Heat Stress Induced Bacterial Tolerance against Phage Facilitates the Evolution of Resistance
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eLife Assessment
This important study analyzes the effect of heat treatment on phage-bacterial interactions and convincingly shows that prior heat exposure alters the bacterial cell envelope, enhancing persistence and bacterial survival when exposed to lytic phages. The study will interest researchers working on antibiotic resistance, tolerance, and phage therapy.
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Abstract
Antibiotic resistance and tolerance present significant challenges in global healthcare, necessitating alternative strategies such as phage therapy. However, the rapid emergence of phage-resistant mutants poses a potential risk. Here, we investigated bacterial persistence against phages, characterized by heterogeneous survival, analogous to antibiotic persistence. We found that heat treatment enhanced persistence and increase bacterial survival under phage exposure, subsequently promoting the evolution of phage resistance. Further experiments demonstrated that heat stress leads to a reduction in envelope components, thereby inhibiting phage DNA injection. Additionally, this heat-induced reduction resulted in systematic alterations in envelope stress responses, rendering bacteria tolerant to the antibiotic polymyxin while making them hypersensitive to pH changes and immune clearance. Our findings provide novel insights into bacteria-phage interactions and highlight potential challenges in implementing phage therapy in clinical settings.
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eLife Assessment
This important study analyzes the effect of heat treatment on phage-bacterial interactions and convincingly shows that prior heat exposure alters the bacterial cell envelope, enhancing persistence and bacterial survival when exposed to lytic phages. The study will interest researchers working on antibiotic resistance, tolerance, and phage therapy.
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Reviewer #1 (Public review):
Summary:
In this interesting and original paper, the authors examine the effect that heat stress can have on the ability of bacterial cells to evade infection by lytic bacteriophages. Briefly, the authors show that heat stress increases the tolerance of Klebsiella pneumoniae to infection by the lytic phage Kp11. They also argue that this increased tolerance facilitates the evolution of genetically encoded resistance to the phage. In addition, they show that heat can reduce the efficacy of phage therapy. Moreover, they define a likely mechanistic reason for both tolerance and genetically encoded resistance. Both lead to a reorganization of the bacterial cell envelope, which reduces the likelihood that phage can successfully inject their DNA.
Strengths:
I found large parts of this paper well-written and …
Reviewer #1 (Public review):
Summary:
In this interesting and original paper, the authors examine the effect that heat stress can have on the ability of bacterial cells to evade infection by lytic bacteriophages. Briefly, the authors show that heat stress increases the tolerance of Klebsiella pneumoniae to infection by the lytic phage Kp11. They also argue that this increased tolerance facilitates the evolution of genetically encoded resistance to the phage. In addition, they show that heat can reduce the efficacy of phage therapy. Moreover, they define a likely mechanistic reason for both tolerance and genetically encoded resistance. Both lead to a reorganization of the bacterial cell envelope, which reduces the likelihood that phage can successfully inject their DNA.
Strengths:
I found large parts of this paper well-written and clearly presented. I also found many of the experiments simple yet compelling. For example, the experiments described in Figure 3 clearly show that prior heat exposure can affect the efficacy of phage therapy. In addition, the experiments shown in Figures 4 and 6 clearly demonstrate the likely mechanistic cause of this effect. The conceptual Figure 7 is clear and illustrates the main ideas well. I think this paper would work even without its central claim, namely that tolerance facilitates the evolution of resistance. The reason is that the effect of environmental stressors on stress tolerance has to my knowledge so far only been shown for drug tolerance, not for tolerance to an antagonistic species.
Weaknesses:
I did not detect any weaknesses that would require a major reorganization of the paper, or that may require crucial new experiments. However, the paper needs some work in clarifying specific and central conclusions that the authors draw. More specifically, it needs to improve the connection between what is shown in some figures, how these figures are described in the caption, and how they are discussed in the main text. This is especially glaring with respect to the central claim of the paper from the title, namely that tolerance facilitates the evolution of resistance. I am sympathetic to that claim, especially because this has been shown elsewhere, not for phage resistance but for antibiotic resistance. However, in the description of the results, this is perhaps the weakest aspect of the paper, so I'm a bit mystified as to why the authors focus on this claim. As I mentioned above, the paper could stand on its own even without this claim.
More specific examples where clarification is needed:
(1) A key figure of the paper seems to be Figure 2D, yet it was one of the most confusing figures. This results from a mismatch between the accompanying text starting on line 92 and the figure itself. The first thing that the reader notices in the figure itself is the huge discrepancy between the number of viable colonies in the absence of phage infection at the two-hour time point. Yet this observation is not even mentioned in the main text. The exclusive focus of the main text seems to be on the right-hand side of the figure, labeled "+Phage". It is from this right-hand panel that the authors seem to conclude that heat stress facilitates the evolution of resistance. I find this confusing, because there is no difference between the heat-treated and non-treated cells in survivorship, and it is not clear from this data that survivorship is caused by resistance, not by tolerance/persistence. (The difference between tolerance and resistance has only been shown in the independent experiments of Figure 1B.) Figure 2F supports the resistance claim, but it is not one of the strongest experiments of the paper, because the author simply only used "turbidity" as an indicator of resistance. In addition, the authors performed the experiments described therein at small population sizes to avoid the presence of resistance mutations. But how do we know that the turbidity they describe does not result from persisters?
I see three possibilities to address these issues. First, perhaps this is all a matter of explaining and motivating this particular experiment better. Second, the central claim of the paper may require additional experiments. For example, is it possible to block heat induced tolerance through specific mutations, and show that phage resistance does not evolve as rapidly if tolerance is blocked? A third possibility is to tone down the claim of the paper, and make it about heat tolerance rather than the evolution of heat resistance.
A minor but general point here is that in Figure 2D and in other figures, the labels "-phage" and "+phage" do not facilitate understanding, because they suggest that cells in the "-phage" treatment have not been exposed to phage at all, but that is not the case. They have survived previous phage treatment and are then replated on media lacking phage.
(2) Another figure with a mismatch between text and visual materials is Figure 5, specifically Figures 5B-F. The figure is about two different mutants, and it is not even mentioned in the text how these mutants were identified, for example in different or the same replicate populations. What is more, the two mutants are not discussed at all in the main text. That is, the text, starting on line 221 discusses these experiments as if there was only one mutant. This is especially striking as the two mutants behave very differently, as, for example, in Figure 5C. Implicitly, the text talks about the mutant ending in "...C2", and not the one ending in "...C1". To add to the confusion, the text states that the (C2) mutant shows a change in the pspA gene, but in Figure 5f, it is the other (undiscussed) mutant that has a mutation in this gene. Only pspA is discussed further, so what about the other mutants? More generally, it is hard to believe that these were the only mutants that occurred in the genome during experimental evolution. It would be useful to give the reader a 2-3 sentence summary of the genetic diversity that experimental evolution generated.
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Reviewer #2 (Public review):
Summary:
An initial screening of pretreatment with different stress treatments of K. pneumoniae allowed the identification of heat stress as a protection factor against the infection of the lytic phage Kp11. Then experiments prove that this is mediated not by an increase of phage-resistant bacteria but due to an increase in phage transient tolerant population, which the authors identified as bacteriophage persistence in analogy to antibiotic persistence. Then they proved that phage persistence mediated by heat shock enhanced the evolution of bacterial resistance against the phage. The same trait was observed using other lytic phages, their combinations, and two clinical strains, as well as E. coli and two T phages, hence the phenomenon may be widespread in enterobacteria.
Next, the elucidation of …
Reviewer #2 (Public review):
Summary:
An initial screening of pretreatment with different stress treatments of K. pneumoniae allowed the identification of heat stress as a protection factor against the infection of the lytic phage Kp11. Then experiments prove that this is mediated not by an increase of phage-resistant bacteria but due to an increase in phage transient tolerant population, which the authors identified as bacteriophage persistence in analogy to antibiotic persistence. Then they proved that phage persistence mediated by heat shock enhanced the evolution of bacterial resistance against the phage. The same trait was observed using other lytic phages, their combinations, and two clinical strains, as well as E. coli and two T phages, hence the phenomenon may be widespread in enterobacteria.
Next, the elucidation of heat-induced phage persistence was done, determining that phage adsorption was not affected but phage DNA internalization was impaired by the heat pretreatment, likely due to alterations in the bacterial envelope, including the downregulation of envelope proteins and of LPS; furthermore, heat treated bacteria were less sensitive to polymyxins due to the decrease in LPS.
Finally, cyclic exposure to heat stress allowed the isolation of a mutant that was both resistant to heat treatment, polymyxins, and lytic phage, that mutant had alterations in PspA protein that allowed a gain of function and that promoted the reduction of capsule production and loss of its structure; nevertheless this mutant was severely impaired in immune evasion as it was easily cleared from mice blood, evidencing the tradeoffs between phage/heat and antibiotic resistance and the ability to counteract the immune response.
Strengths:
The experimental design and the sequence in which they are presented are ideal for the understanding of their study and the conclusions are supported by the findings, also the discussion points out the relevance of their work particularly in the effectiveness of phage therapy, and allows the design of strategies to improve their effectiveness.
Weaknesses:
In its present form, it lacks the incorporation of some relevant previous work that explored the role of heat stress in phage susceptibility, antibiotic susceptibility, tradeoffs between phage resistance and resistance against other kinds of stress, virulence, etc., and the fact that exposure to lytic phages induces antibiotic persistence.
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Reviewer #3 (Public review):
PspA, a key regulator in the phage shock protein system, functions as part of the envelope stress response system in bacteria, preventing membrane depolarization and ensuring the envelope stability. This protein has been associated in the Quorum Sensing network and biofilm formation. (Moscoso M., Garcia E., Lopez R. 2006. Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion. J. Bacteriol. 188:7785-7795; Vidal JE, Ludewick HP, Kunkel RM, Zähner D, Klugman KP. The LuxS-dependent quorum-sensing system regulates early biofilm formation by Streptococcus pneumoniae strain D39. Infect Immun. 2011 Oct;79(10):4050-60.)
It is interesting and very well-developed.
(1) Could the authors develop experiments about the relationship between …
Reviewer #3 (Public review):
PspA, a key regulator in the phage shock protein system, functions as part of the envelope stress response system in bacteria, preventing membrane depolarization and ensuring the envelope stability. This protein has been associated in the Quorum Sensing network and biofilm formation. (Moscoso M., Garcia E., Lopez R. 2006. Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion. J. Bacteriol. 188:7785-7795; Vidal JE, Ludewick HP, Kunkel RM, Zähner D, Klugman KP. The LuxS-dependent quorum-sensing system regulates early biofilm formation by Streptococcus pneumoniae strain D39. Infect Immun. 2011 Oct;79(10):4050-60.)
It is interesting and very well-developed.
(1) Could the authors develop experiments about the relationship between Quorum Sensing and this protein?
(2) It would be interesting to analyze the link to phage infection and heat stress in relation to Quorum. The authors could study QS regulators or AI2 molecules.
(3) Include the proteins or genes in a table or figure from lytic phage Kp11 (GenBank: ON148528.1).
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