An insight into genes responsible for fosfomycin resistance among uropathogens of asymptomatic bacteriuria during pregnancy: A North Indian study

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1. 

   Version published to 10.1099/acmi.0.000623.v5 on Access Microbiology

   Dec 1, 2023
2. 

   Access Microbiology

   Oct 28, 2023

   Thank you for submitting your revised manuscript to Access Microbiology and including the suggested changes. The manuscript will now be accepted for publication. Congratulations!

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3. 

   Version published to 10.1099/acmi.0.000623.v4 on Access Microbiology

   Oct 26, 2023
4. 

   Access Microbiology

   Oct 4, 2023

   Editor's Comments to Author Thank you very much for submitting your revised manuscript to Access Microbiology and implementing the suggested changes. I would really like to commend all authors for the efforts in improving the manuscript. However, there are still some modifications that need to be applied. In particular, there are issues with the representations shown in figure 3. The bar chart represents prevalence in percentages, however the bars, as they are piled up, nearly reach 400% (which makes no sense in this terms). This type of representation makes it really difficult for the reader to ascertain a proportional value to the prevalence of each antibiotic sensitivity for the different pathogens. Furthermore, the table included in this figure shows words split in different lines, as they do not fit in the cells, and values …

   Editor's Comments to Author Thank you very much for submitting your revised manuscript to Access Microbiology and implementing the suggested changes. I would really like to commend all authors for the efforts in improving the manuscript. However, there are still some modifications that need to be applied. In particular, there are issues with the representations shown in figure 3. The bar chart represents prevalence in percentages, however the bars, as they are piled up, nearly reach 400% (which makes no sense in this terms). This type of representation makes it really difficult for the reader to ascertain a proportional value to the prevalence of each antibiotic sensitivity for the different pathogens. Furthermore, the table included in this figure shows words split in different lines, as they do not fit in the cells, and values exceeding the cell limits. I suggest to look for an alternative table format, and present it as a separate table (as it is a table), rather than as part of a figure.

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5. 

   Version published to 10.1099/acmi.0.000623.v3 on Access Microbiology

   Oct 4, 2023
6. 

   Access Microbiology

   Sep 17, 2023

   Thank you very much for submitting your manuscript to Access Microbiology. This revised version has now been reviewed and, although there is a level of improvement, some of the reviewers’ comments have not been addressed. Corrections still needed are: • The title suits better the work presented now, but please consider changing the semicolon by a colon. • Change “&” by “and” throughout the manuscript • Avoid the use of “etc” when the full information can be provided, for example in L45, L69 and L256. • Capitalise “Gram” and italicise gene names, species names (including the “sp.” abbreviation) and the “et al.” abbreviation throughout the text. Examples are L50, L54, L73-74, L102, etc. • L57: correct “prelimanary” to “preliminary” • L63: correct “occurs” to “occur” • Use the Greek symbol  when referring to …

   Thank you very much for submitting your manuscript to Access Microbiology. This revised version has now been reviewed and, although there is a level of improvement, some of the reviewers’ comments have not been addressed. Corrections still needed are: • The title suits better the work presented now, but please consider changing the semicolon by a colon. • Change “&” by “and” throughout the manuscript • Avoid the use of “etc” when the full information can be provided, for example in L45, L69 and L256. • Capitalise “Gram” and italicise gene names, species names (including the “sp.” abbreviation) and the “et al.” abbreviation throughout the text. Examples are L50, L54, L73-74, L102, etc. • L57: correct “prelimanary” to “preliminary” • L63: correct “occurs” to “occur” • Use the Greek symbol  when referring to -lactamases, as in L202. Corrections are needed in L73 and L170. • L75-78: this sentence is very long and complex. Please split and simplify to improve the reader’s understanding. • After the first mention to a bacterial species (Escherichia coli, L67), in the following mentions of it the name can be contracted (E. coli). Correct this in L79 and throughout the manuscript. • L84-85: justifications of lack of funding can be given in the appropriate place in Materials and Methods (if given at all in the text, after answering the reviewer’s comment). Here it seems out of place. • L85-86: “similar works done internationally” needs references and ideally a more elaborated explanation, as requested by the reviewers. • L124: “the following antimicrobial agents” are not mentioned. • L138-139: what does “in ambient air” mean? • L146: correct “molecular grade” to “molecular biology grade” • L149: refer to Table 3 listing the primers. Correct “Deionized Nuclease” to “Deionized Nuclease-free water”. • L151: define the composition of TAE buffer based on concentrations, not absolute amounts, as the reader cannot know to what volume that is referred. • Figure 3: labelling cannot be read in the amoxiclav bar. • L171: “Carbapenem” does not need capitalisation. • Figure 4: italics for gene names are missing in the figure labels and the figure legend. • L227: Enterobacterales needs capitalisation • L256: fosX has not been previously mentioned in the text, then the corresponding citation needs to be added. • L264: “The overall resistance of fosfomycin was low according to our study”. This statement needs something to be compared to, as the definition of “low” can be subjective. • Table legends: Genus and species names need to be in italics.

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7. 

   Version published to 10.1099/acmi.0.000623.v2 on Access Microbiology

   Sep 14, 2023
8. 

   Access Microbiology

   Jul 10, 2023

   In this manuscript, Khatoon et al. describe the antibiotic susceptibility of bacterial strains isolated from asymptomatic bacteriuria during pregnancy. Overall, the content of the manuscript is of interest to the public and has relevance in the clinic. This work has now been reviewed by two experts in the field, who have raised their concerns about the manuscript and made suggestions. As per these suggestions, major improvements need to applied to structure and writing of the text, as well as to the level of detail used in the different sections of the manuscript. Please apply these suggestions and correction thoroughly, especially those concerning: • Expansion and deeper level of detail in the Introduction, and stating a clear aim that is coherent with the rest of the sections. • Providing more methodological details that allow …

   In this manuscript, Khatoon et al. describe the antibiotic susceptibility of bacterial strains isolated from asymptomatic bacteriuria during pregnancy. Overall, the content of the manuscript is of interest to the public and has relevance in the clinic. This work has now been reviewed by two experts in the field, who have raised their concerns about the manuscript and made suggestions. As per these suggestions, major improvements need to applied to structure and writing of the text, as well as to the level of detail used in the different sections of the manuscript. Please apply these suggestions and correction thoroughly, especially those concerning: • Expansion and deeper level of detail in the Introduction, and stating a clear aim that is coherent with the rest of the sections. • Providing more methodological details that allow reproducibility and clarify the specific points raised by the reviewers. • Extensive corrections are needed in the Results section, including the figures. Please also consider the suggestions on alternative data representation to improve the understanding of the reader. • The Discussion section needs to be expanded and the findings and their implications need to be put in context, rather than holding on the repetition of the results. • The abstract needs to be coherent with the rest of the manuscript and substantially improved. • Several suggestions have been made on title changes, please consider them for improvement.

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9. 

   Access Microbiology

   Jul 10, 2023

   Comments to Author

   Introduction * Introduction could be expanded: o Have other studies looked at this? What have they found? o Why were the 4 genes chosen in this study? Are these genes important in resistance? Give background / rationale behind this Methods * Line 101 - was quality control (QC) performed for antimicrobial susceptibility testing (AST) according to CLSI recommendations? What was the control strain used for each bacterial species? * Line 104 - what CLSI guideline was used? * Line 106 - 116: this information would be better presented in a table - include antibiotic category and concentration of antibiotic (μg) used for susceptibility testing of different bacterial species. The CLSI breakpoints for the zone diameter interpretive criteria (mm) to classify isolates as sensitive (S), intermediate (I) and …

   Comments to Author

   Introduction * Introduction could be expanded: o Have other studies looked at this? What have they found? o Why were the 4 genes chosen in this study? Are these genes important in resistance? Give background / rationale behind this Methods * Line 101 - was quality control (QC) performed for antimicrobial susceptibility testing (AST) according to CLSI recommendations? What was the control strain used for each bacterial species? * Line 104 - what CLSI guideline was used? * Line 106 - 116: this information would be better presented in a table - include antibiotic category and concentration of antibiotic (μg) used for susceptibility testing of different bacterial species. The CLSI breakpoints for the zone diameter interpretive criteria (mm) to classify isolates as sensitive (S), intermediate (I) and resistant (R) and the QC results for each antibiotic. * Line 117 - 136: this information could also be summarized in a table (similar to above point) * Line 138 - 140: reword these statements as it is unclear * For disc diffusion and MIC: o What inoculum of bacteria suspension was used? o Incubation: How long, what temperature and what conditions e.g. aerobic/anaerobic? * Line 146 - 150: More information is needed for PCR method: o Was this just for E. coli? o Were all isolates tested for all genes or was it just a selection? o How was DNA extracted for PCR? o Were the reaction volumes / components the same as the references? o What controls were used? o Cycling conditions could for each gene could be included in the table. o References are different for the primers in the table (13,15,16) than those in the text (12, 13) - please correct this. o How were the PCR products visualized (e.g. gel, staining etc)? Results * Figure 1: o Update figure - not clear what colours correspond to what bacterial species and this looks messy with a lot of text - could include some information in a table in supplementary information o What is the * for [n* =259, (22%)]? * Lines 156 - 165: This information should be condensed as it's already mentioned in the figure * Figure 1 and lines 156 - 165: The percentages quoted do not match between the figure and the text - this needs corrected e.g. E. coli 496 (67.7%) in text and 495 (42%) on figure * Line 160 and 164 - Acinetobacter species mentioned twice but different figures - which is correct? * Table 2 and 3 could be presented as stacked bar charts, showing the percentage of isolates that were resistant, intermediate or susceptible. A table with the raw results could be included in the supplementary. * Line 166 -182: At the minute, the results are repeating those in the table and can be improved to give a summary, highlighting the antibiotics that had high sensitivity for different bacterial species and also those that had high levels of resistance. * Figure 2: The bp on the ladder should be labelled on the figure. Figure 3a and b shows poor separation/smearing of the ladder, is a better quality image available? Discussion * Discussion could be improved - results have been repeated on several occasions e.g. line 214-218, 229-230, 245-251, 265-269. The relevance of the results showed be discussed without repeating the results and compared to other work. How will the results be used in the future and will there be future work on this? Were there any limitations to the study? Abstract * This could be improved: o The aim in the abstract differs from the aim in the introduction - may be best to keep these consistent. o Method - scope of writing needs to be improved. Was done was used multiple times, use other phrases e.g. determined etc. o What is the overall conclusion from this study? Were rates of Fosfomycin high - could this be used to treat ASB cases during pregnancy? General points: 1. Remove Title of the article: from the title on website and manuscript 2. Should have MIC in full along with abbreviation in title - consider changing the title of the article? 3. Make sure abbreviations are in full the first time they are used e.g. MIC in title, Line 35 ASB and MIC etc. 4. species does not need to be in italics when mentioning bacteria e.g. line 67 5. Figure 2 - the image for figure 2 has this labelled as figure 3 - please update to correct figure number 6. Line 168 - change percent to % 7. Scope of the writing could be improved throughout the manuscript

   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?

   Yes

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10. 

    Access Microbiology

    Jun 11, 2023

    Comments to Author

    Authors Khatoon et al. explore the distribution of bacterial species found in asymptomatic bacteriuria in pregnancy. The authors then assess susceptibility to a range of antibiotics using both disc diffusion and agar dilution assays, finding high levels of resistance to clinically important antibiotics, including carbapenems and methicillin. Following this, the authors assessed the susceptibility of all isolates to fosfomycin to determine if this is a viable alternative to current treatments, finding high degree of susceptibility (>90%) in all isolates tested. Overall, the manuscript is well written and logically ordered. Figure 1 could be more appropriately be presented with a bar chart rather than pie chart to aid interpretation. However, there are questions around the appropriate use of disc …

    Comments to Author

    Authors Khatoon et al. explore the distribution of bacterial species found in asymptomatic bacteriuria in pregnancy. The authors then assess susceptibility to a range of antibiotics using both disc diffusion and agar dilution assays, finding high levels of resistance to clinically important antibiotics, including carbapenems and methicillin. Following this, the authors assessed the susceptibility of all isolates to fosfomycin to determine if this is a viable alternative to current treatments, finding high degree of susceptibility (>90%) in all isolates tested. Overall, the manuscript is well written and logically ordered. Figure 1 could be more appropriately be presented with a bar chart rather than pie chart to aid interpretation. However, there are questions around the appropriate use of disc diffusion assays for fosfomycin (detailed below) and the interpretation and inclusion of PCR results. Major comments: Define abbreviations throughout at their first usage, including the abstract. Introduction is very brief, resistance to fosfomycin could be explored more here and inclusion of previously published data to support statements included. This is also true of the discussion, although I appreciate this is a short communication. Why was BHI used for agar dilution assays for VRSA and VRE, rather than MHA? Can the authors confirm glucose 6 phosphate was include in the testing for fosfomycin disc diffusion? It is not clear from the methods. Line 225: What is considered as "significant" level of resistance? Line 249-251: Modify this sentence as it states all were found to be resistant by agar dilution, but one isolate was found to be susceptible. Line 260: States Fosfomycin resistance mediated by mutations in murA, glpT or uhpT, however PCR only detects the presence of these genes not mutations within them, which questions the validity of inclusion of this assay. Where the PCR products sequenced to detect mutations? Minor comments: Line 39: Where the Department of Microbiology located? Line 41: What standard methods were used, CLSI or EUCAST? Line 41-43: How was this performed? And why were isolates randomly tested by agar dilution? Line 44 (and throughout): gene names need to be italics. Line 49-51: What species were these isolates? Line 115: Remove around, be specific on numbers What is meant by "high level" in table 2 and 3? Ensure bacterial species names are italicised throughout Ensure Gram is capitalised throughout.

    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

    Read the original source
11. 

    Version published to 10.1099/acmi.0.000623.v1 on Access Microbiology

    May 16, 2023