Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org

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An important question for those with ecological and evolutionary biological interests is when a given situation is of ecological relevance vs. when it might be more of evolutionary biological importance (Abedon, 2022b). Telling the difference can be important for all of us.

Ecology by definition is the interaction of organisms with their environments.

We can describe phage use as antibacterial agents, that is, phage therapy, as an example of community ecology, or more precisely an applied community ecology. This is community ecology because there is more than one species of organism involved, i.e., as making up an ecological community. Minimally this is the phage (species #1) and the targeted bacterium (species #2), but also of importance is the treated body (species #3).

By definition, bacterial resistance to phages is ecological, as it describes a specific type of interaction, in this case between at least two species, the phage and the bacterium. That the resistance ‘interaction’ is one of ‘non-‘ or ‘less-‘ contact by the bacterium with the phage antagonist is only a detail, just so long as this lack of interaction is phenotypic, i.e., as opposed to the phage and bacterium instead just happening to exist in different places.

Bacterial resistance to phages also of course can have evolutionary aspects.

Evolution by definition is a change in allele frequencies in at least one species, or, more precisely, changes in allele frequencies in one population, in either case as observed over time.

Often the changes in allele frequency that we care most about are consequences of the impact of natural selection, and natural selection under most circumstances has a strong ecological component. Indeed, natural selection in most cases can be defined as the impact of ecology on evolutionary biology (and hence, as an aside, the existence of the science of evolutionary ecology).

By definition yet again, changes in the frequency of phage-resistance alleles within a bacterial population is an evolutionary process and typically these changes are a consequence of natural selection. The selective agent would be that phage population that is negatively affecting a bacterial population, resulting in increases in the frequency of whatever bacterial alleles are conferring protection from this phage.

Of interest to phage therapy is that this ecology driving evolutionary biology can in turn drive ecology. Specifically, once the frequency of alleles conferring phage resistance are high enough within a targeted bacterial population, then the applied community ecology of phage therapy can be affected, e.g., phage therapy can stop working.

In addition, if the frequency of a phage-resisting allele is found to be 100% within the bacterial population, following phage treatment (i.e., a frequency of 1.0), then if nothing else this is indicative that bacterial survival – an ecological issue – in this instance likely is a function of the occurrence of phage resistance.

If the frequency of phage resistance instead is 0% following phage treatment (0.0), then if nothing else this is indicative that bacterial survival (again, an ecological issue) was not a function of the occurrence of phage resistance. In fact, any frequency of phage resistance below 100% within a targeted bacterial population means that phage-sensitive bacteria are persisting despite phage treatment. For an example of the latter, see, e.g., Box 2 of Abedon (2022c).

Phage-resistant bacteria may display reduced virulence against bodies or may be subsequently treated with a different phage. Consequently, in some ways phage-resistant bacteria are not necessarily that big of a deal as a midpoint of a phage treatment, and this can be particularly if a diversity of other treatment phages are available. Phage resistance is not desired nor welcome, of course, but evolution of phage resistance also is not a certain indication of phage therapy microbiological failure.

That, by the way, to a degree contrasts with the evolution of antibiotic resistance that can occur over the course of antibiotic treatments, which can indeed be associated with treatment failures with high likelihood. One difference is something called antagonistic pleiotropy – not to be confused with antagonistic coevolution (Abedon, 2022a)!!! – i.e., whether or not resistance alleles are otherwise costly to the carrying organism (Abedon, 2022d). If resistance is both easily attained and not ecologically costly, then, well, that can be problematic, particularly given only mono therapies. Another difference is the sheer abundance of diverse, typically safe-to-use phages that often can be available to phage therapists (Abedon and Thomas-Abedon, 2010).

In any case, the persistence of phage-sensitive bacteria despite phage treatment probably means that, for whatever reason, treatment phages are not able to successful infect targeted bacteria despite those bacteria being phage sensitive; again, see Box 2 of Abedon (2022c). This frankly should be viewed as a big deal as essentially by definition it implies a phage therapy microbiological failure, one that may or may not be easily rectified, or at least a lack of complete eradication of phage-sensitive bacteria. I mean, how does one deal with phages not being able to easily reach and/or kill the otherwise phage-sensitive bacteria they are targeting?

Perhaps, as an answer to that question, some other phage that happens to be able to do a better job of reaching and/or killing those otherwise phage-sensitive bacteria might be available for use. That certainly is possible, but at this point in time we really aren’t all that good at figuring out what might constitute a better phage for phage therapy, other than in terms of host range – though see for example Bull et al. (2002; 2019) – and particularly better than the phage that we started with, presumably assuming that the first phage tried we thought was the better phage for phage therapy, hence why it was used first.

Of course, we almost take it as a matter of faith that phage carriage of extracellular polymeric substance (EPS) depolymerases (Danis-Wlodarczyk et al., 2021b) will solve many problems of phage penetration to targeted bacteria. But whether that is actually true in all instances, e.g., such as phage distribution throughout lungs – yet again, see Box 2 of Abedon (2022c) – is in my opinion just not known.

How might use of a phage cocktail instead result in complete eradication of a phage sensitive bacteria when a monophage does not? Just better odds that at least one of the phages used will be particularly good at achieving this? As another aside (Danis-Wlodarczyk et al., 2021a; Abedon, 2022c), note that it can be helpful to just apply a phage or phages at higher or multiple doses before giving up on a given treatment strategy!

At any rate, not being able to eradicate bacteria from an infection even though those bacteria are sensitive to a given treatment can be a far greater problem than failure that get rid of bacteria that explicitly are not susceptible to a treatment protocol. That is, there exits a basic problem in the applied ecology of treatments if not even phage-sensitive bacteria can be removed in full, just as there is a basic problem for an antibiotic treatment if the antibiotic is unable to fully eliminate even antibiotic-sensitive bacteria, a.k.a., the concept of antibiotic tolerance. For a bit on the latter, see Appendix A1 of in fact yet yet again, Abedon (2022c).

So where exactly am I going with this? At the end of a phage treatment, it is important to know whether the frequency of phage-resistant bacteria among the targeted bacterial population is high (at or approaching 100%) rather than low (near 0%). But high precision in that measurement, e.g., more than just whole percentage-point differences, really is not all that important. Why not?

Especially ecologically, there likely is little difference between 0.1%, 0.01%, or even maybe 10% or 50% of the bacteria being phage resistant, as that will still leave an awful lot of phage-sensitive bacteria having escaped phages during treatment. At some probably higher frequency of phage resistance we might come to feel that the frequency of remaining phage-sensitive bacteria is less important, but exactly where that point lies is difficult to say. My gut feeling, though, is that at the point where we start having to do statistics to tell the difference, i.e., at a point where higher precision in measurements becomes important, the importance of differences in the frequencies of phage-resistant bacteria – 100% or a tiny bit less than 100% – probably are no longer all that relevant. (For consideration of the statistics of plating-based enumeration, see Abedon and Katsaounis, 2021.)

Ah, you are saying, clearly therefore I am leading up to claiming that if we are interested instead in the evolutionary biology phage resistance, then in that case we really should care about measuring resistance frequencies with higher precision. And you would be absolutely right!  Except also maybe not.

The problem here is that a key word in the definition of evolution that we are using is “Change”, and by definition change cannot be measured using only a single data point, or in the case of quantifying evolutionary change, a single time point. Thus, no matter how precisely you measure the endpoint frequency of phage-resistant bacteria, that will not tell you that evolution has occurred in the course of phage therapy treatment, much less how much evolution.

Here is the basis of this latter point: At the start of an experiment, if your population of bacteria ever is going to contain phage-resistant members, then it likely already does contain those mutants (this, by the way, is why only-qualitative determinations that phage resistance is present, e.g., such as following phage treatments, are basically meaningless). Exceptional would be if the starting number of bacteria involved is so low that this number is, e.g., less than the inverse of the rate of mutation to phage resistance. Thus, for every time a bacterium divides, let’s say that there is a probability of 10^-5 that a mutation to phage-resistance will occur. If so, then in a population of 10⁶ bacteria, on average 10 phage-resistant bacteria will be expected to be present, more or less (Abedon et al., 2021).

That last part, “More or less”, is crucial, however, as the frequency with which mutations conferring phage resistance are expected to be present is predicted to somewhat “Fluctuate” about an average (Luria and Delbrück, 1943). In practice, this means that even if you precisely know bacterial rates of mutation to resistance to a given phage, you still will not know how many phage-resistant bacterial mutants will be present prior to the start of treatments. (As yet another aside, actually calculating mutation rates, vs. just mutant frequencies, is a not trivial thing to do.)

Without knowing the frequency of phage resistance prior to the start of treatments, then you are only really guessing whether evolution has occurred in the course of a phage treatment, no matter how precisely frequencies of phage resistance may be measured after a treatment is done.

In short, ecologically, the precision of measures of frequencies of bacterial phage resistance need not be all that high to possess high value in understanding the outcome of phage treatments. I mean, either phage-sensitive bacteria have persisted despite prior treatments or they have not, without a need to describe percentages with precision past the decimal point. Thus, 50.0% vs. 50.1%? Who cares? Indeed, 50% vs. 51%, who cares?

Alternatively, if one really cares about being precise in monitoring the evolution of phage resistance, then the most important place to emphasize that precision actually should be prior to the start of treatments, i.e., prior to initial phage application, and only then should one be measuring frequencies of phage resistance after treatments as well. But don’t forget that you need to have this information for explicitly that bacterial culture that is being treated, since evolutionarily all we really will care about is how a specific bacterial culture as a population changes in allele frequencies over time, and in phage therapy that bacterial population is precisely the one that you are treating.

Even so, how much more than order-of-magnitude precision do we really need in monitoring the evolution of phage resistance during phage treatments? Will we really care for example if the frequency of phage-resistant bacteria have changed from 10^-5 to 10^-5.5? And how hard would we have to try to be sure that such a relatively small change is actually real? I mean, seriously, except for the most hard-core evolutionary experiments, who would really care?

For what it is worth, when I look at the outcome of a phage treatment, if all of the targeted bacteria remaining are phage resistant, then I know what went wrong (clue: the bacteria have evolved resistance to the treatment phages, i.e., an evolutionary outcome). But when I look at the outcome of a phage therapy experiment and a substantial portion of the bacteria remaining are still phage sensitive, then more often than not I can only speculate as to what might have gone wrong, except again for those bacteria that have evolved phage resistance (Abedon, 2022c).  Still, this latter scenario should be viewed at least as an ecologically relevant outcome.

But bottom line: Obtaining an additional decimal place or two in describing the frequency of phage-resisting alleles within the treated bacterial population generally will not greatly aid in improving the precision of our applied ecological speculation.

Afterthought:

You would think that this essay came into existence as a natural outgrowth of the cited publications, particularly Abedon (2022c), but you would be wrong! On the other hand, I did wait a few months until that publication was published and available open access. Thanks for your interest!

Literature Cited:

Abedon, S. T. 2022a. A primer on phage-bacterium antagonistic coevolution, p. 293-315. In Bacteriophages as Drivers of Evolution: An Evolutionary Ecological Perspective. Springer, Cham, Switzerland. https://link.springer.com/chapter/10.1007/978-3-030-94309-7_25

Abedon, S. T. 2022b. Frequency-dependent selection in light of phage exposure, p. 275-292. In  Bacteriophages as Drivers of Evolution: An Evolutionary Ecological Perspective. Springer, Cham, Switzerland. https://link.springer.com/chapter/10.1007/978-3-030-94309-7_24

Abedon, S. T. 2022c. Further considerations on how to improve phage therapy experimentation, practice, and reporting: pharmacodynamics perspectives. Phage 3:95-97. https://www.liebertpub.com/doi/full/10.1089/phage.2022.0019

Abedon, S. T. 2022d. Pleiotropic costs of phage resistance, p. 253-262. In Bacteriophages as Drivers of Evolution: An Evolutionary Ecological Perspective. Springer, Cham, Switzerland. https://link.springer.com/chapter/10.1007/978-3-030-94309-7_22

Abedon, S. T., and C. Thomas-Abedon. 2010. Phage therapy pharmacology. Curr. Pharm. Biotechnol. 11:28-47. https://pubmed.ncbi.nlm.nih.gov/20214606/

Abedon, S. T., and T. I. Katsaounis. 2021. Detection of bacteriophages: statistical aspects of plaque assay, p. 539-560. In D. Harper, S. T. Abedon, B. H. Burrowes, and M. McConville (ed.), Bacteriophages: Biology, Technology, Therapy. Springer Nature Switzerland AG, New York City. https://link.springer.com/referenceworkentry/10.1007/978-3-319-40598-8_17-1

Abedon, S. T., K. M. Danis-Wlodarczyk, and D. J. Wozniak. 2021. Phage cocktail development for bacteriophage therapy: toward improving spectrum of activity breadth and depth. Pharmaceuticals 14:1019. https://pubmed.ncbi.nlm.nih.gov/34681243/

Bull, J. J., B. R. Levin, T. DeRouin, N. Walker, and C. A. Bloch. 2002. Dynamics of success and failure in phage and antibiotic therapy in experimental infections. BMC Microbiol. 2:35. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC138797/

Bull, J. J., B. R. Levin, and I. J. Molineux. 2019. Promises and pitfalls of in vivo evolution to improve phage therapy. Viruses 11:1083. https://pubmed.ncbi.nlm.nih.gov/31766537/

Danis-Wlodarczyk, K., K. Dabrowska, and S. T. Abedon. 2021a. Phage therapy: the pharmacology of antibacterial viruses. Curr. Issues Mol. Biol. 40:81-164. https://pubmed.ncbi.nlm.nih.gov/32503951/

Danis-Wlodarczyk, K. M., D. J. Wozniak, and S. T. Abedon. 2021b. Treating bacterial infections with bacteriophage-based enzybiotics: in vitro, in vivo and clinical application. Antibiotics 10:1497. https://pubmed.ncbi.nlm.nih.gov/34943709/

Luria, S. E., and M. Delbrück. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28:491-511. https://pubmed.ncbi.nlm.nih.gov/17247100/

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org

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Here is a something worth knowing about, from Pires, D.P., H. Oliveira, L.D. Melo, S. Sillankorva, and J. Azeredo. 2016. Bacteriophage-encoded depolymerases: their diversity and biotechnological applications.  Appl. Microbiol. Biotechnol. 100:2141-2151. [PubMed], (calls to figure and table excluded from quote):

Based on our search, the huge majority of phage depolymerases (126 proteins) are encoded in the same open reading frame of phage structural proteins (mostly on tail fibers, base plates, but sometimes also in the neck) or in close proximity to those genes, and were thus considered as structural proteins. Twenty other depolymerases found in this work might be soluble proteins since they are distant from any structural gene.

Depolymerases that are only soluble, that is, not virion attached, presumably are only useful in the immediate vicinity of phage-lysed bacteria, e.g., towards phage burrowing more deeply into biofilms. This perhaps means that phages don’t need depolymerases to initially infect biofilm bacteria (see here for that argument). Depolymerases that are associated with virions, by contrast, presumably are useful as well upon initial phage encounter with a biofilm bacterium.

That the majority of depolymerases are may not be soluble, but instead appear to be associated with virions, is suggestive that depolymerases are employed for the sake of initial encounter between virions and biofilm bacteteria. But this then begs the question of why more phages don’t encoded depolymerases?

Is it that we have trouble recognizing them in sequence data? Is it that bacteria are just too diverse in terms of extracellular polymers produced? (In addition to limiting utility, the latter may also interfere with our ability to detect depolymerase phenotypes during phage growth as plaques.) Is it because for the most part phages can infect biofilm bacteria sufficiently even without depolymerases? Or are there unexplored trade-offs associated with depolymerase encoding, perhaps especially when they are present as structural components of phage virions?

In the three previous paragraphs I am drawing on a tiny bit of past thought as to the role of depolymerases in phage interaction with biofilms, as can be found in my 2011 book, Bacteriophages and Biofilms. In particular, from p. 23 (of the revised pagination version, or p. 27 of the original… don’t ask…):

Ecologically, EPS depolymerases improve phage movement that occurs either adjacent to or distant from a phage’s parental infection. If distant, then movement towards bacteria will be enhanced by physical linkage between virions and depolymerases. Alternatively, for more localized movement, then soluble depolymerases may suffice, such as for phage dissemination out of biofilms [2004]. Scholl et al. [2005] 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. 4872). 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. 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. 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 [I then illustrate this argument with a figure…].

 

 

 

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org

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This quote is from Lee Watkins and J. W. Costerton (1984). Growth and biocide resistance of bacterial biofilms in industrial systems. Chemical Times and Trends (October):35-40.

The article has nothing to do with viruses. The quote is interesting, however, since it speaks to the question of how exactly to employ viruses in the biocontrol of microorganisms, specifically in the biocontrol and indeed elimination of biofilms from surfaces (quotation marks in the original):

lt is important to be able to answer that old question “Shall we slug with a biocide, shall we continuously treat with a biocide or shall we soak with a biocide—what is the best deal for the situation?”

To me these three alternatives are distinguishable in terms of how we might think about treatment of bacteria or biofilms with phages, that is, phage-mediated biocontrol of bacteria, or phage therapy.

The first alternative I interpret as the application of large amounts of biocide over short periods, perhaps in a single dose, i.e., slugging, or what we might describe as passive treatment in the case of phage therapy. Keep in mind that passive treatment should mean that for every bacterium targeted not only should at least 10 phages be added but at least 10 phages should be adsorbing, per adsorbable bacterium.

Continuous application, to me, would imply the application of lower but still minimally effective concentrations of biocide over longer periods. Continuous application represents an extreme of multiple dosing, i.e., where the time gap between applications is reduced to zero. Key here is that something other than overwhelming amounts of biocide is being applied, what many (unfortunately) would describe as something other than high multiplicities of infection (MOI) in the case of phage application. One can view such continuous application a preventive, or prophylactic.

Lastly there is soaking, which could also be viewed as continuous application, though this is an application that takes place over a relatively short period, i.e., days or weeks rather than months or years. This would be equivalent to the application of phages by soaking bandages, soaking various absorbent material (one sees mention of “tampons” in various places in the phage therapy literature, though it’s important to realize that the word has a medical definition), or instead via the application of, e.g., Phage Bioderm (for example, as discussed here).

What’s missing, of course, are any assumptions that the biocide will replicate in situ, i.e., so-called active treatment, which is typically considered to be a hallmark of phage-mediated biocontrol/phage therapy. That absence, though, is not unexpected given that this is from a discussion of chemical and physical anti-biofilm biocides rather than of phages.

Still, it once again is nice to see that there really is little that is new under the sun. When dealing with bacterial infections, particularly chronic ones which are associated with biofilms, it can be important to keep in mind these ideas:

1. We can hit them very hard (literally overkill) over short periods,
2. We can hit them less hard (minimally adequate biocide concentrations) but over long periods, perhaps particularly towards prevention, or
3. We can soak the infections over intermediate periods, presumably with periodic re-invigoration of dosing, using antibacterial levels which, also presumably, are somewhat in excess of what might be viewed as minimally adequate.

Any other approach, unless backed by hard data, should be considered to represent mostly wishful thinking.

Some additional reading:

Abedon, S.T. 2016. Bacteriophage exploitation of bacterial biofilms: phage preference for less mature targets?  FEMS Microbiol. Lett. 363:fnv246. [PubMed]

Abedon, S.T. 2016. Commentary: phage therapy of staphylococcal chronic osteomyelitis in experimental animal model.  Front. Microbiol. 7:1251. [PubMed]

Abedon, S.T. 2016. Phage therapy dosing: The problem(s) with multiplicity of infection (MOI).  Bacteriophage 6:e1220348. [PubMed]

Abedon, S.T. 2014. Bacteriophages as drugs: the pharmacology of phage therapy., p. 69-100. In J. Borysowski, R. Miedzybrodzki, and A. Górski (eds.), Phage Therapy: Current Research and Applications. Caister Academic Press, Norfolk, UK.

Abedon, S. 2011. Phage therapy pharmacology: calculating phage dosing.  Adv. Appl. Microbiol. 77:1-40. [PubMed]

Abedon, S.T., S.J. Kuhl, B.G. Blasdel, and E.M. Kutter. 2011. Phage treatment of human infections.  Bacteriophage 1:66-85. [PubMed]

Abedon, S.T. and C. Thomas-Abedon. 2010. Phage therapy pharmacology.  Curr. Pharm. Biotechnol. 11:28-47. [PubMed]

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org

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Way back in 2010 we (Abedon and Thomas-Abedon) suggested that the growth of phage plaques within bacterial lawns could serve as mimics of bacteriophage interaction with bacterial biofiolms. In fact, we made a rather extensive argument with six Roman numeraled points: (i) constraint of bacterial movement, (ii) bacterial growth within lawns as microcolonies, (iii) inhibition of phage movement, (iv) plaque-like phage growth within actual biofilms, (v) possible temporary shielding of bacteria within lawn microcolonies from phage attack, and (vi) variation in bacterial physiologies again as found within microcolonies within lawns and as potentially equivalent to bacterial microcolonies within biofilms. We concluded that, “Given these similarities, phage plaques as a facile laboratory model therefore could enrich our understanding of phage-bacterial interrelations as they may occur during the phage therapy of biofilm-producing bacterial infections.”

Indeed, we noted as well that Gallet et al. (2009) described phage formation of plaques also as phage growth within a “biofilm-like environment”.

Here I provide a quote from an earlier publication which serves to further these arguments. From Gilbert and Brown (1995) [Mechanisms of the protection of bacterial biofilms from antibacterial agents, p. 118-130. In J. W. Costerton and H. Lappin-Scott (ed.), Microbial biofilms. Cambridge University Press, Cambridge, UK.], p. 119:

The most simple in vitro method of generating biofilms to study antimicrobial sensitivity is to inoculate the surface of an agar plate to produce a confluent growth. Such cultures, whilst not fully duplicating the in vivo situation, have been suggested to model the close proximity of individual cells to one another and the various gradients found in biofilms. In this respect, colonies grown on agar may bе representative of biofilms at solid-air interfaces.

Of course, one cannot claim that bacteria growing within soft agar overlays are perfect representations of naturally occurring biofilm structures. Nonetheless, as we’ve noted previously, e.g., Abedon and Yin (2009), plaque formation within them is a lot more complex than people otherwise may realize.

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org

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Recently I suggested that:

Phage tails, to the extent that they display smaller diameters than phage virions as a whole, might contribute to nonenzymatic virion translocation into EPS [Extracellular Polymeric Substance, i.e., as associated with biofilm matrix], perhaps with longer, narrower tails permitting deeper or faster local penetration to biofilm-surface located bacteria.

Perhaps not unexpectedly, I now find that this was not an entirely original thought. From Wilkinson (1958), p. 68:

Physical blocking of the surface receptor. Can the presence of a capsule protect the cell simply because of its physical properties? Presumably an infective phage must be able to inject its DNA through the cytoplasmic membrane and, therefore, the main body of the phage must be at a distance from the cytoplasmic membrane smaller than the length of the phage tail (rarely longer than 150 mµ [meaning 150 nm]). Therefore, any layer outside the cytoplasmic membrane which is greater in thickness than 150 mµ and is impermeable to phages will act as a nonspecific phage inhibitor. It has already been shown that a capsule is by definition greater than 150 mµ in thickness and that it is probably impermeable to particles of the size of a phage head (about 100 mµ). An additional barrier to the phage might be the high negative charge of the polysaccharide capsular surface. In confirmation of this role, capsulate bacteria have been reported to be generally phage resistant…

In my defense, my suggestion pointed specifically to longer phage tails, and the general thrust of my article was that it is especially less mature aspects of biofilms which may be more vulnerable to phages. Less maturity, in other words, might be associated with less thick biofilm matrix, e.g., as perhaps associated with new growth on biofilm surfaces.

Indeed, Wilkinson discusses further an article, unfortunately which is not in English, suggesting that when polymeric substance material is thinner then successful phage adsorption may be more likely (as continuing directly from the previous quote):

Thus, Kauffmann and Vahlne (82) found that most capsulate strains of E. coli were resistant to phage and that when capsulate strains were attacked, there was a proportionality between the thickness of the capsule and the resistance.

One could speculate therefore that it could be, conversely, that shorter tails would be less able to penetrate thicker “capsulate”.

It should be noted that I am making no claims that phage tails exist solely for the sake of penetrating extracellular polymers towards adsorption of bacteria, though it is entirely possible that such penetration could serve as a benefit of possessing especially “longer, narrower tails”.

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org

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Myth: Freezing phage T7 can select for deletion mutations…

From Clark and Geary (1973), with emphasis added:

ATCC freeze-dries phages for distribution (Clark and Geary, 1969). Davis and Hyman (1971) have reported the existence of at least two authentically different T7 phage strains, T7M (Meselsohn) and T7L (Luria) (ATCC 11303-B₇). They also stated that T7L lyophilized stock from ATCC contained a high percentage of deletions in the DNA molecule, and they attributed this to lyophilization selecting for DNA deletions. However, since this publication, ATCC prevailed upon these authors to test the ATCC broth stock of T7L, which had been maintained at 4° C unlyophilized or unfrozen since its deposit in the Collection by Luria in 1952. They reported in a letter to W. A. Clark that the T7L stock before freeze-drying contained the same deletions as the freeze-dried product.

In other words, the evidence is not consistent with freezing selecting for deletion mutations in phage T7…

REFERENCES

Clark, W. A., and D. Geary. 1969. The collection of bacteriophages at the American Type Culture Collection, p. 179-187. In T. Nei (ed.), Freezing and Drying Microorganisms. University Park Press, Baltimore.

Clark, W. A., and D. Geary. 1973. Preservation of bacteriophages by freezing and freeze-drying. Cryobiology 10:351-360.

Davis, R. W., and R. W. Hyman. 1971. A study of evolution: The DNA base sequence homology between coliphages T3 and T7. J. Mol. Biol. 62:287-301.