Transposable Element Insertions into the Escherichia coli Polysialic Acid Gene Cluster Result in Resistance to the K1F Bacteriophage

ABSTRACT Reviewing the genetics underlying the arms race between bacteria and bacteriophages can offer an interesting insight into the development of bacterial resistance and phage co-evolution. This study shows how the natural development of resistances to the K1F bacteriophage, a phage which targets the K1 capsule of pathogenic Escherichia coli, can come about through insertion sequences (IS). Of the K1F resistant mutants isolated, two were of particular interest. The first of these showed full resistance to K1F and was found to have disruptions to kpsE, the product of which is involved in polysialic acid translocation. The second, after showing an initial susceptibility to K1F which then developed to full resistance, had disruptions to neuC, a gene involved in one of the early steps of polysialic acid biosynthesis. Both of these mutations came with a fitness cost and produced considerable phenotypic differences in the completeness and location of the K1 capsule when compared with the wild type. Sequential treatment of these two K1F resistant mutants with T7 resulted in the production of a variety of isolates, many of which showed a renewed susceptibility to K1F, indicating that these insertion sequence mutations are reversible, as well as one isolate that developed resistance to both phages. IMPORTANCE Bacteriophages have many potential uses in industry and the clinical environment as an antibacterial control measure. One of their uses, phage therapy, is an appealing alternative to antibiotics due to their high specificity. However, as with the rise in antimicrobial resistance (AMR), it is critical to improve our understanding of how resistance develops against these viral agents. In the same way as bacteria will evolve and mutate antibiotic receptors so they can no longer be recognized, resistance to bacteriophages can come about via mutations to phage receptors, preventing phage binding and infection. We have shown that Escherichia coli will become resistant to the K1F bacteriophage via insertion element reshufflings causing null mutations to elements of the polysialic acid biosynthetic cluster. Exposure to the T7 bacteriophage then resulted in further changes in the position of these IS elements, further altering their resistance and sensitivity profiles.

While I agree with earlier reviews that the science seems sound for the overall study, the paper itself still needs some major revision. The introduction has references to figures and other content (for example lines 62-96) that is more appropriate for the results and discussion. The flow of the results subsections is disjointed and hard to follow. The subsections do not seem to be in a logical order. All the information and results surrounding the K1F mutants should be described prior to introducing T7. Likewise, the discussion needs re-organized and should mirror the order in the results section. Moreover, the methods should also mirror the order seen in the results. For example, section 5.2 in the methods describes one of the last experiments mentioned in the results, therefore, it should be moved towards the end of that section. Taken together, an overall reorganization would greatly improve the readability of the study and give greater clarity to its significance.
Line 12 : "This study shows how" instead of "has shown" sounds more appropriate. Lines 62-96: The content in these sentences seem more appropriate for results and discussion as opposed to the introduction. This can be replaced with a concise paragraph that clearly says why the study was conducted and the aims of the manuscript. Lines 66-67: What is the intended meaning of "mechanism of resistance development was highlighted via the long-read sequencing?" Line 105: Replace "and" with "as" Lines 108-109: Mutants 1 and 5 are not labeled on Figure 3, Panel B yet they are being individually denoted in the text. Please ensure the figures can be understood without having to read the main text. Label the mutants and make the captions clear. Line 149 -156 -NCBI accession numbers are not working. Please release on NCBI. Line 160: Should be Figure 7, Panel B instead of Figure 8? Line 177: This study didn't use pathogenic bacteria so can't generalize Line 194: Production of the capsule wasn't significantly costly to the specific strain used in this study -can't over generalize to "a bacterium." Line 202: What is meant by, "with knock on effects?" Lines 205-206: awkward sounding Lines 243-244: verb tenses need adjusting Line 250: Upregulation of what has been associated with challenges? Figure 7, Panel B: Is this referring to DNA and genes or amino acids and proteins? Comments in figure legends and text aren't consistent. Figure 7 needs more work to improve the quality of the figure. Impossible to review due to the small size and inferior quality. Also, it states that Nanopore was used for the assembly but in the methods, there is no mention of Nanopore but Pacbio. Please clarify what sequencing platform was used.

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If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.   Figure 7 needs more work to improve the quality of the figure. Impossible to review due to the small size and inferior quality. Also, it states that Nanopore was used for the assembly but in the methods, there is no mention of Nanopore but Pacbio. Please clarify what sequencing platform was used. Thank you for sufficiently addressing earlier comments and concerns. See below further minor comments.
Lines 45-47: The details of "this study" should be presented in the last paragraph of the Introduction. Please incorporate this sentence into the last paragraph.
Lines 57-59: explanations for using particular strains are usually given in the methods or results. While introductory information about the viruses are ok here, perhaps consider removing "making it a suitable marker for this study." Please move to the methods section.
Lines 60-63 are specific to "this study" and as addressed in comment above, would be better suited at the end of the Introduction. Please incorporate this sentence into the last paragraph.
Combine lines 45 -47, lines 60 -63 and lines 74 -80 to make one concise last paragraph in the introduction .
Lines 45-47: In this study we investigated the K1F bacteriophage (6) and compared it to the T7 bacteriophage (7, 8) and how the E. coli strain EV36 (9) would respond to and develop resistance or sensitivities to these lytic phages. In addition, Lines 60-63: In this study, bacteriophage-resistant EV36 isolates were collected and characterized, through the investigation of relative growth and fitness, as well as a phenotypic analysis using confocal microscopy and the sequencing of entire genomes, to identify the causative genes in the development of phage resistance.
Lines 74-80: This study aims to offer a better understanding of the role of genetics in the competition and equilibrium between bacteria and bacteriophages, showing an insight into the development of bacterial resistances via the movement of insertion sequences (IS) through the bacterial genome. Specifically, we study the natural development of resistance and then returned sensitivity, to the K1F bacteriophage, a phage which targets the K1 capsule of pathogenic Escherichia coli. The genomic flexibility provided by IS movement appears to allow rapid adaptation to the presence versus absence of bacteriophages, removing potentially energy costly mutations once they are no longer needed.
Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
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