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Thank you for editing your manuscript after the reviewers suggesting and once again, congratulations for this Accepted work.
There are a few very minor changes suggested by the reviewer. I encouraged you to edit your manuscript accordingly and submit a revised version.
Please provide producent and country of origin of reagents used in a whole study, ie. lines: 196, 197
Line 202 genome assembly
Fig. 1A lacks x-axis numeric scale (resistance count)
Authors improved the manuscript significantly and added all my corrections and suggestions.
Congratulations on the improvements made with respect to version originally submitted. You will find a number of thoughtful recommendations by reviewer 3, which surely will further improve your work.
the authors have addresses my concerns.
the authors have addresses my concerns.
the authors have addresses my concerns.
In this manuscript, the authors used the whole genome sequencing for 17 S. epidermidis strains and obtained a large package of interesting results. The manuscript is well written, the English have been improved and the aims were clarified. Still, I do have some comments which should be taken into consideration before acceptance.
Overall, the introduction is sufficient but in my opinion authors should also mention the host specific colonization by Staphylococcus strains. The authors truly mention that : “There is no clear genetic distinction between pathogenic and commensal non-pathogenic SE strains, even though nosocomial strains are enriched in virulence and antibiotic resistance genes (..)” and here the literature-based host adaptation should be mentioned. As the species-specific adaptation is associated with MGEs but also with reorganization of the entire genomes and may also be linked to different virulence patterns.
Line 106: This abbreviation INPer should be explained here. If not, please leave only SE.
This study concerns an important topic of genetic diversity of opportunistic Staphylococcus epidermidis bacteria, which can be especially pathogenic for neonates. The research meets the scope of the journal. The methods are appropriate and based on innovative tools although the low number of the used strains is a drawback. Also I recommend more clarification especially how the NGS experiments were performed as it is crucial for valid results.
Line 130: Please provide details how the bacterial strains were cultured prior to genomic DNA extraction
Lines 130-131: Please provide details how the DNA concentration and quality was measured
Lines 131-134: Please provide the Illumina cartridge and buffer system used for sequencing and the produced reads length
Line 132: Please correct: MiSeq unit not MySeq unit
Overall, I find the results novel and interesting. I do have some concerns about figures and tables. Also the MRSE phenotype and genotype should be checked in more detail.
Lines 200-201: Please provide the information how the isolates were identified at the species level – chromogenic growth media, Vitek, MALDI-TOF MS?
Figure 1: All subsections of the Figure 1 (A-H) should have the same scale – 1 -11 antibiotics and the scale at y-axis should be the same in all figure subsections as now the same height of the bars indicate both 10 and 60 strains, please refer
Please ensure that the figure have enough quality as now it is not easy readable, it should have higher quality
Lines 289-290: I think that MIC values should be provided at least for methicillin resistance
Line 293: How the methicillin susceptibility of S10 and S16 was determined since in Table 2 these strains are included as resistant?
Line 294: As indicated in Table 2, the methicillin resistance was observed for 15 strains not 14, please refer
Table 2: Why not the same antibiotics set used for all isolates?
Lines 289-307: Did you check if any of the strains have ACME element?
Lines 302-303: The mecC gene presence should be investigated as S10 and S16 strains demonstrated phenotypic methicillin resistance and lack the SCCmec cassette, and mecA gene
You will see that one reviewer suggested rejection of this manuscript, although I considered worth giving you the opportunity to improve your work by addressing this reviewer concerns. To the best I can see, it might be worthwhile to consider a title that better comprehend your findings, as well as provide the specific details of methods and reference suggested by both reviewers. I invite you then to consider submission of an edited version that fully address both reviewers comments.
Overall, the manuscript is reasonably well written.
The research question is well defined but the study is seriously flawed based on isolate selection. The genetic analysis approaches are comprehensive and well-described. However, a significant bias was introduced into the study by selecting, seemingly at random, a small group of isolates that were antibiotic resistant and recovered over a seven year period.
In my view, many of the findings are not valid for the reasons I have provided under general and specific comments below.
GENERAL COMMENTS: This study undertook a very comprehensive investigation of 17 selected clinical isolates of Staphylococcus epidermidis recovered in a tertiary care hospital in Mexico between 2006 and 2013 in order to investigate whether specific traits could be assigned to isolates from cases of infection. Overall the analysis of the isolates is very thorough and involved whole genome sequencing (WGS) and comparison with selected genomes of S. epidermidis in GenBank. The detailed genetic analysis is impressive. Eight multilocus sequence typing (MLST) sequence types (STs) were inferred from the WGS data, six with a worldwide distribution. Many antimicrobial agent resistance genes were also identified and a number of virulence genes. The population of isolates investigated was found to have a clonal structure. High rates of recombination were also identified and an uneven distribution of insertion sequences, prophages and CRISPER Cas immunity systems.
In my view, even though the present study is comprehensive, it is limited by the small number of isolates investigated, the basis of their selection (i.e. antibiotic resistant) and the timeframe (several years) over which the isolates were recovered. I have provided a number of specific comments below for the authors’ consideration, which are intended to be constructive.
(1). In my view it would have been prudent to investigate a much larger number of S. epidermidis clinical isolates recovered over a shorter period of time (e.g. a year) in the same hospital without biasing the population of isolates selected based on their being resistant to methicillin. This suggested approach would provide a more accurate population of S. epidermidis isolates to accurately determine whether specific traits could be associated with isolates responsible for infection.
Very little information is provided on the basis of isolates selection in the Materials and Methods section, yet the first few lines of the Results state that isolates were “selected according to its outstanding antibiotic resistance profile”. Such a choice (pre-selection) introduces a major bias into the study. This choice is reflected by the not unsurprising finding of many antibiotic resistance genes in the isolates WGS sequences. Many S. epidermidis (both commensal and from infection) harbour antibiotic resistance genes, including mecA encoding methicillin resistance. In the present study, 15/17 isolates were methicillin resistant.
(2). The group of isolates selected for study were recovered over a seven-year period. This is far too long a span to suggest the isolates “were representative” of the S. epidermidis circulating in the hospital concerned. As S. epidermidis is a ubiquitous skin colonizer of humans and many animals, the population circulating in a hospital over many years would be vast.
(3). A total of 15/17 isolates investigated wee found to harbour an SCCmec type IV element, five of which also harboured SCCmec type V element. Isolates S12 and S15 were found to harbour SCCmec II, IV and V elements. SCCmec IV is the most common SCCmec element identified previously in S. epidermidis. The authors should provide a detailed figure showing the structural organization of the SSCmec structures relative to each other identified in the five isolates with multiple SCCmec elements. I presume one element was inserted in orfX and subsequent elements were inserted in tandem for each of these five isolates?
In this manuscript the authors isolated and sequenced 17 antibiotics-resistant SE strains, and found some interesting results like non-pathogenic SEs from patients and impacts of recombination on genome evolution of Staphylococcus. It's a generally a good study. But its writing needs be improved greatly.
line 184-186 264-266 All of the SE strains were first isolated based on their ability to resist antibiotics. If it is so there should be more details regarding how this is done.
line 173: only identical sequences were recorded in CRISPR-Cas identification. But was it too stringent? At least some reasons or citations should be put here.
line 251: few or 'a few'? This impacts that paragraph a lot.
line 409-411: the authors said that the lack of ica is suggestive of non-pathogenic strains. This is an important conclusion. But are there any possibilities that they are actually pathogenic strains that use different virulence factors? Besides it might be good to label the presence of ica and other virulence factors in the phylogeny of all strains, for instance in figure 1.
line 68-70 and line 78-80: I think the same sentence is repeated. besides, do you mean phage or prophage (also in line 384 and throughout the manuscript)?
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