in publications in a manner that allows replication of results by others Similarly antibiotics or EPS depolymerases that could be included in formulations should be shown to display biofilmdisrupting ability Similarly for bacteria that exist as clumps or arrangements the concept of the killing titer will apply not to phage impact on individual bacteria but instead to phage impact on these multiple aggregated bacteria unless those bacteria are separated from each other prior to their enumeration Similarly increased secondary adsorption like greater phage adsorption rates in general should help to counter the proposed increase in infection numbers Similarly it can be difficult to think through problems without some means of framing them in reasonably familiar terms and a good model will provide just enough simplification and familiarity that novel insights may be obtained Similarly the potential for biofilms to remove viruses from water should be a function in part of biofilm quantity Similarly this would be equivalent to multiple overlapping plaques growing within a single lawn Similarly though EPS could serve to slow phage movement between bacteria it seems unlikely that EPS could have evolved as a means by which bacteria protect other bacteria Similarly too low bacterial densities may be insufficient to prevent phage and therefore focus extinction Since compromise between conflicting strategies may be required for effective phage exploitation of biofilm bacteria the result could be less vigorous phage impact on biofilms than the high bacterial densities as found within biofilms themselves would intimate Since e-0 Since phage interactions with bacteria are interspecific these are considered to be community ecological Stephen T Size-exclusion (gel-filtration) chromatography as a model for non-specific virus-biofilm interaction Size-exclusion chromatography (Figure p Smaller particles (solid line center-left in figure) enter the bead matrix and follow a complex path through this matrix So too can bacterial defenses against phages be classified with CRISPR mechanisms notably analogous to adaptive immunity since this resistance ?adapts? in response to phage exposure So too replication fidelity might be sacrificed for the sake of virion-maturation rapidity or brevity of the phage eclipse So too the phage generation time should be shorter when bacterial densities are higher except to the extent that bacteria physiologically decline in their ability to support phage infections when they are found at very high bacterial densities (i Some capsules or additional EPS as noted can be protective against at least some phages thereby serving as a barrier Some fraction of phage-progeny produced diffuse into overlying bulk water to potentially found new foci of infection Some phage might escape to the bulk liquid where they could infect planktonic host cells Son et al Specifically the concept of multiplicity of infection as a ratio of phages added to bacteria is misleading especially when bacterial densities are low Specifically the Poisson distribution supplies two key predictions: The number of bacteria that avoid phage infection and the number of bacteria that are multiply adsorbed both for a given ratio of adsorbed Stephen T Specifically they use the term ?settlement? as equivalent to ?attachment? to describe the initial phage-biofilm interaction Stars are illustrated as relatively tiny in radius to suggest that the early post-burst diffusive behavior of virions is more important in determining the likelihood of adsorption to the parental microcolony than later behavior Stated as unambiguously as possible: Low phage densities or singledosing protocols should not be relied upon to clear bacterial cultures unless of course such treatment strategies have been shown to be efficacious based upon rigorous experimentation Stephen T Stephen T Stephen T Stephen T Stephen T Stephen T Stephen T Stephen T Stephen T Stephen T Stephen T Stephen T Still if lower phage doses do not achieve complete or at least near-complete removal of a biofilm then it may be prudent to at least explore the potential for higher doses to achieve this removal i Strategies that instead intervene early in rather than prior to biofilm formation especially that involve active treatment should be viewed as more equivalent to vaccination Strictly speaking however we would not expect biofilm EPS to have evolved as means of non-specifically trapping viruses study study simply may have failed to hit upon an equivalent time point to that seen by Curtin and Donlan in which both densities were the same with versus without phage treatment Subsequent EPS depolymerization occurs along with dissemination of released free phages (initial stages of the latter is shown as black arrows) Subsequent modeling of the impact of the phage adsorption constant on plaque size however failed to support the ?negative impact? hypothesis instead suggesting that adsorption constant increases could augment plaque growth rates up to a point but changes once adsorption rates are high enough would have no impact Subsequently adsorbing phages are ?secondary? phages though many authors use the terms ?superinfection? as a synonym Subsequently phages have been employed towards treating bacterial disease as a means of elucidating the basic principles of molecular genetics to detect fecal contamination and towards identification and detection of bacterial strains Subsequently though adherent bacteria increased in number without phage treatment whereas with phage treatment they decreased Substantial breadth of activity therefore may be readily engineered into formulations consisting of both phages and soluble enzymes assuming local application to readily disrupted biofilms subtilis and P Such adsorption at least hypothetically will have the same impact on phage viability as adsorption to already-infected bacteria Such competition between foci though should be less likely the lower the prevalence of phages within biofilms (Chapter 3) Such dosing is easy to achieve if the ratio of the toxic density to the effective density is high meaning that much more drug must be supplied to cause damage than is necessary to achieve efficacy Such expedience is of greater utility to phages given greater availability of bacteria Such immediate productivity can occur upon infection by either temperate phages lysogeny-incapable filamentous phages or of course obligately lytic phages Such media in either case consists of gels liquid on which solid-like spatial structure is imposed Such passive-only phages meanwhile can be quantified without plaquing via killing titer determination (Appendix 3) Such targeted killing may be achieved given ?efficient phage application particularly at high titers so that passive treatment is achieved Such treatments will have to be continued for as long as a wound continues to be susceptible to contamination and infection Suddenly in other words plaques were a complex ecological phenomenon with subtle spatial heterogeneity even within genetically homogeneous lawns Summarizing: Higher concentrations of viruses in the overlying water along with higher attachment rates to biofilms should increase rates of virus trapping whereas higher detachment rates from biofilms along with lower inactivation rates within biofilms should increase virus densities returning to bulk water Summary of forces impacting plaque wavefront velocity particularly as bacterial lawns mature Summary of proposed local dynamics of plaque formation Superinfection strictly is the coinfection of a single cell by more than one phage where there is a time differential between the addition of the two phages Table Table Table Table Tait et al Taking advantage of these tendencies biofilters can be created to non-specifically remove viruses during water treatment (see ?Membrane bioreactors? ?Wastewater treatment? and ?Use of wetlands? Table 3) Taking up the entire bottom of the field of view is a phage T7 plaque (?) Tellingly the syllabus to the Cold Spring Harbor Phage Course as published in 1950 notes ?Since adsorption of phages is never 100% the actual multiplicity has to be determined for each experiment? (p Temperate phages that instead display chronic productive infections also exist the most notable of which is phage CTX? of Vibrio cholerae which has the distinction of encoding the toxin responsible for the disease cholera Temperate phages for example can display either productive or lysogenic infections Terms Definitions Prophage A phage genome as it exists during a lysogenic infection; different prophages can exist either inserted into bacterial chromosomes or as cytoplasmically located plasmids Pseudolysogenic infection A phage infection in which initiation of either productive or lysogenic infection is delayed and during which phage genome replication does not take place Reduction to lysogeny The process whereby a newly infecting temperate phage establishes a lysogenic infection (lysogenization); the generation of a reductive lysogenic infection (sensu ?reduction into prophage? p That and described multiplicities no greater than That bacteria typically are physiologically heterogeneous within biofilms is also a concern That frequency may be viewed as a conservative estimation if we take into account that 4/15 is nearly three times that value and that phage virions are visible within infected bacteria for only a portion of the total latent period resulting in detection biases towards false negatives That I would write a book on this subject came to me as a complete surprise That interpretation however seems to contradict the movement of ex situ-labeled phage particles throughout the biofilm (above) so it is possible that successive bacterial lysis was more a function of infection access to nutrients rather than solely phage penetrated to underlying bacteria i That is at some point biofilm densities may be reduced sufficiently that phages cannot be produced faster than they are lost to bulk water That is at the same time that it Stephen T That is earlier adsorbing phages will produce new phages sooner That is even with phage densities as high as 107/ml the resulting killing of bacteria can occur at rates that are about four hours per decimal reduction (230 min) and even then only if phage densities are not allowed to decline over time That is given abortive infections phages are sequestered by bacteria with both infecting phages and infected bacteria are destroyed That is given sufficient time the ratio of added phages to bacteria present will come to equal the number of adsorbed phages to bacteria or at least will do so up to the adsorption capacity of bacteria and ignoring the potential for both phages and bacteria to fluctuate in their numbers over time That is it should be advantageous to phages for enzymes to be present at the point of phage adsorption by being virion attached rather than present only immediately following the lyses of phage-infected bacteria (Figure 5) That is just as selection should favor sooner lysis over greater per-infection fecundity if new bacteria are easily acquired That is Kochs molecular postulates were not applied to this system though see the work of Rice et al That is not all bacteria making up a biofilm may be found within the host range of a given attacking phage That is not only is an explicit assumption made that phages will be amplified in situ to sufficient densities to achieve biofilm control but this amplification is assumed to produce sufficient phage densities following only a single dosing That is phage production such as may occur within a biofilm or in overlying bulk water or media is not required That is presence of phage densities necessary to allow active phage amplification (unless of course phage amplification is achieved via infection of planktonic bacteria) That is prevention tends to require ongoing vigilance applied to large otherwise minimally affected populations rather than targeted ?after-thefact treatment of only those individuals who or entities which are demonstrably affected That is so long as phages are able to effect lysis then even reproduction-incompetent infections may be able to effect active penetration given provision with sufficient phage numbers That is these phages are settlers rather than scouts That is unless populations are either increasing or decreasing in number then each clonally established bacterial microcolony will on average produce only a single bacterium that goes on to establish a new microcolony (Figure 9) The advantage of food treatment is that in principle very large numbers of phages may be applied so that passive treatment is achieved The advantage of lysogen existence within a biofilm would be the production through binary fission of lysogen progeny (step 4b Figure 6) The alternative to immediately productive or previously established lysogenic infections is the process of reduction to lysogeny by a temperate phage that is a phage infection that forms a lysogen The answer isnt even so small as to be effectively zero at least as seen from a phage therapy perspective The argument behind the latter claim is that only outward phage diffusion contributes to plaque-size increase whereas the infection process itself cannot directly do so (bacteria-infecting phages that is are assumed to be as stationary as their hosts) The arrow showing this second burst is dotted to indicate that it occurs well after (at least one latent period) that of the first burst shown to its left The attachment component conversely may or may not positively impact rates of plaque development and in fact might negatively impact those rates The authors also speculate that lysis inhibition may have occurred in the course of phage infection potentially giving rise to a delay in lysis of bacteria as induced by multiple phage adsorption per bacterium The authors also suggest that all of the cells in the biofilm became phage adsorbed The authors supply an alternative explanation : ?This might be due to the lower number of cells that remained on the surface after phage infection which results in a more difficult contact between the adhered hosts sic cells and the phages present in the flow The bacterium so-infected is then assumed to produce a full burst of phages though with the caveat that were the arrangement to consist of more than two bacteria then further phages may be lost to secondary adsorption The burst found to the left presumably produces phages with a greater propensity to infect adjacent surface-located bacteria The calculation for the top graph is (fraction bacteria killed) = POISSON(0 MOIactual False) using Microsoft Excel® while the calculation for the bottom graph is (fraction bacteria survived) = POISSON(0 MOIactual False) = e M where M = MOIactual The combination of a need for ongoing phage presence and requirements for passive treatment to actually prevent biofilm formation should place an emphasis on more ?efficient phage use The complication on these considerations occurs when ingredients are able to enhance the functioning of other ingredients but without demonstrable efficacy if used in isolation The concept of ?deep is more ambiguous describing distance from a biofilms upper surface rather than necessarily distance from nutrients etc The concept of killing titers stems from two considerations The concerns about active versus passive treatment (Figure 1) are also not as severe for biofilm eradication The cycle is completed by ?emigration or diffusion of the newly emerged phages out from the current focus of infection into liquid carrier for translocation to new biofilm patches The discrepancy is suggestive that phage production resulted in additional phage acquisition of bacteria The early history of phage biology was even marred by controversy over the degree to which phages were distinct from their bacterial hosts and over two decades elapsed between the discovery of phages and their first viewing as distinct entities via electron microscopy (contrast other particularly non-virus microorganisms which often were viewed in some detail through microscopes before they were otherwise characterized in vitro ) The easiest way to determine the fraction of bacteria corresponding to these latter numbers is via the POISSON function of Microsoft Excel® The efficacy of phage treatment was measured in terms of membrane performance with substantially greater retention of permeability given phage treatment The exception to that statement is when protected bacteria are clonally related to EPSproducing bacteria The exit in particular gives rise to an expanding bubble of net-outwardly diffusing phage virions and these virions randomly encounter bacteria found in their vicinity (Figure 13) The explorer phages with their vertical movement in turn are involved in both emigration and subsequently settlement The extent of diffusion away from the parental microcolony potentially affects the likelihood of secondary adsorption of infected bacteria already occupying the parental microcolony with less outward diffusion resulting in more secondary adsorption The extent of these losses may be enhanced via the application of greater numbers of phages (towards passive treatment; Figure Chapter and Chapter 5) or by limiting the potential for phage diffusion away from biofilms Stephen T The first argument stems from concerns that too-high phage doses even if phages are inherently non-toxic could result in the generation of bacterial lysis products that in fact are toxic The first concern is the duration of treatment which for prevention will necessitate repeated or continuous dosing for as long as a surface is subject to possible colonization The first is that any bacteria-free corridors between bacterial microcolonies likely become narrower (Figure 13) The first is that upon phage-induced lyses of overlying bacteria the previously interior but now surface-exposed bacteria may again acquire nutrients and oxygen plus Stephen T The first is the phage multiplicity of infection (MOI) and the second is the value that corresponds to the number of bacteria adsorbing (or infecting) a specific bacterium The first mechanism involves phage-virion acquisition of bacteria found adjacent to an infection within the same biofilm as equivalent to phage plaque formation (step 4a Figure along with Figure 7) The first steps in debugging phage-mediated antibacterial protocols thus should involve questioning both dosing and penetration adequacy as basic pharmacological principles The first and more important is understanding how to do the various manipulations required to both dilute and titer phages The five-fold jump from lowest to highest phage density gave rise to a two-to-three-fold increase in the rate of bacterial removal which could be seen as evidence for the potential utility not just of passive phage application but also passive treatment at very high phage densities The following discussion should be seen as complementing various critical MOI discussions found elsewhere The fraction of bacteria of only those that have been phage adsorbed can be more relevant to the study of phage biology however since in many cases only those bacteria that are phage adsorbed are immediately relevant e The grander points are that there is much to learn about phage plaque formation that a diversity of phage-bacterial combinations as well as plaquing conditions may be explored and that at least some of what is discovered may provide insight into phage-bacterial Stephen T The immediate environment thus is relatively easily reached whereas especially more distant environments can be more difficult to reach The immune system of especially vertebrate animals is commonly distinguished into innate and adaptive components The impact of the phage adsorption constant on plaque wavefront velocity is more complicated than the role of either burst size or latent period The implication from these calculations is that realistically phage therapy will not be highly effective even if phage numbers are replenished (adsorption with phage production) until phage densities are in the range of 108/ml or possibly higher unless long delays between phage addition and substantial bacterial killing one finds acceptable The implication essentially is that much lower phage multiplicities were achieved than were expected given the high phage densities employed placing changes in observed killing within an approximately linear range as a function of multiplicity as seen in the upper plot (Figure 12) e The importance of the second mathematical concept that of the Poisson distribution is more subtle as well as more narrowly applicable The important question is how far on average a particle/phage can flow/diffuse before encountering a bead/microcolony to which it attaches/adsorbs with the latter impacted by particle-bead affinity as equivalent to the phagebacterium adsorption constant: The lower the ratio of bead surface area to column volume along with the lower particle affinity for beads then the longer the distance particles should diffuse (see also Appendix 5) The initial and important observation was that phage adsorption as indicated by increased bacterial fluorescence occurred at a greater rate when more phages were supplied The initial stages of plaque formation are similar to phage population growth as it occurs in the presence of planktonic bacteria except that phage diffusion is somewhat reduced The interiors of microcolonies as well as their bases for example can display reduced metabolic activity or even bacterial death e The interpretation might be that very low adsorption rates negatively impact plaque growth rates but that once some sufficient rate of adsorption has been attained then adsorption rates no longer are relevant The key in these instances is distinguishing between phage amplification as it might occur within biofilms themselves which is a legitimate phenomenon when treating biofilms with phages and phage amplification as it might occur in planktonic bacteria overlying biofilms The killing of resuspended and disaggregated biofilm bacteria was similar to that of planktonic stationary phase bacteria and also was not substantial The larger colonies seen towards the bottom of the field of view presumably are phage T7- resistant bacteria while the large number of dots that can be seen throughout the field of view (lighter dots seen against darker backgrounds) especially above these larger colonies I interpret to be bacterial microcolonies The latter approach requires not just the killing of bacteria and perhaps their lysis The latter as found in combination with bacteria-produced EPS are described as biofilms The latter calculations determine the fraction of bacteria multiply infected of the total number of bacteria both adsorbed and not adsorbed rather than the fraction of bacteria of only those that have been phage adsorbed The latter can either remain more or less distinct within competing microcolonies or at the other extreme can be built up via intra-specific cellular adherence The latter can include such phenomena as mutual exclusion where one phage upon coinfection blocks the production of another phage or the depressor effect where an infections burst size is reduced given coinfection by two dissimilar phages The latter gives rise to what I describe as an effective burst size which is not the number of phages initially produced by an infection but instead the number that survive to produce infections of their own The latter includes involvement in the editing of three volumes one as contributing editor (?with editorial assistance by The latter is a means of demonstrating infection of an unknown phage host the preadsorbed center of infection by plating that infected host on a plaquing-permissive bacterial lawn The latter is particularly relevant under circumstances where phage application must occur over large volumes or areas such as with phage treatment of plants The latter is sufficient to effect passive treatment where sufficient phage densities to acquire most or all bacteria present are supplied exogenously (shown as multiple arrows middle of figure) The latter is to be expected since bacterial densities were low close to 105/cm2 at the point of switch over from passive to active treatment while the likelihood of rapid phage loss from these biofilms to bulk water was high (plus phage adsorption rates to bacteria may have been somewhat low; above) The latter for most phages occurs as the bacterial lawn enters stationary phase The latter microcolonyexploitation process is indicated by smaller dashed lines involving the settler phage generation of more settler phage The lawn consists of wild-type E The level of complexity I propose is greater than what can be found using cellular automata or especially wavefront models of plaque formation The lighter oval is a lysing bacterium The likelihood of phage movement through bacteria-free corridors during plaque formation could similarly vary as a function of position in or on a microcolony from which phage release following bacterial lysis occurs The lysis of the infected bacterium releases phages which since they are in the immediate vicinity of the other bacterium likely will adsorb that bacterium with perhaps more than one phage adsorbing to the second bacterium (hence the indication that the original burst is the normal burst size less some amount that is potentially greater than one phage) The major development pathway (solid line) consists of explorer phage vertical movement out of biofilms (a form of emigration) which is followed by settlement scout phage formation settler phage formation and subsequent phage amplification especially by settler phages resulting in substantial explorer phage production The majority of members of domain Eukarya as well as the majority of kingdoms making up domain Eukarya are neither animals plants nor fungi but instead are microorganisms combining to make up a large fraction of what formerly was described solely as kingdom Protista The majority of phages also possess dsDNA genomes including all tailed phages (order Caudovirales consisting of families Podoviridae Myoviridae and Siphoviridae) The majority of phages though possess tails which they use as their adsorption organelles i The manuscript began as a chapter called simply ?Bacteriophages and Biofilms? to be written for a book on biofilms but for which at first I had only modest ambitions The method used can be described as ?inefficient (see Chapter 5) as it involved the co-immersion of phage (>109/ml) target bacteria (~106/ml) and stainless steel surfaces The microcolonies formed by biofilm bacteria can be complex in shape taking on appearances reminiscent for example of mushrooms The middle burst also would infect surface-located bacteria but would be biased towards either encountering such bacteria within the surrounding void or at least potentially entering biofilm ECM lining the void The mixing of phages with foods before their packaging for example can be viewed as a biofilm prevention strategy and in fact one that is fairly ?efficient to the extent that fluid volumes overlying foods tend to be small The model leaves a number of unanswered questions The most common such phenotype is the display of superinfection immunity against similar phages though also common is the expression of bacterial virulence factors such as exotoxins The most obvious constraint is in terms of their movement relative to the surface to which they are attached with biofilm bacteria remaining attached to these surfaces through multiple bacterial generations The most obvious places where tradeoffs or antagonistic pleiotropy apply are in terms of conflicts between phage latent period and burst size as well as conflicts between phage host range and infection vigor The multi-arrowed burst on the upper-left of the r = microcolony illustrates with arrows a sampling of the directions that virions may initially travel The negative impact of secondary adsorption on plaque wavefront velocity thus should increase with lawn bacterial density The number of bacteria that are expected to have become phage infected therefore is e-0 The number of multiply adsorbed bacteria of those bacteria that have been adsorbed at all similarly is only 40-fold greater for MOI versus MOI The one determinant of phage burst size that is fairly simple in its impact is the length of the phage post-eclipse period The ongoing ?goal of these initially adsorbing phages indeed of the resulting focus itself is to produce virions that at least occasionally acquire bacteria found in different biofilms or specifically which establish new foci of infection The organismal characteristics of phages can be generalized as consisting of those things impacting the duration of the phage infection period (latent period) those things that impact the phage per-infection fecundity (burst size) and those things that impact phage acquisition of new bacteria (adsorption rate) The other scenario is that the broader a phages host range then the less likely that they may be able to effectively infect all of the bacteria found within their host range resulting for example in reduced adsorption ability longer latent periods or reduced burst sizes in comparison to phages infecting a more narrow range of bacterial strains The parental microcolony is that within which a phages parental infection is located The penetration of phages into biofilms may actually take place to a larger extent in the short term if phages are unable to specifically adsorb to the bacteria making up a biofilm The phage interaction with conspecifics is described as population ecology those interactions with different species are studied by community ecologists and interactions with the abiotic environment are considered under the auspices of ecosystem ecology The phage T4 lysis inhibition strategy perhaps could be portrayed as a mechanism of burst size enhancement that could be advantageous under just such circumstances (see Preface) The phages involved in these processes are explorer scout and settler phages The phages so described should be viewed as clonally related members of the same population or alternatively an approximation of a superorganismal colony where as with Hymenoptera insects (e The phages that adsorb bacteria at this distance should generate the phage progeny that will serve as a plaques leading edge that is provide the bulk of phage particles that come to diffuse outside of a plaques existing perimeter The phages that collide with close-by bacteria will on average display shorter generation times than the phages that collide with more-distant bacteria The phenomenon may be viewed as equivalent to the retention of colloidal substances during size-exclusion chromatography: Particles tend to move from the carrying buffer and into a stationary matrix to a greater extent if particle availability is higher within the buffer and lower within the size-exclusion matrix The potential dearth of such infections would be because while the diversity of both phages and bacteria can be great the host range of phages often is relatively narrow The problem of biofilms being genetically heterogeneous either across time from biofilm to biofilm or within a single biofilm greatly complicates presumptive treatment that is treatment that is commenced prior to the identification of the bacteria involved along with their phage or antibiotic susceptibilities The process of eradicating biofilms even if phage mediated need not be limited solely to phage application The prophage in many instances is found inserted into the bacterial chromosome though for some phages or circumstances the prophage instead consists of a plasmid The prophages found within lysogens also can contribute to the morphology as well as function of biofilms The pseudolysogenic state is then thought to come to a close upon introduction of limiting nutrients such that bacterial metabolic rates increase The qualification ?infected? even if relaxed to mean simply adsorption nonetheless is crucial to the meaning of the concept of multiplicity of infection The reason for this claim is that lysogens tend to be immune to infection by the same phage types that they are able to produce The reason for this is that the fewer bacteria present the more variable will be the delay until the initial plaque-founding adsorption occurs resulting in variation in the duration of formation of individual plaques and therefore in plaque size The reason as we came to find though have yet to publish is that despite the small size of the actual clearing associated with wild-type plaques their phage-excluding power an apparent ability to block phages found in other plaques from accessing surrounding bacteria appears to reach nearly as far into the bacterial lawn as does the bacterial lysis associated with r-type plaques The reduction to lysogeny of a biofilm bacterium by a temperate phage (step 3b Figure 6) is not of guaranteed utility to that phage notwithstanding potential advantages to lysogens of becoming established within biofilms The result can be a great deal of heterogeneity within even clonal biofilms with biofilm complexity as well as heterogeneity increasing dramatically given the inclusion of multiple bacterial species The result can be a loss not only of a potential for independent replication but even loss of potential for understanding published experiments The result can be an excessive reliance on active rather than passive biofilm treatment which is my next consideration The result can be an expansion of the spectrum of activity of formulations to impact either multiple biofilm types or multiple bacterial inhabitants of individual biofilms (see ?Mixed biofilm treatment? Table 4) The result is a combination of less diffusion before encountering bacteria proportionately fewer phages that are productively infecting and perhaps greater virion inactivation than were phages instead diffusing away from just individual bacteria The result is a decline in productivity per individual adsorption since most multiply adsorbed individual bacteria even if infected by more than one phage will still tend to produce only a single bursts worth of phages The result is a potential for phages in biofilms to be simultaneously rare and persistent The result is a reduction or at least holding steady of the fraction of common bacterial types that are susceptible to the phages to which they are currently exposed The result is formation of natural biofilms on surfaces that otherwise are artificially residing in environments The result is that observation of high levels of phage infection within unperturbed bacterial biofilms might be rare The result is that the term e-M (EXP(-M) in Excel®) can Appendices be used to estimate the fraction of bacteria that have survived following phage adsorption at multiplicity M that is NM/N0 where N0 is starting bacterial density and NM is the density of uninfected bacteria following adsorption of M phages per bacterium The result is the prospect of longer-term survival for the lysogen lineage both within and beyond that biofilm The result is three contrasting strategies of subsequent phage dissemination The result of perhaps a combination of mechanisms is that viruses readily enter into biofilms but only slowly escape The result I feel is an effort that starts with the original chapter and then expands that material into quite a bit more The result potentially is a trapping of infections in the pseudolysogenic state The result potentially is insight into phagemicrocolony interactions during plaque formation that could help to inform our understanding of phage-microcolony interactions as they occur within biofilms The result Wild type contrary to prediction was competitive or at least nearly so no matter the plaque density The resulting adaptations could be helpful towards phage-mediated eradication of biofilms The resulting clumped dispersion could also affect bacterial predation: Biofilms may be less frequently encountered via random predator movement but provide large numbers of bacteria when encounter does occur The resulting infections they describe as plaque-like (pp The resulting plaques are so turbid towards their periphery that they typically are described as ?small? though in fact may be nearly as large in terms of lawn bacteria that have become phage infected as plaques made by lysis inhibition-defective mutants (unpublished observation; however for evidence see Figure The results are multifold The reward for successfully infecting all of the bacteria making up a microcolony however should be greater levels of phage production per focus of infection i The role of penetration and lateral movement is to create larger pools of phage progeny per focus so that more phages may be released to seek out bacteria through bulk water: The more phages produced per focus then presumably the greater the potential that new foci may be subsequently established The role of settlers instead is to maximize the generation of explorers a role for which greater infection economy should be advantageous i The same cannot be said for loss of uninfected bacteria to phage adsorption The same opportunity may not be available to phages generated via induction of lysogens already making up biofilms The scenario for less-frequent dosing in combination with using fewer phages assumes that active treatment is sufficient to achieve efficacy The scout phages with their lateral movement are involved in emigration particularly as within a biofilm and give rise to settler scout and explorer phages The screening device has the greatest potential for fouling by inorganic particulate matter oxidation of minerals (e The second concern is that active treatment is less of an option since it requires that phage amplification occur in overlying planktonic bacteria which may not always exist in sufficiently high densities to achieve this amplification The second is phage-virion release from the biofilm to potentially initiate new foci of infection (step 5a Figure as well as Figure 8) The settler phages with their minimal movement are involved in penetration into microcolonies and give rise to settler and explorer phages but to a much lesser extent to scout phages due a lack of proximity of most settler phages to the periphery of Phage Plaques as Models of Biofilm Exploitation a focus of infection The simpler of these two is movement since the faster phages diffuse for example then both the sooner adsorption should occur at a given distance from the center of a plaque and the faster plaques should grow in size The solid arrows represent the path of successful focus reproduction i The study does not provide proof of a causative relationship between phage production and cell dispersal indeed phage activity was determined unambiguously solely in terms of phage presence in biofilm effluent so it is possible that both phenomena instead are consequences of changes in bacterial physiological state The study is notable for their questioning why biofilm-associated EPS failed to block phage infection The study is unusual in that biofilms were seeded with up the three bacterial species: A The study of what phages do can be differentiated into two conceptually distinct but nevertheless overlapping aspects what I will call the molecular and the organismal The technique also makes no effort towards being ?efficient in terms of phage use The third mechanism involves bacterial division i The three basic steps of phage exploitation of biofilms thus are settlement emigration and penetration the latter particularly as into microcolonies The time from burst to burst is one phage generation The trend not surprisingly is that greater biofilm removal was achieved with higher phage densities The typical morphology of a plaque however involves a central clearing that is potentially followed going from plaque center outward by increasing turbidity until a turbidity equal to that of the bacterial lawn is reached The use of MOIinput to calculate dosing requirements during phage therapy as a consequence can be problematic The use of phages as biofilm-preventing disinfectants also would be limited to specific locations e The utility of explorer phages thus is the initiation of new foci of infection The utility of scouts is to increase the area over which a focus of infection expands to increase the number of microcolonies that are phage infected (one aspect of production sensu Gallet et al The value ?mean? here is MOI The value of ?cumulative? is either False or True (which is equivalent to or respectively) The value to the left defining the column values MOI is the average multiplicity of infection (or adsorption) where MOI = MOIactual The value ?x? is the number etc The virions of tailless phages can be either icosahedral filamentous or pleiomorphic The word ?soluble? is meant to imply an association only with the lysing parental infection Their experiments however show limits on the potential for phages to reduce biofilm densities in this case to about remaining bacteria/cm2 of biofilm: This density may be too small to generate phages faster than they can diffuse out of the biofilm Their prominent observation is that deletion of prophage genes results in an increase in biofilm formation implying that these prophages exert a negative impact on this formation Therapy can involve for example either whole phages or phage products such as extracellular polymeric substance (EPS) depolymerases or alternatively purified phage endolysins There are additional reasons for plaque cloudiness and/or failure of phages to either penetrate into or otherwise eradicate bacterial microcolonies There are at least three categories of phage-encoded hydrolytic enzymes that have a potential to affect biofilms There are at least three perspectives from which one can understand the problems with the ?modern definition of multiplicity of infection one based on ratios of added rather than adsorbed phages There are two alternative scenarios to this latter assertion: One which can be used to argue for both more frequent and lower-density dosing and the other which can be used to argue for a combination of less frequent along with lower-density dosing There are two potentially related ways around this situation There is also expectation that plaques will grow in size faster the more phages that are produced per bacterial infection that is the larger the phage burst size There is no inherent reason that active penetration should require also active treatment since lysis should also allow exogenously supplied phages access to lytically exposed bacteria at least so long as sufficient phage numbers are supplied and lysis is sufficiently thorough There the distance a particle moves is a function of levels of bead packing which defines the amount of column volume unoccupied by beads but also of the degree of bead affinity for particles There thus is good reason to expect that phages readily productively and continuously exploit natural biofilms but not necessarily in high numbers There was no subsequent decline in bacterial numbers following treatment for up to four days and subsequent bacterial recovery during that time was observed unless phages were initially applied at a higher temperature (37°C for min) before incubation at 4°C There via diffusion flow or some combination of both they are able to encounter and then infect bacteria Therefore as a rule one should view any dosing expressed solely as either ?MOI? or ?ratios of phages to bacteria? as a red flag as I point out repeatedly in discussion of individual biofilm eradication studies (Chapter 6) Therefore it is possible to at least speculate that abortive infection mechanisms can serve bacteria as microcolony-protective innate immune functions Therefore lytic infections of biofilm bacteria may not be readily observed except during or immediately after the fact These agents include prions (all-protein infectious agents of animals and fungi) and viroids (all-RNA infectious agents of plants) but consist especially of viruses These and other aspects of phage ?organismal? biology are summarized in Figure on p These are also exactly the circumstances that would be expected to drive the development of steady-state equilibria in biofilms These are semantic historical and mathematical which I consider in order These are the single-celled eukaryotes as well as a number of small colonial forms such as volvocine algae These are: (i) Enzymes that hydrolyze biofilm EPS involved for example in the structural integrity of biofilms but which otherwise do not fully block nonspecific viral diffusion These areas include ? Treatment of foods using phages particularly just prior to packaging; ? Association of phages with implantable medical devices; ? Prophylactic treatment of wounds These bacteria can be actively released in large numbers as planktonic often motile cells (swarming or seeding dispersal) that frequently are associated with microcolony hollowing These ?biofilms? where the quotes are meant to suggest that EPS/ECM were not necessarily prevalent were formed by one-hour exposure of stainless steel or polypropylene surfaces to ~108 bacteria/ml and a Stephen T These biofilters can also remove phages (see ?Phage references? Table 3) implying that phages may be readily taken up by biofilms even in the absence of bacteria serving as permissive hosts These calculations are based on only two values These can include not only enzymes that degrade different varieties of EPS associated with the same bacterial species but also enzymes that act against different bacterial species These centers are surrounded to varying degrees by ?zones of reduced turbidity? where either intact bacterial cells or substantial bacterial debris persists These clues come from knowledge of phage biology phage ecology and the modeling of phage population growth in semi-solid media i These complex structures are supported by EPS produced by the biofilm bacteria These complications in part are consequences of the complexity of the adsorption constant itself which actually represents an amalgam of three or more factors: (i) The rate of phage movement e These conditions are particularly violated when phages are employed as biocontrol agents of bacteria These conflicts can be portrayed generally as tradeoffs or more specifically in terms of what can be described as antagonistic pleiotropy i These consider: (i) The Poisson distribution which describes the default probability that underlies the interaction between free phages and bacteria and which is key to understanding the potential for bacterial populations to survive even following extensive phage adsorption These considerations are complicated with biofilms however given the potential for either hindered phage penetration to bacteria (which should limit bacteriakilling efficacy) or development of high phage densities only locally (which may enhance such efficacy at least locally) These consist of long-term associations between phage genomes there called prophages and the bacterial cell an association that can span multiple generations (e These depolymerases can be exogenously supplied or phage expressed (as soluble and/or virion-associated enzymes) These differences in durability may be viewed as having an impact on the phage effective burst size that is the number of phages of a burst that survive to obtain new bacteria to infect These difficulties can be responsible at least in part for a commonly observed laboratory phenomenon the cloudy or turbid phage plaque These dilution and titering calculations are straightforward ?Dilutions are just ratios ? as I like to say These discuss Poisson distributions the commonly misunderstood concept of multiplicity of infection the related ideas of phage killing titers rates of phage-bacterial adsorption and additional discussion of phage plaques These environments can be differentiated into biotic and abiotic components These findings are suggestive that the isolated phages were capable of bypassing lysogen superinfection immunity functions though I have to dispute the suggestion that the phages necessarily ?caused lysis of P These I consider separately beginning with the E These I describe as mechanisms that give rise to reduced infection vigor (e These ideas are complicated when competition exists both within and between populations These include (in the order presented): ? EPS blocking phage movement; ? Phage sequestration or inactivation by (presumably) non-bacterial substances; ? Phage sequestration by biofilms (presumably ones not containing susceptible bacteria); ? Masking of phage receptors found on biofilm bacteria such as by EPS; ? Hydrolysis of virion proteins by biofilm-associated proteases; ? Modification of receptors and/or ?intracellular defense responses? (p These include phages involved in the exploitation of immediately local resources (settlers) phages that instead take on the task of local territory expansion Phage Plaques as Models of Biofilm Exploitation (scouts) and lastly phages that are responsible for non-local dissemination (explorers) These ingredients need not be limited to phages These latter strategies may be favored especially when bacteria are less available such as within lowdensity planktonic populations or perhaps due to substantial separation of individual biofilms across environments These lawns typically grow in or on solid or semi-solid media These limits can be evaded microscopically so long as otherwise densely packed bacteria are well separated e These mixed-indicator plaques to some degree might be useful towards modeling the development of phage foci during infection of mixed-strain biofilms These of course are all basic pharmacological concerns These other things include most notably bacteria These phages can encounter biofilms resulting either in bacterial infection or nonspecific trapping within the biofilm ECM both of which I will describe consistent with Gallet et al These problems of biofilm prevention in many ways are equivalent to the use of preventative strategies in general These progeny phage would in turn infect other biofilm cells These progeny like biofilm bacteria that are not lysogenized could disseminate to found new biofilm microcolonies (step 5b Figure 6) These properties in turn will control how long a column must be to effect a desired degree of particle retention These r mutants with their larger plaque size in this case would be able to reach uninfected lawn bacteria before adjacent wild-type competitors These Stephen T These two populations consist of those phages that have a relatively high likelihood of acquiring new bacteria to infect on the periphery of a plaque (the edge of a plaque-forming focus of infection) and those phages for which the likelihood of reaching that periphery is relatively low These variants display a combination of phage release (including release also into the biofilm effluent) greater biofilm-forming ability including in terms of formation of voids as seen by Webb et al These vertically disseminating phages call them explorers likely are generated throughout foci They also are not unlike biofilms in terms of their spatial structure along with microcolonial basis and have been addressed to at least a limited extent theoretically They also can kill especially Gram-positive bacteria when supplied from without plus can be used against biofilm They also determined densities of planktonic bacteria which were reduced to about 10/ml but there was no equivalent measurement of biofilm removal They contribute to the ecology of their predators such as phages while also displaying vulnerability to phagemediated eradication They employed an iron-oxidizing bacterium (S They nonetheless can vary physiologically depending on the access that individual bacteria have to nutrients or oxygen or the relative absence in the vicinity of these bacteria of metabolic waste products e They obtained approximately one-log reductions (i They suggest that this lysis could supply nutrients for other cells reduce crowding (?competition for nutrients with nearby cells? p Thicker arrows indicate more likely outcomes This ?modern concept of multiplicity of infection however represents a simplification indeed an over-simplification of a more meaningful ?historical definition which is equivalent to MOIactual also using the terminology of Kasman et al This active-infection prevalence implies a productive infection rate in the vicinity of 10-4 This book represents my attempt to explore how phages might interact with naturally occurring spatially constrained bacteria that is with biofilm bacteria This ?bred? phage was employed to prevent biofilm formation on stainless steel and its efficacy was compared with that of lactic acid a lessspecific disinfectant This can be described as a passive treatment (Figure 1) This can be followed by slower movement within biofilms (see ?Diffusion within biofilms? Table 3) as well as slower diffusion out of the matrix into overlying bulk water This can be given dense populations of planktonic bacteria or follow phage encounter with biofilms This cloudiness or turbidity can be concentrated towards the outside of plaques found especially towards the center (as seen with temperate phages) or involve more complex arrangements such as alternating rings of clearing and turbidity This colorimetric approach thus holds promise as a firstapproximation means of exploring phage potential to inhibit biofilm formation but only given revision of protocols so that biofilm prevention can be differentiated from the more complicated as well as extraneous broth-culture dynamics of phages and bacteria This compared to reduction of only This consideration comes up when comparing especially the biology of infections involving multiple adsorptions with those involving only single adsorptions This constant rate of development is perhaps what one would expect for phage propagation within biofilms at least for less spatially heterogeneous biofilms This degradation can be mediated by phages (see ?Lysis or reduction…? Table next page) and their EPS depolymerases (Chapter 3) as well as by antagonistic bacteria (via bacteriocins bacteria-produced antibiotics or even predation ) This degree of resistance was not compared with that found in the E This diffusion likely is limited if phage-adsorbable bacteria are prevalent since bacterium encounter would result in phage adsorption giving rise to at least temporary cessation of such diffusion (see e This distinction has relevance to phage-biofilm interactions to the extent that one may distinguish between phage infection of biofilm bacteria and the generation of macroscopically visible lesions (i This estimation is based on a calculation in which MOI is assumed to be equal to Pkt where P is phage density k is the phage adsorption constant and t is time This expectation of 40% of bacteria becoming phage infected is rather than the 85% indicated This function is of the form POISSON(x mean cumulative) This gives these phages an ability to display what I have termed bacteria-like versus phage- (or virus-) like population growth This greater vulnerability stemming from a multi-celled existence is not unique to bacteria of course but instead is a general problem of multicellularity whether in animals plants fungi or biofilms This idea of diffusion between microcolonies as driving plaque spread is similar to a proposal by Fort et al This idea that MOIinput may be reasonably used only under a narrow set of well-defined conditions should come as no surprise given that it is or in any case should be standard practice in microbiology to normalize all indirect estimations before use such as the use of culture turbidity as a gauge for bacterial viable counts plus to modify normalizations whenever experimental conditions are changed This immunity allows phage adsorption but blocks subsequent infection meaning that bacteria that are clonally related to the induced lysogen will not serve as permissive hosts This interaction is described as giving rise to ?phage production? which is indicated to be ?in terms of plaque size and productivity? (p This interest he pursues from both basic science and applied perspectives organismal evolutionary adaptation and the use of phages as ?antibiotics phage therapy respectively This interval is short because the natural tendency will be for frequencies of phagesusceptible bacteria to decline given exposure to phages This is a process reminiscent of the pseudolysogeny phenomenon This is an expansion of the concept of emigration of Gallet et al This is an expected outcome given random phage collision with bacteria that is assuming operation of mass-action principles as seen when bacteria are planktonic and it points to the utility of supplying greater phage numbers per dose when seeking biofilm eradication (see Appendices and 4) This is because a phages effective burst size may be higher if bacteria are more readily acquired assuming that virion particles otherwise would decay over time This is because a variety of pathogens may be of concern the window during which contamination is possible may be longer and keeping phage densities sufficiently high at surfaces to effect passive treatment (and thereby truly prevent biofilm formation) may be difficult to achieve This is because bacterial densities are already relatively high once biofilms are noticed implying that there is at least some potential for phage amplification to sufficient densities to bring bacteria under control This is because even such scout phages will still experience a decline in further scout production as released phage progeny adsorb to the parental microcolony This is because it generally is preferable to produce more offspring on a relative scale beating another individual or species during direct competition and doing so even if this is done at the expense of overall productivity than it is to produce more progeny on an absolute scale but nonetheless to produce fewer offspring than ones competitor This is because not all bacteria may be immediately available to phage adsorption within biofilms though may become available subsequently due to the lysis of overlying bacteria i This is because plaques are relatively easy to examine and ubiquitously available for study This is because the more a bacterium is isolated from the world found outside of the microcolony it inhabits then the more its physiology resembles stationary phase This is because with passive treatment we should expect sufficient phage presence that both planktonic and any surface-adherent bacteria would be immediately targeted This is because as the authors note experiments involving phage T4 are complicated due to that phages ability to display lysis inhibition which might slow phage penetration into biofilms due to the resulting greatly extended latent period This is because except under the most controlled conditions bacterial densities constitute an uncontrolled variable This is because per unit time the fraction that is equal to the expression (free phages adsorbed) / (initial number of free phages) increases at a rate that is equal to (total bacteria) ? (adsorption constant) = Nk where N is bacterial density and k is the phage adsorption constant This is because under various conditions temperate phages predominantly display immediately productive infections meaning that only a fraction of infections result in reduction to lysogeny This is especially important because indication of ratios of added phages to bacteria as the sole description of dosing levels often mistakenly described as multiplicity of infection or MOI can deprive the reader of that key information This is especially so in food microbiology where phages may be used as biocontrol agents of bacteria that are found at very low densities or for biofilm prevention in general (Chapter 6) This is followed by infection and burst This is only about 30-fold more singly adsorbed bacteria of those bacteria that are adsorbed at all given this 100-fold lower MOI This is particularly so since abortive infections and reduced infection vigor may be relatively easily distinguished by comparing EOP determinations with what can be described as ECOI determinations (for Efficiency of Center of Infection a process involving preadsorption of phages to a potentially nonpermissive hosts that is followed by free phage removal and then infective center plating using a known permissive host as indicator) This is particularly so under circumstances where treatment failure is costly such as in the treatment of acute high morbidity or mortality bacterial infections of humans This is rather than as the randomly dispersed isolated cells giving rise to mass action adsorption kinetics that models of plaque development have assumed This is the column labeled as ?0? meaning x = This is the fraction of multiply adsorbed bacteria of those bacteria that have been adsorbed This is to say that a phage infecting any one bacterium upon release of phage progeny will have a greater potential to acquire neighboring bacteria than would be the case were bacteria not physically associated This is to say that phages historically have been viewed by many including by phage biologists in terms of how phages either inform or affect other things rather than from the perspective of phages themselves This latter process involves a ?hollowing as motile cells evacuated from the microcolony interiors? (p This latter statement comes with the caveat however that any savings associated with avoiding phage concentration must be balanced against costs of repeated dosing This latter zone should be assumed to be both quite thin and constantly moving outward what we described as a plaques ?periphery? which is to say the very outer reaches of a plaque before entering into a phage-less bacterial lawn This lower availability diminishes the likelihood of virus collision from within the biofilm with the biofilm-bulk water interface This may be particularly so to the extent that phages might be employed to increase biofilm susceptibility to these non-phage products since biofilms otherwise can be resistant Stephen T This may occur because adsorption to stationary bacteria serves to inhibit phage diffusion (for more on the impact of phage adsorption rates on phage diffusion particularly as during plaque formation see Appendix 5) This means that the ratio of phages added to bacteria and the ratio of phages adsorbing to bacteria can be similar This modification of host lifestyle could potentially have ecological consequences for the prophage This movement within biofilms apparently occurs without specifically interacting with biofilm-embedded bacteria plus is not barred by biofilm ECM This observation indicates that radial diffusion (convective transport) rather than downstream flow (advective transport) of progeny phage away from lysing infected cells is predominant in the relatively stagnant boundary layer surrounding a Phages as Anti-Biofilm Agents biofilm This observation is pertinent because of its suggestion that prophage induction might contribute to biofilm structure as well as as the authors speculate to bacterial dispersal from microcolonies This observation along with the fact that substantial increases in culture optical density occurred absent phage application suggests that the authors conclusion that ?Both planktonic and biofilm-grown cells demonstrated low rates of killing by phage K? in fact is not warranted This occurs because after one unit of time such as one minute (free phages adsorbed) = (initial number of free phages) ? (total bacteria) ? (adsorption constant) which rearranges to (free phages adsorbed) / (initial number of free phages) = (total bacteria) ? (adsorption constant) = Nk This occurs due to growth of biofilm bacteria to replace bacteria lost to phage infection either as phage-resistant bacteria or alternatively via regrowth by sensitive bacteria that have managed to avoid phage infection This overloading can be avoided by keeping the matrix-to-sample ratio high which is accomplished by using wider columns (thus increasing the column capacity) or by applying less sample per run This penetration as an aspect of biofilm exploitation I take up in detail in the following chapter This perspective actually is slightly more complex than as indicated since adsorption timing like latent period contributes to phage generation time while phage survival impacts burst size This presumptive phage reproduction provides a hypothesis for the lower susceptibility of higher-initial-density biofilms to phages and that is that these other biofilms Phages as Anti-Biofilm Agents may not have supported phage reproduction and subsequent lysis as well as lower-initialdensity biofilms This ratio of adsorbed phages to total bacteria is called multiplicity of infection or MOI a This recovery however was blocked when a cocktail of five phages was used rather than a single-phage (M4) treatment This result is suggestive that though initiation of plaques occurred with low efficiency once those infections commenced then subsequent EPS depolymerization presumably facilitated phage migration towards adjacent bacteria to complete plaque formation This results in biases towards congregation in regions where organisms are less active This selection for greater expedience may come to triumph over selection for greater economy but only to the extent that there are multiple foci present within a biofilm that are competing for the same bacteria with faster focus outward growth allowing lawn bacteria to be reached before the phages associated with other foci reach them This separate- or independent-application approach may be particularly useful to the extent that it is difficult to identify or engineer phages that contain both all necessarily EPS depolymerase activity and all necessary bacterial infection ability i This should be sufficient to reduce low-level bacterial contamination of food to zero or nearly so though with the caveat of so long as all bacteria are physically available to phage adsorption and physiologically available to phage-mediated killing This speculation however should be rigorously tested in the course of formulation of phage cocktails as too should the testing of potential antagonistic interactions between phages and any antibiotics or disinfectants that also may be employed together This stasis is suggestive of the establishment of a bacterial steady state i This steady state can be viewed from the perspective of individual phages but also from the perspective of individual foci This substantial increase however is suspicious and perhaps indicative that phage ?S1s adsorption rate under the conditions used was quite low This task is perhaps less arduous to the extent that not all biofilm components necessarily must be targeted in order to prevent control or eradicate biofilms This technique results in the formation of large numbers of phage-resistant microcolonies as well as cloudy plaques if a plated phage can only infect one of the two indicators used This temporal heterogeneity to a first approximation should slow plaque wavefront velocities as bacterial lawns mature This tendency to display productive infections explains why temperate phages that can reductively infect a particular host often also can form plaques on that host albeit plaques with cloudy or turbid centers that are the result of lysogens formed early during plaque formation This they followed up with experiments showing that purified phage particles (polyethylene glycol precipitated) were capable of reducing the viscosity of EPS specifically alginate as a consequence of virion-associated depolymerase activity This was followed by phages reportedly to ?inactivate released planktonic bacteria This we can speculate will tend to limit the ability of phages to diffuse large distances prior to colliding with bacteria i This we differentiated into two regions one where infection is actually taking place what we called a ?zone of infection? but which might better be described a ?zone of scout-phage infection? and a more peripheral zone in which phage diffusion is occurring but not yet phage infection This will slow phage acquisition of specific bacteria This work was supported financially by an Ohio State intramural grant awarded to Jeff LeJeune Brian McSpadden Gardener and myself This work would not have been possible without the help of Paul Hyman my frequent collaborator who kindly covered my classes while I was otherwise engaged in writing This again is the essence of ?Kill the winner?: If phage infections are sufficiently common then that commonness alone may reduce the future frequency of phage infections Those bacteria can exist either in planktonic states or as surface-attached cells Those nearest to the periphery (edge of oval shape) called scouts are charged with expanding the volume occupied by a phage clonal population (focus of infection or plaque rendered as a quasi-three-dimensional oval) Those phages that adsorb to their parental microcolony will not contribute extensively to the outward phage migration being biased instead towards infecting bacteria that are nearer rather than farther away Those phages that diffuse laterally or vertically scout phages or explorer phages respectively I describe collectively as emigrating phages that is which emigrate away from their place of origin particularly away from their parental microcolony Those phages that do not leave the parental microcolony but which instead penetrate into that microcolony further but fail to reach a plaques periphery I describe as settler phages Though a number of claims are made in the abstract little supporting data is presented Though an elegant approach it may be simpler in many instances to physically rather than genetically mix different soluble enzymes with different phages into a single formulation Though foci can be thought of as plaque-like in terms of within-biofilm phage propagation (Figure 7) in terms of phage infection they may not involve sufficient bacterial numbers to result in macroscopically observable biofilm eradication Though not shown the authors also indicate (p Though physiological differences can exist among these different phage types with the phage T4 lysis-inhibition phenotype perhaps best exemplifying such differences in fact spatial differences likely are more relevant in determining which phages play which roles in focus development reproduction and dissemination Though seemingly a simple solution to biofilm control involving a ?nipping of the problem in the ?bud in fact prevention in certain ways can be more demanding than outright eradication Though useful killing titers have limitations Thought it would seem obvious that active treatment and active penetration would be synonymous in fact so long as phages are supplied in sufficient densities then lysis can at least potentially expose underlying bacteria to exogenously added phages (passive treatment) as effectively as they might become available to endogenously generated phages (active treatment) Three weeks later I had created a working draft of the chapter but which included only a single paragraph from that older material (see p Thus it may be more competitive within communities to produce only progeny after min if new bacteria are readily acquired but more competitive between communities to instead produce progeny but take min to do so if substantial delays are expected before phages reach bacteria to infect e Thus one could argue that a relative lack of phage toxicity could allow one to reduce the repetition of phage application via the use of higher phage densities as achieved in situ that is higher densities than otherwise would be necessary to effect bacterial removal at some minimum effective rate Thus a phage perspective is one that asks not what phages can do to others but instead what phages can do for themselves Thus and despite appearances suggesting otherwise r mutants may reach uninfected lawn bacteria little faster than do wild-type phages Thus as noted prevention of biofilm formation using phage treatment may be of only limited utility though not of non-existent utility Thus by producing phages an infection is more likely to achieve rare acquisition of bacteria than by producing phages while phages available after min will reach common bacteria faster than phages that instead are available only after min Thus certain phages may be prevented to at least a degree from easily reaching otherwise susceptible bacteria found within biofilms Thus for example if phages adsorb to bacteria with a multiplicity of then 37% of bacteria will be expected to remain unadsorbed (since Thus for example POISSON(2 True) = POISSON(2 False) + POISSON(1 False) + POISSON(0 False) Thus for example while ants can have worker and soldier castes so too can localized phage populations consist of individuals that play different roles in the reproduction of the focus Thus for half of bacteria surviving then -ln(0 Thus I suggest that microcolony formation during plaque growth can mitigate the impact of ongoing lawn-bacterial reproduction on plaque wavefront velocities as summarized in Figure (next page) Thus in principle a drug for which this ratio is very large could be supplied in a single very large dose Thus it could be simply that a combination of planktonic and biofilm bacteria the former in part as generated via glycolic acid addition supported increases of phages to densities sufficient to eventually eliminate bacteria from the system resulting in an absence of the regrowth noted with chemical treatment alone as well as better penetration to bacteria than seen with phage addition alone Thus it is possible to envisage phage plaque formation within maturing bacterial lawns as entailing phages skipping through corridors or otherwise upon microcolony surfaces from one relatively physiologically permissive bacterium to another i Thus it might be more conservative to assume that lysogeny provides temperate phages with more options to disseminate from biofilms rather than necessarily superior options to those available to obligately productive phages Thus it would seem to be wasteful for a phage to fail to infect a substantial fraction of bacteria found in microcolonies Thus M = ln(NM/N0) Thus MOI = Thus multiplicity of infection means literally the ratio of phages to bacteria counting only those phages that have attached to and then infected bacteria Thus neither biofilm prevention nor sterilization was achieved Thus not only are rates of bacterial adsorption (and infection) by phages essentially independent of bacterial densities and especially so at lower bacterial densities thereby invalidating phage-tobacterium ratios as a legitimate descriptor of the pharmacodynamics of biocontrol but phage-to-bacterium ratios in fact are often operationally unknowable during phage therapy or biocontrol Thus on average no matter how many phages a focus produces nor how long it is productive a single focus will give rise to only a single additional focus of infection unless a phage (or focus) population is either growing or declining in size Thus one can envisage phage movement whose potential for transmission to adjacent microcolonies is affected by the vertical position of a lysing bacterium relative to the biofilm within which it is found Thus perhaps all molecular aspects of phage biology can be differentiated into those impacting phage acquisition of bacteria (adsorption) phage generation time (latent period but also lysogen doubling time) and the per-generation fecundity of a phage (burst size) Thus POISSON(0 False) = Thus scout phages increase the number of bacteria infected by increasing the number of microcolonies acquired while settlers are involved primarily in phage penetration into already-acquired microcolonies that is into their parental microcolonies Thus slower killing and lysis of bacteria could be desirable in at least certain circumstances Thus strictly speaking biofilm prevention preventing bacterial colonization and infection via passive phage treatment is more similar to the prevention of food spoilage using chemical preservatives than it is to vaccination Thus the historical meaning of multiplicity of infection (as MOIactual) at some point was altered creating a new tradition (MOIinput) that continues into modern times Thus there are animal viruses plant viruses fungal viruses protist viruses algae viruses viruses that infect members of domain Archaea and of course viruses that infect members of domain Bacteria Thus there is an expectation that on their own accord phages may be capable of reducing bacterial presence within biofilms down to certain minimal densities below which rates of phage production cannot even balance rates of phage loss i Thus though a natural tendency may exist for establishment of at least approximations of foci-biofilm steady states steady states are by no means the only focus-bacterial dynamic that is possible Thus though seemingly a trivial consideration I would like to stress the importance of explicitly indicating phage doses in experiments as phage titers i Thus to summarize the advantages of preventing biofilms rather than treating them after formation include that ? Biofilms may be physiologically more susceptible to phages earlier during development; ? Bacteria are never present as biofilms if biofilm formation is prevented meaning that any damage to a patient or system associated with biofilm formation simply will not occur given successful prevention; ? Any downstream contamination that could result from biofilm presence also will be blocked Thus under certain conditions a relatively small quantity of phages may be periodically added to environments with the hope of decimating high-density planktonic bacteria and then biofilm populations Thus while biofilm formation may be controlled when employing lower phage densities it likely will not be completely prevented Thus while it is easy for us to picture for example humans charismatic megafauna large trees and various domesticated plants and animals as important unto themselves it is perhaps more difficult to view mere viruses as existing for any reason other than their impact on others Thus while ongoing slowing in plaque wavefront velocity may be predicted to occur as lawn bacteria reproduce to higher densities due to increased secondary adsorption bacterial aging or greater limitation on virion diffusion experiments instead are more consistent with a constant pre-stationary-phase velocity To achieve uniformity in plaque size especially if initial densities of lawn bacteria are low it is best to initiate plaques as phage-infected bacteria (i To avoid confusion note that what I refer to as a ?modern usage of MOI as MOIinput Kasman et al To do this I modify the definition of emigration though not from its more basic meaning of movement from one area to another and avoid the use of production except as a more general term meaning an increase in phage numbers To more fully explain the discrepancy between theory and observation I propose a view of phage-plaque development that explicitly takes into account that lawn bacteria grow as microcolonies To put things bluntly: MOIinput = MOI should never be employed without both explicit description of intended meaning (such as by explicitly using the construct MOIinput) and unambiguous discussion of the limitations of this usage To the extent that bacteria or microcolonies remain intact but nonetheless still phage susceptible then this is the realm of settler phages (by contrast once bacteria are gone such as in the zone of clearing then only explorer phages remain) To the extent that microcolony exploitation is important to phage transmission in order to form new foci of infection settler phages producing explorer phages then it might be expected that evolution would favor phages that are better at microcolony penetration To the extent that phages display a low toxicity as well as a relatively low rate of inactivation then supplying phages in a single dose should be feasible at least so long as that dose contains large numbers of phages (e To the extent that steady-state equilibria occur at lower versus higher bacterial densities then one might suppose that improving adsorption rates can play a larger role in improving phage fitness at least in the short term than reducing phage latent periods To the extent that we can view biofilm-inhabiting bacteria as ?established then it might be preferable for a temperate phage to initiate lysogens during biofilm infection (step 3b Figure 6) To the left is shown a normal singly infected bacterium To the right represents the adsorption of one phage to one bacterial arrangement or microcolony indicated here as a diplococcus To them evolutionarily the world exists as two distinct Stephen T Too this book can be viewed as an informal second edition to the original chapter Topical phage use in conjunction with other approaches particularly systemic antibiotic application could however provide more robust protection than just phage use alone Total reductions in biofilm viable counts even with this additional killing however were only about two-log Towards calculating just how many phages are necessary to achieve this killing I provide discussion of Poisson distributions (below and Appendix 1) Towards gaining a better understanding of the biology of phages interacting with biofilms in this monograph I provide an overview of the subject divided into five areas: (i) The many facets of phage-biofilm interactive biology including consideration of virus trapping phage hydrolytic enzymes such as EPS depolymerases infection of biofilm bacteria and phage prevalence within natural biofilms Towards reconciliation of these various terms I provide the following as a description of the life cycle of a focus of infection: Explorer phages are found in bulk water overlying biofilms Towards that end they provide a number of terms to describe the process Towards that end in chapter I consider how phages might ?view? the world from their own perspective which is in terms of selective pressures acting on phage populations TRADEOFFS Adding to the complexity of phage evolution towards greater Darwinian fitness certain adaptations can be in conflict Trapping and Release of Viruses and Virus-like Particles by Biofilms Attributes References Phage references (reviews indicated with ) as cited by as cited by Non-phage virus references as cited by Beads/microspheres/particles Trapping1 by biofilms Diffusion within biofilms Detection of viruses or virus-like particles within biofilms TRAPPING OF VIRUSES IN BIOFILMS Biofilms can act as filters (see ?Biofilm removal…? Table 3; next page) Treatment additionally was not ?efficient Turbidity in both of these latter zones should be effectively identical to that of the bacterial lawn thus implying that these are more regions of phage presence and infection (edges of the plaque focus of infection) than demonstrating the macroscopic clearing that more strictly defines a plaque Two phage T4 r48 plaques (lysis-inhibition defective) are shown upper left (? and ?) while two T4 wild-type plaques are shown to the upper right (? and ?) Two to five hours post phage application the biofilm had been reduced over three logs in terms of viable count (down to bacteria/cm2) Typically these stages are considered as they occur macroscopically especially in terms of the rate of outward progression of the plaques outer edge or wavefront or alternatively may be modeled using cellular automata Ultimately a similar argument can be made for the products of immediately productive infections: Once such phages have finished exploiting a single microcolony there may not be other microcolonies consisting of similar phage-susceptible bacteria in their vicinity unless bacteria of similar phage-susceptibility types tend to congregate into the same biofilms Ultimately not much is conclusively understood about phage exploitation of biofilms particularly on microscopic scales Ultrasound also may be employed as a biofilmdisrupting/ phage delivery approach ; see for description of ultrasound use against biofilms in conjunction with the antibiotic gentamicin Under the latter circumstances especially where bacterial infection is unlikely (such as due to bacterial rarity) then selection should be predominantly towards greater survival capacity or more generally a greater effective burst size Under what circumstances should phage treatment nonetheless be potentially useful as a means of biofilm prevention Three areas come immediately to mind all of which are either already being used or are under development Understanding phage ecology or optimizing phage therapy thus requires an appreciation of phage interactions with biofilms as this book considers Unfortunately killing in this case is shown on a linear rather than exponential scale making it impossible to ascertain the full extent of biofilm eradication Unfortunately there does not appear to be any means by which bacterial densities may be calculated nor is there any mention of what fraction of initially added phages succeeded in adsorbing Unfortunately phage characterization in terms of adsorption rates were not performed Unfortunately phage dosing against biofilms in this study is expressed in ratios of added phages to bacteria so it is impossible to calculate actual doses Unfortunately phage dosing is presented as ratios of added phages to bacteria so actual phage titers used are not known Unless phages can preferentially target biofilms containing susceptible bacteria or susceptible bacteria are especially abundant then encounter between a particular phage and a particular biofilm may not give rise to any infection Upon lysis of infected cells at the surface of the biofilm the fluid boundary layer above the biofilm would tend to retain progeny phage Using more phages better adsorbing phages or more time potentially could have resulted in greater biofilm eradication Using phages only overall bacterial reduction was two-to-four log in one hour Usually in plaque formation phages make initial contact with bacteria prior to bacteria either initiating or progressing terribly far towards microcolony formation Various augmentations in addition to increased repeated or more frequent phage dosing instead may be employed Verma et al Vertical movement is out of biofilms lateral movement is within biofilms and minimal movement is as relative to the parental microcolony Virus trapping within biofilms in particular can give rise to an extreme variance in the timing of infection initiation following initial phage encounter with a biofilm i Viruses can be released from cells either continuously (a Viruses can be trapped for substantial periods within biofilms e Viruses in fact display a kind of kinesis with regard to biofilms Viruses that are pathogenic to humans similarly may be released from biofilms during drinking water distribution through pipes (see ?Drinking water? Table 3) Viruses in their virion state are constructed of nucleic acid that is surrounded by a proteinaceous capsid and which in some viruses also can be associated with lipids along with additional complexities We can also consider certain phage resistance mechanisms such as abortive infection systems as being especially efficacious against naturally occurring phage infections if those bacteria are located within microcolonies acting as I argue in the following section as the equivalent of a primitive innate immunity We can also consider phage-abiotic interactions in terms of phage ability to cope which is a more ?organismal? perspective We can also view these different strategies as ones that emphasize either expediency in phage reproduction or instead economy in that reproduction We might also consider that phage transmission between biofilms in certain circumstances may be episodic rather than continuous such as occurring in greater numbers with biofilm sloughing (see ?Release upon sloughing? Table 3) or coinciding with substantial rehydration of an environment which may occur in rainsaturated soils We might even speculate that these various destructive changes can have the effect of increasing the vulnerability to phage attack of what otherwise would be biofilm interiors Webb et al What drives the tendencies for particles to move into and then inhabit biofilms In part this movement should be particle-concentration dependent: Higher densities of free particles in the bulk fluid surrounding biofilms results in greater collision rates going from the outside in versus from the inside out What emerges is a representation of phage-biofilm interaction that is built upon ideas of phage movement within biofilms phage enzymatic impact and the bacterial microcolony as the primary focus of phage exploitation (Figure 24) What fraction of bacteria are multiply adsorbed (x > 1) given MOI = or What fraction of bacteria would then be expected to not have been phage adsorbed The answer is not zero What I saw when I looked through those dissection scopes were clearly visible bacterial microcolonies (see for example the last figure presented in this book found on p What is not legitimate is to conclude that such ratios of addition versus of infection should be equivalent under all circumstances and in fact it is especially under conditions of low bacterial densities or poor phage adsorption that multiplicity of ?adsorption? and starting phage-to-bacteria ratios will be most different What may not be appreciated though is just how much time can be required before MOIactual comes to resemble MOIinput WHAT PHAGES WANT To understand phages from a phage perspective one must first picture phages as organisms in their own right When bacteria are especially available then the primary selective pressure acting on phage populations is towards more-rapid population growth such as may be achieved given shorter latent periods When graphed on a linear scale increasing multiplicity (MOIactual; see Appendix 2) increases the amount of killing of bacteria approximately linearly only when multiplicities are relatively low (top graph) When temperate phages exist as components of lysogenic bacteria then they are able to take advantage of both the growth and survival aspects of their bacterial hosts Where biofilm bacteria are directly targeted even if done so ?efficiently early stage biofilm formation will not be expected to be blocked unless phage densities sufficient to effect passive treatment are employed Whether or not phage infection occurs within the vicinity of that maximum turbidity is an open question Whether or not phage production occurred however was not determined and an alternative explanation for the eradication efficacy could have been due to EPS degradation since the phage employed was known to produce a depolymerase enzyme While artificial as well as natural biofilms certainly ought to be employed I believe that phage plaque formation as a model for phage-biofilm interactions should not be overlooked While depolymerase enzymes also can disrupt biofilms (below) phages or other bactericidal agents are a necessary component of anti-biofilm formulations if killing of bacteria is desired e While many of these consequences could be affected by either immediately lytic infections or induced productive infections in this section I focus on the latter While one can view these aspects broadly as consisting of a combination of survival and reproduction as introduced above I take a more phenotypic perspective While outwardly diffusing phages from a central focus of infection may acquire additional microcolonies (scout phages) such outward movement may require only the infection of Phage Penetration into Bacterial Microcolonies physiologically permissive bacteria found on microcolony surfaces (Chapter 8) While there might be no reason for this approach to not be effective it is my opinion that prudence would dictate repeated dosing even if active treatment is expected While with phages the idea of a population can be complicated by the phage potential to recombine with a variety of entities other than genetically similar individuals plus the notion of just what a phage species consists of is not straightforward nonetheless that phages can exist as populations is not controversial Wild-type T4 plaques are small and turbid due to lysis inhibition while r-type plaques are large and clear With ?inefficient phage application there can be a tendency to scrimp on phage dosing With ?inefficient strategies by contrast phages instead are diluted throughout the overlying fluid resulting in the addition of more phages than would be needed if such dilution could be avoided With active treatment phages are not supplied in sufficient densities to effect substantial killing of bacteria but instead must reproduce in situ to produce a satisfactory ?killing titer? (Appendix 3) With active treatment by contrast sufficient phage densities to directly achieve anti-bacterial efficacy are not supplied often intentionally so With destructive infections phages die indicating a display of phage resistance by infected bacteria With distance EPS depolymerases would be expected to separate from virion particles unless the two entities are physically attached (i With greater numbers of phages produced or greater fractions released there may be a larger potential for acquisition of more distant bacteria i With high bacterial densities rates of phage adsorption are also high resulting in a rapid depletion of free phages With passive treatment this concern (in theory) is eliminated because phages are supplied in sufficient densities to give rise to sufficient killing of bacteria (however ?sufficient? is defined) rather than counting on in situ amplification which is inherently less effective at lower bacterial densities With phage-biofilm interactions deviations should be expected as noted With reduced infection vigor by a lytically infecting phage the bacterial host dies and so too does the phage at least operationally by failing to form a plaque With reductive infections phages take on the bacterial life cycle as their own (bacteria-like phage population growth or simply survival) while with productive infections phages rapidly alternate between infecting and diffusing to infect (virus-like phage population growth) With regard to phage exploitation of biofilms the key take-home message is that not all EPS will serve as absolute barriers to phage diffusion e With regard to phage treatment of biofilms especially using contrived experimental models it can sometimes be difficult to envision how active treatment could achieve consistent efficacy given scale-up of protocols towards use outside of the laboratory With restriction typified by the bacterial expression of restriction endonucleases that cleave phage DNA the host survives With simplification of course comes the danger of oversimplification With standard antimicrobials (or drugs in general) dosing is designed to achieve two specific outcomes: maintenance of sufficient drug density to achieve efficacy (the minimum effective concentration) while at the same time not applying so much drug that it causes damage to the treated individual or entity (keeping levels below the minimum toxic or damaging density) With the combined treatment a startling reduction in biofilm densities was observed essentially to zero from about bacteria/cm2 (presumptive units i With treatment of the dual-species biofilms it is clear that P With vaccination subsequent infections are not necessarily prevented just disease Within a biofilm therefore there may be no additional bacteria to successfully infect that are found in the vicinity of inductively produced temperate phages that is unless similar but not clonally related bacterial types have congregated close by due to related ecological preference Within an environment one can distinguish between the utility of producing numerous offspring versus the utility of producing offspring sooner which is a population-wide phenomenon that is equivalent to per-infection conflict between latent period and burst size Within biofilms pseudolysogenically infected bacteria may gain access to nutrients as a consequence of lysis of overlying lytic infections resulting in transition from pseudolysogenic to productive infections Within plaques though nutrient availability will decline as the lawn matures towards stationary phase while metabolic wastes build up Within these structures bacteria are piled on top of one another resulting in differences in cell access to nutrients and therefore physiological differences across biofilms Without rigorous demonstration of efficacy either alone or in conjunction with other components a given phage or other ingredient should be assumed to be superfluous Wood et al Wounds that are not yet apparently infected can be treated with phages through a variety of methods including continuous irrigation the soaking of bandages with phage formulations the application of phages within semi-solid creams or the use of phages impregnated within solid-phase materials (i You and Yin suggest that any declines in wavefront velocity that could occur as a consequence of increased bacterial densities can be countered to some degree by an increasing number of phage infections producing greater total numbers of phages Zegans et al Zones of phage plaque formation (or foci of infection)