Fluoroquinolone resistance does not facilitate phage Φ13 integration or excision in Staphylococcus aureus
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Prophages of the ΦSa3int family are commonly found in human-associated strains of Staphylococcus aureus where they encode factors for evading the human innate immune system. In contrast, they are usually absent in livestock-associated methicillin-resistant S. aureus (LA-MRSA) strains where the phage attachment site is mutated compared to the human strains. However, ΦSa3int phages have been found in a subset of LA-MRSA strains belonging to clonal complex 398 (CC398), including a lineage that is widespread in pig farms in Northern Jutland, Denmark. This lineage contains amino acid changes in the DNA topoisomerase IV and the DNA gyrase encoded by grlA and gyrA , respectively, which have been associated with fluoroquinolone (FQ) resistance. As both of these enzymes are involved in DNA supercoiling, we speculated that the mutations might impact recombination between the ΦSa3int phage and the bacterial chromosome. To examine this, we introduced the FQ resistance mutations into S. aureus 8325-4 attB LA that carry the mutated CC398-like bacterial attachment site for ΦSa3int phages. When monitoring phage integration and release of Φ13, a well-described representative of the ΦSa3int phage family, we did not observe any significant differences between the FQ-resistant mutant and the wild-type strain. Thus our results suggest that mutations in grlA and gyrA do not contribute to the presence of the ΦSa3int phages in LA-MRSA CC398.
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Dear Professor Hanne Ingmer, thank you for addressing the comments. I am pleased to tell you that your article has now been accepted for publication in Access Microbiology. This study would be a valuable contribution to the existing literature and the methods used are very rigorous.
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Dear Authors Thanks for submitting a revised version of your manuscript. After going through the revisions and the responses to the reviewers, I kindly ask you to add a sentence in the discussion to address Reviewer 2 concerns about conducting the study with a lab phage (phi13) rather than one derived from the study-specific isolates. I understand the suggested work would imply a whole new set of experiments and overcoming the experimental difficulties as you have stated into the response to the reviewer. While this is understandable, it would be useful to at least address in the discussion of the study the concerns and limitation of using phi13 instead of a isolate-derived phage.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature.
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Comments to Author
The manuscript "Fluoroquinolone resistance mediated by mutations in gyrA and grlA does not facilitate phage integration in Staphylococcus aureus" by Leinweber et al. characterises the impact of fluoroquinolone resistance mutations identified in livestock-associated (LA) Staphylococcus-aureus isolates on phage integration and release. Sa3 phages are rarely found in livestock, however a significantly larger proportion of mutants in gyrA/grlA were found to contain the Sa3 phage compared to non-resistant isolates. The authors reconstructed the relevant resistance mutations in a phage-cured lab strain that also had the modified attB site found in LA-isolates. Using a kanamycin-marked phi13 the authors then assessed the phage's ability to lysogenise into this host strain. The authors further look at the …
Comments to Author
The manuscript "Fluoroquinolone resistance mediated by mutations in gyrA and grlA does not facilitate phage integration in Staphylococcus aureus" by Leinweber et al. characterises the impact of fluoroquinolone resistance mutations identified in livestock-associated (LA) Staphylococcus-aureus isolates on phage integration and release. Sa3 phages are rarely found in livestock, however a significantly larger proportion of mutants in gyrA/grlA were found to contain the Sa3 phage compared to non-resistant isolates. The authors reconstructed the relevant resistance mutations in a phage-cured lab strain that also had the modified attB site found in LA-isolates. Using a kanamycin-marked phi13 the authors then assessed the phage's ability to lysogenise into this host strain. The authors further look at the ability of the newly lysogenised, fluoroquinolone resistant strains to release phage progeny either spontaneously or after mitomycin C induction of the SOSO response. General comments: Overall, the manuscript is well written and easy to follow. However, I am less convinced by the generalised conclusions of the authors that there is no impact on lysogenisation or phage release. My main concern is that the authors used a lab phage (phi13) and not a phage derived from their isolates to assess the impact. Even though integration is facilitated into the Sa3 attachment site by the same integrase, this does not necessarily mean that the phages are the same and additional phage-encoded factors might also play an important role in the observed association of prophage and fluroquinolone resistance. The authors should provide a comparison of the actual phage(s) found in the isolates and their model to support their hypothesis. Because the authors only test a single phage, they should modify their title and conclusions accordingly. I would also like to see a rational for the selection of an MOI of 1. At this MOI, almost 37% of the cells in the assay would never be infected with a phage. Could a higher MOI reveal a difference in terms of lysogenisation rates? Specific comments: Materials & Methods: Strain constructions are described incompletely and the counterselection steps against the temperature sensitive plasmid have not been described. Line 158: typo "incubatiion" Lines 192 - 196: The authors state that there is a growth defect in the FQ-R strain compared to its sensitive parent (doubling time 41 min compared to 30 min, respectively). Is this a genuine growth defect of the mutant strain or the presence of the antibiotic causing this reduction? If it is not a genuine growth defect, why did the authors choose to maintain antibiotic selection throughout their experiments for a chromosomal mutant that should be stable? Wouldn't it be more comparable to use the same media for selection and determination of lysogens to rule out a confounding impact of growth conditions? When the authors state "However, the colony forming units on kanamycin-free medium were similar for wild type and mutant cells (Error! Reference source not found.) under the conditions used for lysogenization", does this refer to the reported colony numbers or did the authors perform an additional experiment omitting the antibiotic and observing identical CFU numbers? Line 195: Reference missing Figure 1: - Numbers of strains in text and in figure do not match. In introduction (lines 69-71) referring to figure 1 state 20 isolates with Sa3 phage were resistant, however, in the results section (line 182) only 17/70 strains contained the prophage. Also, it is not quite clear how the numbers in Figure 1 (141 isolates) match to the sum of strains with or without phage in the results section (line 182: 26+70 = 96). - Are there additional prophages in these isolates? Figure 2 and Table 4 should be combined as they represent the same data. It would be better to plot table 4 with individual datapoints for each replicate and provide statistical analysis of both the raw (table 4) and derived (figure2) data. Also, it might make sense to plot the lysogenisation frequency on a logarithmic scale rather than a linear scale. Figure 4: Why did the authors assess spontaneous and induced release after only 2 hours of incubation with or without mitomycin C. Do they have time resolved data showing that this is the best time-point to assess this? Many staphylococcal phages will release their progeny later than this and differences might become more evident after longer incubation. Also, the high OD at the start of the experiment might be detrimental for the induction process.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Summary of the work: The authors have studied the role of fluoroquinolone resistance mutations in grlA/gyrA on the integration of Sa3int into the genome of Staphylococcus aureus. Sa3int phages are common lysogens of human associated lineages of S. aureus but are rarely found in livestock-associated genes. In this work the authors found a subset of strains, belonging to the CC398 clonal complex, that harbour a Sa13int phage. They found that these strains all carried mutations in grlA/gyrA, which render them resistant to fluoroquinolones. The mutations in grlA/gyrA were constructed in a CC8 strain of S. aureus, which already had the CC398 attB site introduced to the hlb gene. They found that the mutations in grlA/gyrA had no impact on lysogeny of the prophage but that it did slow the growth rate of …
Comments to Author
Summary of the work: The authors have studied the role of fluoroquinolone resistance mutations in grlA/gyrA on the integration of Sa3int into the genome of Staphylococcus aureus. Sa3int phages are common lysogens of human associated lineages of S. aureus but are rarely found in livestock-associated genes. In this work the authors found a subset of strains, belonging to the CC398 clonal complex, that harbour a Sa13int phage. They found that these strains all carried mutations in grlA/gyrA, which render them resistant to fluoroquinolones. The mutations in grlA/gyrA were constructed in a CC8 strain of S. aureus, which already had the CC398 attB site introduced to the hlb gene. They found that the mutations in grlA/gyrA had no impact on lysogeny of the prophage but that it did slow the growth rate of this strain. Similarly, they found that the mutations in grlA/gyrA had no impact on the frequency of phage excision and release. Overall, this is a well-presented paper that explores an interesting question regarding the conditions required for Sa3int phage's to lysogenize in a CC398 strain. The experimental work is strong and I have added some suggestions regarding discussion of the literature. I believe a little restructuring and expansion of the introduction/conclusion will make the experimental work more understandable to a wider audience in the context of the literature. Major Revisions: Line 205-207 - The conclusions drawn are unclear. The authors hypothesised that the Sa3int phage could lysogenise in the CC398 lineage due to FQ resistant mutations. They have shown that these mutations have no effect on phage lysogeny or excision. They then state that this does not explain the distribution of FQ resistance in the pig population. However, the Sa3int phage is stated to be beneficial for infection of humans, not animals, and therefore it is unclear how the authors have come to this conclusion if Sa3int does not benefit infection in livestock. The focus of this paper has been understanding how the Sa3int phage can integrate into CC398 strains. A discussion relating to alternative hypotheses and gaps in the knowledge would be beneficial to the readers understanding. When addressing this, authors should also discuss how this then links into the question of FQ resistance in the pig population. For example, the authors have not discussed the attB mutations and how these effect lysogeny or the sequence diversity of the prophages lysogenized in CC398 strains in comparison to other Sa3int phages. Minor Revisions: Line 65 - It is stated that the mutation in the attB site possibly reduces phage binding but has not been the focus of the study of this paper. The authors use 8325-4 with an attB mutated to have the sequence of the CC398 attB in the hlb gene. However, they do not assess if this has any effect on Sa3int phage integration/excision. Perhaps this will be the focus of another study or it is been tested previously, however, I feel like a greater discussion of the relevant literature is required on this topic, as it is possible that the attB sequence is not responsible for reduced Sa3int phage integration in CC398 strains. Line 66 - stated that spill over events are related to acquisition of IECs but in Fig. 1 the authors show that most of the lineage that is found to have the gyrA/grlA mutations are human isolates that do not harbour these elements. Greater clarity on livestock associated S. aureus and its association with humans/ human disease may help to clarify this. Line 102 - human isolates of S. aureus referenced again but most of the CC398 analysed are isolated from humans? (78% in the lineage studied). As above, expanding in the intro the role of LA infections in humans and how this links to FQ resistance and to IEC carriage may help to clarify this. Line 69 - The authors have identified 20 CC398 strains in the same lineage that have a Sa3int prophage but only disclose FQ resistance as a difference. I understand that other difference in these strains may be the focus of further study and therefore cannot be disclosed, but if the authors can expand on the extent of the differences between the strains eg. Is FQ resistance the only consistent difference or are there other major differences such as insertions of large mobile elements etc. The authors could also comment on the sequence homology of Sa3int phages that infect CC398 strains. Are these identical to Sa3int phages that infect other lineages of S. aureus or do they have distinct differences that may affect host range? Spelling and Grammar: Line 56 - Duplication of 'and' Line 117 - S. aureus not italicised Line 122 - unnecessary new line Line 158 - 'incubation' misspelt Line 195 - Ref error? Line 31/77 - Topoisomerase V is referenced in abstract but IV in introduction? Line 92 - 'been' not needed in the sentence Throughout - units throughout should be preceded by a space. Eg '100 mM' but most units have not followed this.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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