Characterisation of Staphylococcus argenteus in Christchurch, New Zealand, and comparison to global strains.
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Staphylococcus argenteus (SARG) was discovered in 2009 as part of the Staphylococcus aureus (SAUR) complex and has been documented from various locations worldwide. In this article we describe the genomic features of five strains of SARG found in Christchurch, New Zealand. Isolates were first detected in 2019 using MALDI-ToF identification, and their identities confirmed using whole genome sequencing (WGS). Genomic features, including antimicrobial resistance markers and virulence factors were compared to other SARG sequences in NCBI GenBank. Four isolates belonged to ST2250 and one isolate to ST2793. Phylogenetic analysis based on core genome analysis revealed that all 5 isolates were phylogenetically distinct with four isolates clustering in the ST2250 clade. Three isolates contained staphylococcal cassette chromosome mec (SCCmec) type IV 2Bc, harbouring the mecA gene conferring resistance to beta-lactam antibiotics. All five strains shared many of the virulence genes found in SAUR but no TSST-1 or PVL pathogenic genes were detected. This publication contributes additional data on global occurrences and genomic features of SARG.
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Firstly I commend the authors for adding in all of the published genomes into this analysis and re-graphing to compare NZ with the global strains. There are two reviewer comments (below) that have not been actioned/addressed that I think are important. The introduction focuses on how SARG and SAUR are difficult to tell apart and describes several features that have been used, but your introduction doesn’t give any rationale/objective for this study. The data presented are a comparison of NZ SARGs to global SARGs, and not a comparison between SARG and SAUR for the purposes of differentiating them. The data presented are nice descriptive analysis of SARG genome content, especially with the addition of all published SARG genomes to the analysis, but the context for the study isn’t clear, especially when you comment that comparing gene …
Firstly I commend the authors for adding in all of the published genomes into this analysis and re-graphing to compare NZ with the global strains. There are two reviewer comments (below) that have not been actioned/addressed that I think are important. The introduction focuses on how SARG and SAUR are difficult to tell apart and describes several features that have been used, but your introduction doesn’t give any rationale/objective for this study. The data presented are a comparison of NZ SARGs to global SARGs, and not a comparison between SARG and SAUR for the purposes of differentiating them. The data presented are nice descriptive analysis of SARG genome content, especially with the addition of all published SARG genomes to the analysis, but the context for the study isn’t clear, especially when you comment that comparing gene carriage between SARG and SAUR is not part of the scope of this publication, but it is the focus of the introduction. Specific reviewer comments from the previous review; “the introduction lacks a major objective/major goal of the study. What was the purpose for analysis of the 5 strains, in what context” “Lines 318-323: Conclusion needs to further highlight that advances in differentiating SARG from the rest of the SAUR complex are required” Please amend the introduction and discussion to focus on what this study adds to the literature. New graphs: The visual representation of the data could be a lot clearer. Points to address; Figures 1 & 2 only have one axis. These graphs should have two labelled axis. Fig 1 is a histogram when it should be a bar chart. Please amend so bars are not touching as the data are discrete not continuous. The text is very small (denoting NZ or Global) and messy having it written on the graph repeatedly. A better way to represent that would be to have NZ being filled bars and Global being unfilled bars. The shades of blue used in figures 1and 2 are not distinct enough to reliably denote the different categories they are representing. Please amend to make the distinctions more pronounced (using different colours rather than shades of the same colour for discontinuous data). One reviewer suggested that the data in figure 1 would be better conveyed by grouping the genes together by antibiotic class, rather than alphabetically. I agree with this suggestion for both figures 1 &2- please group them together based on antibiotic class/ virulence factor type rather than alphabetically. Alternatively, the data in figures 1 & 2 could be easily represented as tables without loosing any of the information. It might be a clearer way to present this data, without the need for the % figures above the bars. If you go for this option, again group the categories of genes by function rather than alphabetically. Figure 4 is nice. Please change the colour scales used to denote MLST and Agr group as they are not distinct enough (especially the blues). Make the text of the strain names bigger as they cannot be read.
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Comments to Author
Anderson et al., wrote a very relevant, interesting, well-written, concise and easy to read paper about S. argenteus found in New Zealand. It is still difficult to reliably identify S. argenteus with MALDI-tof analysis, therefore the use of WGS is very important and information about globally spread S. argenteus lineages contributes to the understanding of the genomic epidemiology. The following major issues should be addressed prior to publication: 1) the introduction lacks a major objective/major goal of the study. What was the purpose for analysis of the 5 strains, in what context, and what groups are compared? 2) You analyzed a selection of international isolates and added the five isolates from New Zealand. Why did you chose to combine all isolates in 1 dataset? What did you wanted to show with …
Comments to Author
Anderson et al., wrote a very relevant, interesting, well-written, concise and easy to read paper about S. argenteus found in New Zealand. It is still difficult to reliably identify S. argenteus with MALDI-tof analysis, therefore the use of WGS is very important and information about globally spread S. argenteus lineages contributes to the understanding of the genomic epidemiology. The following major issues should be addressed prior to publication: 1) the introduction lacks a major objective/major goal of the study. What was the purpose for analysis of the 5 strains, in what context, and what groups are compared? 2) You analyzed a selection of international isolates and added the five isolates from New Zealand. Why did you chose to combine all isolates in 1 dataset? What did you wanted to show with that? Would it not be more logic to compare the 5 New Zealand isolates with the international S. argenteus isolates in the figures to see if they have genetic characteristics that the international isolates lack or that your isolates lack? A clear description of the international S. argenteus isolates included is required. 3) You cited the Witteveen et al., S. argenteus paper, but did not included the WGS data of those isolates and added them to the international ones. In the Witteveen et al., paper quite a lot of S. argenteus isolates and specially ST 2250 isolates (n=29), were uploaded to SRA. Please include these isolates in your dataset for global comparison of ST2250 4) A number of antibiotics were tested phenotypically. Are those the most used antibiotics for Staphylococcus aureus/argenteus treatment? Why did you chose to test those? In addition, you mentioned briefly the mupA gene (line 279), but did you also test for mupirocin resistance? And did you perhaps look at the ileS gene mutation V588F (pointfinder) that seems to be able to induce some resistance against mupirocin? 5) Figure 3: what is meant to show here? What are the differences in the pangenome? 6) what is the ratio S. aureus/MRSA : S. argenteus/MRSArg in New Zealand? 7) You mentioned the presence of mecA gene in tree isolates and absence in two isolates, did you noticed a difference in gene content/genetic characteristics between the MRSArg and the MSSArg, except for mecA and drfG (which is on the SCCmec element alike mecA)? 8) Line 193 mentions plasmid analysis and that no rep types were found in 1 isolate, thereby indicating absence of plasmids, but is that true? Or is it that there is no known rep gene found, but not conclusive that the isolate has no plasmid? Long-read sequencing can be performed to confirm this. Minor comments: - Abstract line 19: S. argenteus is not new, it's first description is in 2009? - Line 22: whole genome sequenceing should be whole-genome sequencing, please change throughout. - Line 33: whole-genome sequencing has been introduced in line 22. Please use and introduce abbreviation WGS from here. - Line 136: needs a reference. - Line 138: specify the selection of S. argenteus genomes from the public domain. - Line 150: S. aureus should be in italics - Lines 154-157: gene names should be in italics - Combine table 1 phenotype with table 2 genotype. Do the phenotype and genotype match? - Line 235: mlst should be MLST - The gene names in the figures 1 and 2 need some small adjustments they are all in capitals.
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?
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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I have now received two comprehensive reviews of this submission. I consider most of the comments to be minor rewriting issues, but there are a couple that require expanded analysis to be performed hence a decision of major revisions. In your revisions please include a statement about ethical approval to use clinically derived strains, or state why this is not required for this study. Please address each review comment point by point and submit a track changes document to highlight the changes made.
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Comments to Author
This study investigates Staphylococcus argenteus (SARG), a newly discovered Staphylococcus aureus complex (SAUR) species and highlights the difficulties in differentiating SARG. The five New Zealand SARG isolates in this study are compared to a publicly available global dataset of 168 SARG genomes. While the data and message in this study are important, this study lacks cohesiveness and there is a lot of important information missing. Additionally, there are some issues with confusing sentence structure and numerous minor grammar errors. Major issues: Lines 112 - 115: Doesn't specify what the alignment created was: i.e. was it a whole genome alignment, or a SNP alignment? And was it whole genome SNPs or core genome SNPs? Line 130: Where are the WGS results? I.e. genome size, number of contigs, number …
Comments to Author
This study investigates Staphylococcus argenteus (SARG), a newly discovered Staphylococcus aureus complex (SAUR) species and highlights the difficulties in differentiating SARG. The five New Zealand SARG isolates in this study are compared to a publicly available global dataset of 168 SARG genomes. While the data and message in this study are important, this study lacks cohesiveness and there is a lot of important information missing. Additionally, there are some issues with confusing sentence structure and numerous minor grammar errors. Major issues: Lines 112 - 115: Doesn't specify what the alignment created was: i.e. was it a whole genome alignment, or a SNP alignment? And was it whole genome SNPs or core genome SNPs? Line 130: Where are the WGS results? I.e. genome size, number of contigs, number of plasmids, number of CDSs, number of tRNAs, GC content etc? Do genome size and number/type of plasmids differ between SARG and SAUR? Could add the SAUR plasmid rep types results from section 193-198 here. A paragraph discussing these results needs to be added to the Discussion section. Section starting on Line 131: What about fluoroquinolone resistance (FQR) and gyrA and parC acquired mutations that conference FQR? FQR is found in SAUR. Line 143: How do these results differ compared to the percentages of SAUR isolates that typically carry these ARGs? Line 147: Would make it easier to interpret the results if Tables 1 and 2 were merged. Line 201: Mentions that all NZ isolates are ST2550, however Figure 4 clearly shows that one isolate: 22CHL-3048L is ST2793 and the other four isolates are ST2250. Section starting at Line 200: (a) Nothing is mentioned about what the accessory genes are, and where they may be found in the genome (i.e. phages, genomic islands, plasmids etc). (b) It would be interesting to include another Pangenome figure in Figure 3 with strain 22CHL-3048L (ST2793) removed to see how much of the core-genome is conserved in ST2250 strains. (c) The study would have more impact and be of more interest to the readers if the differences in AMR and Virulence gene presence (Figures 1 and 2) was correlated with MLST and the phylogeny in Figure 4. Lines 201-204: Is it common for a SAUR complex species to only have a small number of STs as seen in the analysis of publicly available SARG genomes? Line 207-208: Sentence incorrect suggest rewrite. In the previous sentence it is stated that the 5 isolates share ~80% core genome homology. Therefore, they are related by core genome analysis. Secondly, the five isolates don't have the same MLST, strain 22CHL-3048L is ST2793. Line 208: Why and how were these 78 genomes chosen? Why couldn't the phylogenetic tree be made with all 168 RefSeq genomes? Lines 295-310: There are numerous results mixed in with the discussion. These results belong in the Plasmid Analysis results section on Lines: 193-198. Lines 309-310; Mentioned the need for long read data, but didn't mention that this is required for identifying the genomic context of all AMR and virulence genes and reconstruction of all mobile genetic elements, not just plasmids. Line 313: Incorrect. Strain 22CHL-3048L is ST2793. Change: "ST2550" to: "ST2250". Line 314: Is this a core genome analysis? This is not mentioned anywhere in the Methods or Results. Line 315: This sentence is not reflected in Figure 4. Four of the isolates are ST2250 and the branch lengths are so small as to be unable to see clustering. The fifth isolate is ST2793 and due to the differences in MLSTs, appears in a different cluster. Without bootstrap information in Figure 4 to show clade support, the sentence on Lines 315-316 is unsupported by the data. Lines 318-323: Conclusion needs to further highlight that advances in differentiating SARG from the rest of the SAUR complex are required. Figure 1: Could be improved by grouping and colouring resistance genes based on the antimicrobial they provide resistance to. Figure 4: Missing scale, missing information on size of alignment. Was this tree created with bootstrapping? This is a phylogram, not a dendrogram. Suggestion: Make the tree substantially larger within the figure so that the actual branches of the tree can be seen, as opposed to dotted lines linking to strain names. Minor issues: Line 43: Define MLST before first use. Line 44: Define CC. Line 47-49: Suggest minor rewrite, poor grammar. Line 68: Sentence is confusing. Line 143: Change to 72 isolates and 18 isolates. Line 146: Define abbreviations. Line 177: Is it cap8H-K or cap8(A-P) as seen in Table 3? Line 177: Mentions that there were variations in gene presence in cap8H-K, however, this is not shown in Table 3. Line 176 - 178: Suggest adding in the name of the virulence factors and a minor change to the sentence structure: "Most virulence factors were common to all five isolates with variations seen in the genes encoding the capsular polysaccharide (cap8H-K), Type VIII Secretion System (esaC), Cell Membrane Protein (esxB), Staphylokinase (sak), Staphylococcus complement inhibitor (scn), serine-aspartate repeat containing proteins (srdC/D/E), and von Willebrand factor-binding protein (vWbp)." Figure 2: Which of the genes in the capsule locus are variable in the 168 SARG strains and what do they encode? This is an important distinction that could be mentioned in the Virulence Factor results section starting on line 174. Line 193: There was no mention of whether there are any differences in plasmid types between SAUR and SARG. Line 249: Change to: such as the nuclease (nuc) gene PCR. Line 255: Change ctrM to: ctrM/N/P/Q Line 256: Need to add to sentence that staphyloxanthin is a major SAUR virulence factor used for phenotypic identification of SAUR. Line 258: Sentence confusing as it appears ctrM and nuc genes have been mixed up. Suggest a rewrite. Line 260: Change: "such as" to: "associated with". Line 264: Change: "that" to: "the" Line 264: Change "pathogenic genes" to: "virulence factors" Line 269: How common are they in SAUR? Line 272: Which mobile genetic elements? Given the following sentence on Line 276-277, I assume its plasmids? Lines 281-294: Specify the actual strain names when discussing the differences between the 5 NZ isolates.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Poor
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?
No: Nothing was mentioned about Ethics in the manuscript, however these isolates were obtained from clinical samples.
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