Staphylococcus aureus associated with surgical site infections in Western Kenya reveals genomic hotspots for pathogen evolution
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Objectives. Staphylococcus aureus is one of the most common pathogens attributed to hospital infections. Although S. aureus infections have been well studied in developed countries, far less is known about the biology of the pathogen in sub-Saharan Africa.
Methods. Here, we report on the isolation, antibiotic resistance profiling, whole genome sequencing, and genome comparison of six multi-drug resistant isolates of S. aureus obtained from a referral hospital in Kakamega, Western Kenya.
Results. Five of the six isolates contained a 20.7 kb circular plasmid carrying blaZ (associated with resistance to β-lactam antibiotics). These five strains all belonged to the same sequence type, ST152. Despite the similarity of the plasmid in these isolates, whole genome sequencing revealed that the strains differed, depending on whether they were associated with hospital-acquired or community-acquired infections.
Conclusion. The intriguing finding is that the hospital-acquired and the community-acquired isolates of S. aureus belonging to the same genotype, ST152, formed two separate sub-clusters in the phylogenetic tree and differed by the repertoire of accessory virulence genes. These data suggest ongoing adaptive evolution and significant genomic plasticity.
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Thank you for addressing the concerns raised by the reviewers in your revised version. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community.
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while you have addressed very thoroughly the comments from Reviewer 1, I have not been able to find how or if you addressed the comments from Reviewer 2 in your point by point response. Could you please include this. "The research work is scientifically sound. However, the title should emphasize SSI rather than wound infections. The article is required to be well written and of an appropriate length, especially the abstract and introduction sections. The research objectives are clearly stated at the end of the introduction section and met with results. But there was not an adequate review of the published literature. The methods are sufficiently elucidated to enable someone to replicate the tests, but the authors have not used appropriate statistical tools to address the findings. How these findings, as presented in the article, …
while you have addressed very thoroughly the comments from Reviewer 1, I have not been able to find how or if you addressed the comments from Reviewer 2 in your point by point response. Could you please include this. "The research work is scientifically sound. However, the title should emphasize SSI rather than wound infections. The article is required to be well written and of an appropriate length, especially the abstract and introduction sections. The research objectives are clearly stated at the end of the introduction section and met with results. But there was not an adequate review of the published literature. The methods are sufficiently elucidated to enable someone to replicate the tests, but the authors have not used appropriate statistical tools to address the findings. How these findings, as presented in the article, represent a breakthrough in knowledge and comprehension is not made explicit." I also noticed that in several sections there appears to be an error with the reference linking (see l109 and l 281-282)
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Dear authors, you will need to provide the raw sequence files as specified in the platforms open data policy (see Sequences section under https://www.microbiologyresearch.org/open-data). Assembled reads alone are not sufficient. Please also address the other concerns raised by the reviewers, in particular regarding discussion of literature and connecting it with your findings.
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Comments to Author
The research work is scientifically sound. However, the title should emphasize SSI rather than wound infections. The article is required to be well written and of an appropriate length, especially the abstract and introduction sections. The research objectives are clearly stated at the end of the introduction section and met with results. But there was not an adequate review of the published literature. The methods are sufficiently elucidated to enable someone to replicate the tests, but the authors have not used appropriate statistical tools to address the findings. How these findings, as presented in the article, represent a breakthrough in knowledge and comprehension is not made explicit.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate …
Comments to Author
The research work is scientifically sound. However, the title should emphasize SSI rather than wound infections. The article is required to be well written and of an appropriate length, especially the abstract and introduction sections. The research objectives are clearly stated at the end of the introduction section and met with results. But there was not an adequate review of the published literature. The methods are sufficiently elucidated to enable someone to replicate the tests, but the authors have not used appropriate statistical tools to address the findings. How these findings, as presented in the article, represent a breakthrough in knowledge and comprehension is not made explicit.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly 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
This is an interesting paper with interesting findings, however I feel that some of the conclusions and findings could be expanded upon. Methodological rigour, reproducibility and availability of underlying data Line 22: No reads available only assemblies. For future reference, it is best practice to produce one BioProject and assign each BioSample to that project, rather than make a new BioProject for each sample. Line 44: As above, there are no reads available only assemblies. Line 145: What parameters for trimming? Line 147: What parameters for assembly? Line 148: Why was ATCC BAA-39 chosen as the reference? It is a different ST and has an SCCmec (which presumably the isolates in this study do not have?). A better reference would be a high quality assembly of the same ST and without an …
Comments to Author
This is an interesting paper with interesting findings, however I feel that some of the conclusions and findings could be expanded upon. Methodological rigour, reproducibility and availability of underlying data Line 22: No reads available only assemblies. For future reference, it is best practice to produce one BioProject and assign each BioSample to that project, rather than make a new BioProject for each sample. Line 44: As above, there are no reads available only assemblies. Line 145: What parameters for trimming? Line 147: What parameters for assembly? Line 148: Why was ATCC BAA-39 chosen as the reference? It is a different ST and has an SCCmec (which presumably the isolates in this study do not have?). A better reference would be a high quality assembly of the same ST and without an SCCmec if the isolates don't contain SCCmec. Line 150: Parameters for the Unipro UGENE consensus sequence algorithm implementations. Line 151: What parameter for annotation, also the annotation that is present on NCBI appears to be PGAP, rather than Prokka. Line 153: It would be good to see the stats for the assemblies before using the mapping and reordering approach. Line 153: It is also unclear the precise process used to get the complete assemblies. What role to read mapping to a reference have? Was it to generate a sequence that could be compared to the de novo assembly? Hence the consensus sequence algorithm analysis? Was the consensus sequence then considered to be the final sequence? Was this final sequence contigs, that were reordered with Mauve? What method was used to confirm the contig boundaries were correct? Line 156: Parameters for Mauve In general, for the assemblies, what QC was carried out to check the quality of the "complete" genomes? Such as BUSCO, QUAST or mapping the reads back to the assemblies to check they map as expected. Line 160: Parameters for CARD-RGI Line 161: What parameters were used with VFDB? Were genome sequences used or extracted amino acid sequences for annotated genes? Line 163: How were plasmid sequences identified/extracted? Line 168: Parameters for MUSCLE and Gblocks Line 170: Custom scripts? Can these be shared? Line 172: Parameters for Neighbour Line 175: Parameters Gene Island Sniffer Line 177: Parameters for Barcoder Also note, as of the 23/12/23 the website for Gene Island Sniffer (www.bi.up.ac.za/SeqWord/sniffer/index.html) is unavailable, as is Barcoder (http://bargene.bi.up.ac.za). 2. Presentation of results Line 202: All 24 isolates seem to be multi-drug resistant. It would be good to know why the isolates that were sequenced were selected (is it because they are all resistant to oxacillin?). It would have been good to have sequenced all the isolates to get a complete picture, but I understand that isn't always possible. Line 213: How were the genes "translated"? Or were the annotated CDS used? Line 222: Would be good to mention that the gene content of the genomic islands will be mentioned later, as this seems lacking in detail. Line 226: Class A Beta-lactamases are generally found on plasmids, so good to see. Line 230: What was the degree of similarity? Top hits is a little vague, would be good to have percentage identity and coverage (for the plasmids). Line 236: What were the Staphylococcal enterotoxin genes? Line 242: Not all of the resistance genes present are mentioned, for example tet(38), which OD001 and ID029 appear to have. It would also be nice to link the AMR genotype with the AMR phenotype. Table S2, should specify it only lists AMR genes from the chromosome, however I think a table listing all the AMR genes, with annotation to say Chromosome or Plasmid would be more useful. Table S2, should specify that it is mutations (and the specific mutations) for gyrA, murA and parC. Table S2, would also be good to know the coverage and percentage identity of the AMR genes identified. Line 248: Not necessary but I feel it would be nice to have more specific references for SA and gyrA mutations (such as: 10.1128/AAC.42.2.236), some of the ones listed are about E. coli and Enterobacteriaceae, when it would be more useful to have ones for Staphylococci. Line 249: Not sure what the actual annotation should be for this kind of deletion, but I feel something like gyrA521_559del and gyrA821_831del would read better. Line 253: None of the references listed (Jaktaji &Mohiti, 2010, Weigel et al., 1998 and Johnning et al., 2015) are about S. aureus ATCC BAA-39 and focus more on E. coli or Enterobacteriaceae, which is not as relevant as not all of the mutations in gyrA are universal (a point that is made in the Weigel paper). Line 255: The S84L mutation has also been particularly well-characterised in S. aureus (see reference shared above). Line 257-258: "directly analogous" in what way? Line 259: "likely to give rise to glycopeptide resistance" reference for this statement and the mutations identified? Line 262: The OD001 isolate is missing mgrA, which is regulated by arlR, what impact would that have? It also has tet(38), which is negatively regulated by mgrA. Line 267: Isolate ID029 has norA but no mgrA, what impact might that have? Line 271: Table S3, It would be good again to have coverage and percentage ID for the identified virulence genes. In addition, an accession for the reference sitB used. For example, it seems that the sitB reference is from E. coli as listed from UPEC and APEC. Line 271: I don't think Table 2 is required. It seems the majority of the virulence genes are within the chromosome, separate from the Genomic Islands. In addition, if these are just genes within the "conserved" chromosome, does that mean there are more virulence genes not mentioned? Line 273: This might be true for IB011 but not necessarily true for the other isolates with only a few virulence genes present in GI or plasmids. It really depends what those genes are and what impact they could have. Line 276: I was unable to find a sitB homologue in the plasmid with the information provided here. Please include the accession number of the reference gene/protein you are using to identify sitB on the plasmids. Line 279: Except the enterotoxins weren't listed earlier, though their presence was. Also, the annotation of the plasmid on NCBI assigns these proteins to R, J, T and S. Line 282: Redundant or additional, rather than "spare". Or say a homologue of icaR. How much homology does the plasmid copy share with the chromosome copy? What's the gene name for the plasmid copy, as it isn't annotated as icaR. Line 283-284: Is there evidence in the literature that the plasmid copy would interact the same way as the chromosome copy would? Line 296: Would be good to include references more relevant to S. aureus, such as https://journals.asm.org/doi/10.1128/mmbr.00091-21. In addition, what about uvrC? As the NER system works in a complex with UvrABC. Table 1: The Isolate IDs don't match those listed on NCBI. Table 1: CDS doesn't match NCBI record. I think this table was made from the Prokka annotation which isn't the uploaded annotation. Table 2: Legend, says Figure 2 and I think it means Figure S1. Figure 1: Concatenated homologous proteins or core proteins? Figure 1: No bootstrap information. Figure 2: Plasmid for ID001 (which I think should be OD001) is missing annotation of the first virulence gene. Figure 2: What does the green and red blocks represent? Consensus? Is red inverted? This information should be included. Figure 3: The legend says the tree was made from concatenated alignments of the two proteins, but we have two trees, should there not be just one? Figure 3: Quality could be better, hard to read and there is no bootstrap data. Figure S1: All the isolates seem to share a GI, position 1.45 Mbp. What is this? It isn't discussed at any point but this is potentially interesting. Figure S1: There also seems to be some more GI that appear to be shared amongst the isolates but this isn't mentioned? Table S1: Really nice table. Notes on Table S2 mentioned before but repeating to keep together with other figures: Table S2: should specify it only lists AMR genes from the chromosome, however I think a table listing all the AMR genes, with annotation to say Chromosome or Plasmid would be more useful. Table S2: should specify that it is mutations (and the specific mutations) for gyrA, murA and parC. Table S2: would also be good to know the coverage and percentage identity of the AMR genes identified 3. How the style and organization of the paper communicates and represents key findings Organisation and style of the paper is fine. It has a reasonable layout for conveying the findings. More relevant figures and tables could be used, for example Table 2 isn't particularly informative over just having a sentence stating the information in the table. 4. Literature analysis or discussion Line 306: Aitken, missing from reference list. Line 307-308: Can this really be implied from two studies? Line 310: ID029 has mecA 104730..106713bp. If the annotation is to be believed there is a frameshift in it. Line 311: Evidence that shows this, as it isn't clear from the references. Line 350: Reference that UV-radiation activates this system? The references listed often don't seem relevant to the point being made (as stated previously). In addition, more in-depth literature analysis could be carried out, such as the impact of a deletion of a global regulator on genes associated with antimicrobial resistance. There's no mention of SCCmec, which I think is an oversight, given the isolates are resistant to oxacillin. This is particularly relevant as I was able to identify the presence of mecA in the following isolates: ID003, IB011 and ID029 (possible frameshift in all). 5. Any other relevant comments Abstract: Line 34: Typo 20.8 Kbp, as mentioned elsewhere this way Line 36: Typo, I think "Despite the similarity of the plasmid these isolates," should read "Despite the similarity of the plasmid," or "Despite the similarity of the plasmid between these isolates," Assemblies: I am a little concerned there may be errors within the assemblies and as no QC was carried out (or if carried out it wasn't shared), it is hard to gauge if these are errors or genuine features of the assemblies. As the reads for the assemblies aren't available, I was unable to check. For example, isolate ID029 seems to have large genomic duplication at 32800 bp, while isolate OD001 seems to have three copies of rlmH (Staphylococcus aureus generally have one). I think it is important for the authors to carry out QC on their complete assemblies to ensure that they are free of errors and are genuine. Some of the conclusions mentioned may differ if there are errors in the assemblies. Assuming the assemblies are correct, then the conclusions are fine but if the assemblies are wrong and contain errors, then the analysis carried out here would need to be repeated. Throughout the paper reference is made to genes/proteins belonging to the isolates but with no reference to the annotated gene/protein ID. It would be good to include this, as it makes it easier for someone reading the paper to find the referenced gene or protein. The references are in order of reference but are name references rather than numbered references. This makes it hard to find the reference in the reference list. The listed references don't always seem relevant to the point being made in the paper e.g. papers discussing E. coli but referenced as if they mention S. aureus.
Please rate the manuscript for methodological rigour
Satisfactory
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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