Epidemiology and characterization of Providencia stuartii isolated from hospitalized patients in southern Brazil: A possible emerging pathogen
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This study aimed to characterize the virulence factors and antimicrobial resistance of Providencia stuartii, an opportunistic pathogen that causes human infections. We examined 45 isolates of P. stuartii both genotypically and phenotypically by studying their adherence to HeLa cells, biofilm formation, cytotoxicity, and antimicrobial resistance, and analyzed their genomes for putative virulence and resistance genes. This study found that most isolates possessed multiple virulence genes, including fimA, mrkA, fptA, iutA, ireA, and hlyA, and were cytotoxic to Vero cells. All the isolates were resistant to amoxicillin plus clavulanic acid, levofloxacin, and sulfamethoxazole plus trimethoprim, and most were resistant to ceftriaxone and cefepime. All isolates harbored extended-spectrum beta-lactamase coding genes such as blaCTX-M-2 and 23/45 (51.11 %) of them also harbored blaCTX-M-9. The gene KPC-2 (carbapenemase) was detected in 8/45 (17.77 %) isolates. This study also found clonality among the isolates, indicating the possible spread of the pathogen among patients at the hospital. These results have significant clinical and epidemiological implications and emphasize the importance of a continued understanding of the virulence and antimicrobial resistance of this pathogen for the prevention and treatment of future infections.
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All editor and reviewer comments have been sufficiently addressed and we welcome your manuscript for publication in Access Microbiology.
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While most criticisms have been sufficiently addressed, some minor issues raised by reviewers persist in the revised manuscript. Please be sure to completely correct for and address the following: -Author response to reviewer 1 in relation to how similarity was determined between isolates was sufficient, but the explicit description of how similarity was determined was not added into the actual manuscript text. Please add this description to the manuscript for reader clarity. -In the absence of sequencing data to support this claim, the statements in lines 385-389 are not appropriate to include in the manuscript. Please remove this section or provide additional data that shows evidence of within-hospital transmission. -Minor English grammar issues are still present throughout the text. Please carefully revise the manuscript as a …
While most criticisms have been sufficiently addressed, some minor issues raised by reviewers persist in the revised manuscript. Please be sure to completely correct for and address the following: -Author response to reviewer 1 in relation to how similarity was determined between isolates was sufficient, but the explicit description of how similarity was determined was not added into the actual manuscript text. Please add this description to the manuscript for reader clarity. -In the absence of sequencing data to support this claim, the statements in lines 385-389 are not appropriate to include in the manuscript. Please remove this section or provide additional data that shows evidence of within-hospital transmission. -Minor English grammar issues are still present throughout the text. Please carefully revise the manuscript as a whole to remove these errors. If additional help is needed, we offer a discounted translation service, Editage (https://www.editage.com/; see https://www.microbiologyresearch.org/prepare-an-article#13 for more information).
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The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. The language used is poor, which can cause ambiguity at times. Please carefully rewrite it. We offer a discounted translation service, Editage (https://www.editage.com/; see https://www.microbiologyresearch.org/prepare-an-article#13 for more information).
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Comments to Author
The study "Epidemiology and characterization of KPC-2-producing Providencia stuartii isolated from hospitalized patients in southern Brazil: A possible emerging pathogen" describes that P. stuartii presents a high frequency of virulence genes commonly found in other Enterobacterales species. The authors demonstrated that increased adhesion capacity to HeLa cells, citotoxicity in Vero cells, hemolytic activity and can form biofilm. Major comments: The exeriments were well designed and are well described, although some minor questions could be completed. The results were well presented. In the discusion section, the authors could improve by trying to correlate the genes found with the results of the adhesion, citotoxicity and biofilm tests. A minor english revision is suggested. Minor comments: The …
Comments to Author
The study "Epidemiology and characterization of KPC-2-producing Providencia stuartii isolated from hospitalized patients in southern Brazil: A possible emerging pathogen" describes that P. stuartii presents a high frequency of virulence genes commonly found in other Enterobacterales species. The authors demonstrated that increased adhesion capacity to HeLa cells, citotoxicity in Vero cells, hemolytic activity and can form biofilm. Major comments: The exeriments were well designed and are well described, although some minor questions could be completed. The results were well presented. In the discusion section, the authors could improve by trying to correlate the genes found with the results of the adhesion, citotoxicity and biofilm tests. A minor english revision is suggested. Minor comments: The title indicates that only KPC-2 isolates would be assessed regarding epidemiology and characterization, while all isolates were actually evaluated. "CTM-M" in the abstract should be corrected to "CTX-M". Line 87: please, clarify which E. coli and K. pneumoniae strains were used. Lines 90-91: how were the primers designed? In Results section, it is suggested to report the absolute number, for example "33/45 (73.33%)". When refering to genes, the nomeclature should be corrected, such as blaCTX-M (with bla in italics and CTX-M underscript), blaKPC-2 etc. If suitabible, it would be interesting to add another columm to figure 2 with the patient. In the Conclusion section, the percentage of KPC-2 positive isolates is different from the one reported in the abstract and in the Results section.
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?
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
Guidone and colleagues present an analysis of clinical Providencia stuartii isolates including for the presence of antibiotic resistance genes and virulence factors, and cytotoxicity, adhesion and biofilm formation. They also examine the presence of VFs in the genome of a model P. stuartii genome. Overall, I think their work is interesting and is suited to Access Microbiology. I think it has relevance to clinical microbiologists. I do however have several major concerns that will need to be addressed: 1. The Results is lacking raw data in the form of tables or figures. It is okay to use the text to summarise the data, but I would like to see it all presented so that it can be analysed independently by the reader. I have included some specific suggestions for each section below. 2. The authors refer …
Comments to Author
Guidone and colleagues present an analysis of clinical Providencia stuartii isolates including for the presence of antibiotic resistance genes and virulence factors, and cytotoxicity, adhesion and biofilm formation. They also examine the presence of VFs in the genome of a model P. stuartii genome. Overall, I think their work is interesting and is suited to Access Microbiology. I think it has relevance to clinical microbiologists. I do however have several major concerns that will need to be addressed: 1. The Results is lacking raw data in the form of tables or figures. It is okay to use the text to summarise the data, but I would like to see it all presented so that it can be analysed independently by the reader. I have included some specific suggestions for each section below. 2. The authors refer repeatedly to the clinical isolates being 'resistance to X/Y/Z antibiotics' or having 'resistance phenotypes'. However, to the best of my knowledge the authors have not conducted any antibiotic susceptibility testing in vitro, and therefore cannot make these claims. They instead need to refer to the data they are presenting, i.e. the presence of resistance genes found using PCR with specific primers. 3. The authors appear to suggest in the Abstract and Discussion that they have evidence of within-hospital transmission. I am not happy that they are showing this with the data presented. I would need to see whole genome sequencing as a minimum, with e.g. analysis of single nucleotide polymorphisms, to be able to make this claim. Minor comments and further details below: Abstract: Would benefit from a little more detail e.g. reference the CTX-M genes as extended-spectrum beta-lactamases, KPC-2 as a carbapenemase to help readers who might not be familiar with gene names. CTX-M genes referred to as CTM-M several times. Introduction is concise and covers all relevant information. Methods: Line 71-73: Please provide a brief overview of the criteria used to categorise the isolates as MDR, alongside reference 11. Section 2.1: Do the authors have genome sequences for the clinical isolates? Are they short/long read, 16S? Please provide details and ensure that the sequences are publicly available, if appropriate. Line 86 - 87: How related is P. stuartii to E. coli and K. pneumoniae i.e. how relevant are these as comparisons? Line 90-91: Provide more details of the DNA extraction protocol so that it could be replicated easily. Results: Line 183: Please list e.g. in a supplementary table the putative virulence factors in ATCC 33672 and their similarity to known VFs. Is this the first time this particular species has been interrogated for the presence of VFs? This could be made clearer. Section 3.1: It is not clear from the text whether the authors have looked for VFs in silico or in vitro. Please provide more details in text and an accompanying figure e.g. table of putative VFs or gel images. Line 198: The legend for Figure 1 is not clear - 'Adhesion of P. stuartii in HeLa cells' is repeated unnecessarily and is confusing. It would be useful to include the treatment in the legend e.g. the addition of D-mannose supplementation to the cells in panel B. Line 200-202: Please provide a figure or table to support the biofilm results (could be Supplementary) e.g. the raw data. Percentages on their own are not adequate without an idea of numbers tested. Line 205-206: Please provide example images showing the hemolysis or other examples of the raw data. Section 3.3 (line 208): As far as I can tell, the authors have not conducted in vitro antibiotic susceptibility testing. This section describes isolates as being 'resistant to amoxi/clav…etc' (lines 209-212). If the authors are inferring this from the presence of antibiotic resistance genes then this should be made clear, else details of MIC testing should be provided in Methods. Line 208: The section title is misleading - from what I can tell there is no phenotype information being given, only the presence of resistance genes. A more appropriate section header could be something like 'Presence of resistance genes in clinical isolates' (or something more informative, if the authors choose). Figure 2: The legend is not as informative as it could be - for example, are the six VFs mentioned in the table the only ones found by bioinformatic analysis, or are they a subset chosen by the authors? The cytotoxicity (against what target) should be mentioned. Please also made the +/- signs a little larger for ease of reading. Why is the adhesion data in the presence of D-mannose not provided here? Line 218: How has this measure of similarity been determined? How were the clonal complexes determined? Line 217: I believe this should be section 3.4, rather than 3.3 Line 218-235: "The phenotypic characteristics varied/were similar' - which characteristics are these? There is a lot of information being presented here so please be clear Line 218-235: Is there a characteristic common to all isolates in a given sub-cluster? From Fig 2 there seems to be a lot of intra-cluster variability, so I am intrigued to understand how they were clustered in this way. Discussion: In contrast to the Introduction, the Discussion is lacking structure and focus. It reads more like an informative passage on VFs rather than a discussion of the results presented in the manuscript. I strongly encourage the authors to consider a re-write to focus on what their results mean in a clinical context. Line 342: What phenotypic characteristics of ESBL producers are the authors referring to? Line 363-356: This is a bold claim, and would benefit from more information being provided to support it e.g. which antimicrobials were being used that could have acted as a selection pressure. Line 366-374: I am not clear whether the authors are merely repeating claims from other papers, or whether they are suggesting that they have also found evidence of within-hospital transmission of P. stuartii. If the latter, I am not convinced that they are able to state this from the data provided. I would expect to see whole genome sequences and SNP analyses to be able to make such a claim.
Please rate the manuscript for methodological rigour
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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