Comparative analysis of salivary antimicrobial resistance genes in dental students: A PCR and questionnaire study

Marwan Mansoor Mohammed; Priyadharshini Sekar; Jahida Al Jamal; Lujayn Abu Taha; Asma Bachir; Sausan Al Kawas

doi:10.1371/journal.pone.0315450

Peer Review History

Original SubmissionAugust 9, 2024
22 Sep 2024 Decision Letter - Farah Al-Marzooq, Editor PONE-D-24-33161Comparative Analysis of Salivary Antimicrobial Resistance Genes in Dental Students: A PCR and Questionnaire StudyPLOS ONE Dear Dr. Mohammed, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact. Please submit your revised manuscript by Nov 06 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript by Marwan Mansoor Mohammed et al., examining the prevalence of antimicrobial resistance (AMR) genes in the saliva of dental students, aims to fill a gap in the literature regarding AMR in the UAE by utilizing PCR techniques and questionnaire data. Despite its relevant topic, the study struggles with significant issues, including methodological weaknesses, poor data interpretation, and a lack of innovation, which undermine its scientific credibility and suitability for publication in PLOS ONE. The absence of a clear rationale for gene selection, inadequate statistical analysis, superficial interpretation of results, and insufficient integration into the wider AMR discourse highlight the urgent need for extensive revisions to elevate the manuscript to the standards expected by reputable scientific journals. Reviewer #2: The present manuscript deals with the prevalence of some important antibiotic resistance genes in saliva from healthy dental students. The authors used real-time PCR to calculate the nrelative concentration of the AMR genes in saliva samples of 40 5th-year dental students and 40 ist-year students. Tha manuscript is well written and oraganized and the results were discussed in a proper manner. Only two points raised by the reviewer: 1) There have been currenty a big study regarding AMR genes in the oral microbiome: The oral microbiota is a reservoir for antimicrobial resistance: resistome and phenotypic resistance characteristics of oral biofilm in health, caries, and periodontitis. Anderson AC, von Ohle C, Frese C, Boutin S, Bridson C, Schoilew K, Peikert SA, Hellwig E, Pelz K, Wittmer A, Wolff D, Al-Ahmad A. Ann Clin Microbiol Antimicrob. 2023 May 13;22(1):37. doi: 10.1186/s12941-023-00585-z. The results of the authors should be compared to the results püresented by the aforementioned study. 2) Some disinfectants like the frequently used chlorhexidine digluconate could influence the AMR prevalence in human saliva (see: Chlorhexidine digluconate mouthwash alters the oral microbial composition and affects the prevalence of antimicrobial resistance genes. Bartsch S, Kohnert E, Kreutz C, Woelber JP, Anderson A, Burkhardt AS, Hellwig E, Buchalla W, Hiller KA, Ratka-Krueger P, Cieplik F, Al-Ahmad A. Front Microbiol. 2024 Jun 25;15:1429692. doi: 10.3389/fmicb.2024.1429692. eCollection 2024.) Again, the authors should also consider discussing their resulty taking the aforementioned study into account. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: review.docx https://doi.org/10.1371/journal.pone.0315450.r001
Revision 1
6 Nov 2024 Author Response Response to Journal Requirements and Reviewers Comments Manuscript ID: PONE-D-24-33161 Title: Comparative Analysis of Salivary Antimicrobial Resistance Genes in Dental Students: A PCR and Questionnaire Study A. Response to Journal Requirements 1. Ensuring Compliance with PLOS ONE Style Requirements: We have revised the manuscript to fully comply with PLOS ONE’s style guidelines, including file naming conventions. The format of the title page, abstract, and manuscript body follows the guidelines provided at the links shared. 2. Grant Information Consistency: We have updated the grant information to ensure consistency between the Funding Information and Financial Disclosure sections. The correct grant number for the awards received for this study are now included in the Funding Information section. 3. Funding Information in the Acknowledgements: As requested, we have removed all funding-related text from the Acknowledgments section. The revised Acknowledgments now only mention non-financial contributions. For the Funding Statement, we propose the following updated statement: "This research was supported by the Research Funding Department, University of Sharjah, Sharjah, UAE. (Grant No. 2101100144)" Please update the online submission form accordingly. 4. Data Availability Statement: We have reviewed our data-sharing plan. We will ensure that the data is freely available upon acceptance, in accordance with PLOS ONE's open data policy. We confirm that there are no participant privacy concerns that would restrict the deposition of the data. The updated Data Availability Statement is as follows: "All relevant data will be made publicly available upon acceptance in a suitable data repository. There are no restrictions to data sharing." B. Response to the Reviewers’ Comments Reviewer #1: The manuscript by Marwan Mansoor Mohammed et al., examining the prevalence of antimicrobial resistance (AMR) genes in the saliva of dental students, aims to fill a gap in the literature regarding AMR in the UAE by utilizing PCR techniques and questionnaire data. Despite its relevant topic, the study struggles with significant issues, including methodological weaknesses, poor data interpretation, and a lack of innovation, which undermine its scientific credibility and suitability for publication in PLOS ONE. The absence of a clear rationale for gene selection, inadequate statistical analysis, superficial interpretation of results, and insufficient integration into the wider AMR discourse highlight the urgent need for extensive revisions to elevate the manuscript to the standards expected by reputable scientific journals. Firstly, the study fails to provide a rationale for selecting specific AMR genes for analysis. The absence of this critical justification undermines the scientific foundation of the research, casting doubts on the relevance and scope of the study. This omission is particularly problematic as it hinders the reader’s ability to fully understand the intentions and potential impact of the research within the broader field of AMR studies. By addressing this issue, the study could potentially make a significant contribution to the field of AMR research in the UAE. Moreover, the manuscript's results section needs robust statistical analysis, omitting essential details such as the statistical tests, p-values, or confidence intervals. This lack of rigorous statistical documentation is a severe shortfall, rendering the findings unreliable and their conclusions untrustworthy. This oversight must meet the stringent standards for a journal committed to rigorous scientific scrutiny. The manuscript also presents a wealth of data on the distribution of various AMR genes among dental students but does so without adequate analysis of how these data correlate with other variables like health status or antibiotic use history. The absence of in-depth analytical insight into these potential relationships means the research does not fully utilize its data to advance understanding of AMR dynamics, which is necessary for impactful research. A more comprehensive analysis is needed to fully understand the implications of the data. Additionally, discussing findings related to different resistance genes needs more contextualization within the broader AMR research landscape. The data are presented in isolation, without comparative analysis to existing literature, diminishing the manuscript’s scientific value. By not linking its findings to the wider discourse on antimicrobial resistance, the study misses an opportunity to make a meaningful contribution to the field. The manuscript frequently notes the high prevalence of various ARGs, suggesting their potential for dissemination within the oral microbiome. However, it needs to contextualize these findings within global research trends or epidemiological data. This lack of a nuanced discussion that critically examines how the results compare with global trends in AMR prevalence, especially given the unique context of the UAE, further limits the manuscript’s impact. Functional assays or additional validation do not support the interpretation of findings, particularly the speculative conclusions drawn from the PCR data regarding the hydrolytic activities of enzymes like the blaCTX-M variants. This overinterpretation relative to the methodological rigor presented leads to conclusions not substantiated by the data. The speculative nature of these interpretations, especially the reliance on Ct values to indicate gene concentration and their clinical relevance, is scientifically unsound. Response to Reviewer #1 Thank you for your thorough and constructive review of our manuscript. We appreciate the time and effort you put into evaluating our work and providing valuable feedback. Below, we provide a point-by-point response to your comments, along with a summary of the revisions made to address each concern. Comment 1: Absence of a clear rationale for gene selection Reviewer’s comment: The study fails to provide a rationale for selecting specific AMR genes for analysis. This omission undermines the scientific foundation of the research, casting doubts on the relevance and scope of the study. Response: We agree that a clear rationale for the gene selection was needed. In the revised manuscript, we have added a section in the Introduction where we explain the selection criteria for the AMR genes included in this study. Specifically, the genes were chosen based on their known prevalence in clinical and environmental samples, particularly in the context of oral bacteria, as well as their documented significance in previous AMR research. Additionally, we focused on genes that are prevalent in Gram-negative bacteria and have been linked to resistance in the human microbiome. These genes are also relevant in the UAE context, as few studies have been conducted in this region, and they represent a growing concern for global public health. Comment 2: Inadequate statistical analysis Reviewer’s comment: The manuscript lacks robust statistical analysis, omitting essential details such as statistical tests, p-values, and confidence intervals. Response: We have now enhanced the statistical analysis in the Results section to address this concern. We have included detailed information on the statistical methods used, including One-way ANOVA with Tukey’s multiple comparison test or unpaired t test was used where applicable. In addition, we have provided p-values and confidence intervals to demonstrate the significance of our findings. These revisions ensure that the data is presented with the appropriate rigor expected for publication. Comment 3: Superficial interpretation of results Reviewer’s comment: The manuscript presents a wealth of data on the distribution of various AMR genes among dental students but lacks adequate analysis of correlations with other variables, such as health status or antibiotic use history. Response: We appreciate this suggestion and have expanded the interpretation of the results accordingly. In the revised Results and Discussion sections, we now provide a more comprehensive analysis of potential correlations between Beta lactamase and aminoglycoside resistance gene prevalence and participant characteristics, including health status, smoking habits, and antibiotic usage by chi-square test. This has allowed us to explore more in-depth relationships and implications of the findings, providing a clearer picture of how these factors may influence the prevalence of AMR genes in dental students. Comment 4: Lack of contextualization within broader AMR research Reviewer’s comment: The data are presented in isolation, without comparative analysis to existing literature, diminishing the manuscript’s scientific value. Response: We agree that placing our findings within the broader AMR research landscape is crucial. We have revised the Discussion to compare our results with recent studies on AMR gene prevalence, particularly those examining the oral microbiome in different populations. We now reference several key studies, including those conducted in other geographical regions, to demonstrate how our findings contribute to the understanding of AMR in the oral microbiome. This comparison also highlights the unique aspects of our study in the context of the UAE population. Comment 5: Lack of global contextualization and epidemiological data Reviewer’s comment: The manuscript lacks a nuanced discussion that compares the findings with global trends in AMR prevalence, especially given the unique context of the UAE. Response: We have expanded the Discussion to include a broader analysis of global AMR trends, particularly focusing on the oral microbiome as a reservoir for AMR genes. By comparing our findings with global epidemiological data, we show how the high prevalence of certain AMR genes in the UAE fits within international patterns. This comparison strengthens the significance of our study, emphasizing the potential for oral microbiota to act as a reservoir for antimicrobial resistance, not only in clinical settings but also in non-clinical populations such as dental students. Comment 6: Overinterpretation of PCR data without functional validation Reviewer’s comment: The interpretation of findings is not supported by functional assays, particularly the conclusions drawn from PCR data regarding the hydrolytic activities of enzymes like the blaCTX-M variants. Response: We have revised the Discussion to clarify the limitations of our study, particularly with respect to the interpretation of PCR data. We no longer speculate on the hydrolytic activity of blaCTX-M variants based solely on the PCR results. Instead, we focus on the detection of gene presence and relative concentration, acknowledging that functional assays are required to confirm the enzymatic activity of these genes. We have also added a section in the Conclusions that suggests future studies should include functional validation to substantiate these findings. We believe that these revisions significantly improve the manuscript and address the concerns you raised. Thank you again for your valuable feedback. We hope that the revised version meets the high standards of PLOS ONE. Reviewer #2: The present manuscript deals with the prevalence of some important antibiotic resistance genes in saliva from healthy dental students. The authors used real-time PCR to calculate the relative concentration of the AMR genes in saliva samples of 40 5th-year dental students and 40 1st-year students. The manuscript is well written and organized and the results were discussed in a proper manner. Only two points raised by the reviewer: 1) There have been currently a big study regarding AMR genes in the oral microbiome: The oral microbiota is a reservoir for antimicrobial resistance: resistome and phenotypic resistance characteristics of oral biofilm in health, caries, and periodontitis. Anderson AC, von Ohle C, Frese C, Boutin S, Bridson C, Schoilew K, Peikert SA, Hellwig E, Pelz K, Wittmer A, Wolff D, Al-Ahmad A. Ann Clin Microbiol Antimicrob. 2023 May 13;22(1):37. doi: 10.1186/s12941-023-00585-z. The results of the authors should be compared to the results presented by the aforementioned study. 2) Some disinfectants like the frequently used chlorhexidine digluconate could influence the AMR prevalence in human saliva (see: Chlorhexidine digluconate mouthwash alters the oral microbial composition and affects the prevalence of antimicrobial resistance genes. Bartsch S, Kohnert E, Kreutz C, Woelber JP, Anderson A, Burkhardt AS, Hellwig E, Buchalla W, Hiller KA, Ratka-Krueger P, Cieplik F, Al-Ahmad A. Front Microbiol. 2024 Jun 25;15:1429692. doi: 10.3389/fmicb.2024.1429692. eCollection 2024.) Again, the authors should also consider discussing their results taking the aforementioned study into account. Response to Reviewer #2 Thank you for your positive comments and for highlighting the two main points in your review. We appreciate your suggestions, which have helped us improve the manuscript. Below is our response to each of your comments. Comment 1: Comparison with Anderson et al. (2023) study on AMR genes in the oral microbiome Reviewer’s comment: There have been currently a big study regarding AMR genes in the oral microbiome: The oral microbiota is a reservoir for antimicrobial resistance: resistome and phenotypic resistance characteristics of oral biofilm in health, caries, and periodontitis. Anderson AC, von Ohle C, Frese C, Boutin S, Bridson C, Schoilew K, Peikert SA, Hellwig E, Pelz K, Wittmer A, Wolff D, Al-Ahmad A. Ann Clin Microbiol Antimicrob. 2023 May 13;22(1):37. doi: 10.1186/s12941-023-00585-z. The results of the authors should be compared to the results presented by the aforementioned study. Response: We appreciate your suggestion to compare our findings with the recent study by Anderson et al. (2023). In the revised Discussion section, we have now incorporated a detailed comparison of our results with those of Anderson et al. (2023). Specifically, we discuss similarities and differences in the prevalence of key AMR genes in the oral microbiome, as well as how the resistome differs between healthy individuals and those with oral conditions like caries and periodontitis. We believe this comparison adds depth to our discussion and situates our findings within the broader context of recent AMR research. Comment 2: Consideration of chlorhexidine’s impact on AMR prevalence Reviewer’s comment: Some disinfectants like the frequently used chlorhexidine digluconate could influence the AMR prevalence in human saliva (see: Chlorhexidine digluconate mouthwash alters the oral microbial composition and affects the prevalence of antimicrobial resistance genes. Bartsch S, Kohnert E, Kreutz C, Woelber JP, Anderson A, Burkhardt AS, Hellwig E, Buchalla W, Hiller KA, Ratka-Krueger P, Cieplik F, Al-Ahmad A. Front Microbiol. 2024 Jun 25;15:1429692. doi: 10.3389/fmicb.2024.1429692. eCollection 2024.) Response: We agree that the impact of disinfectants like chlorhexidine on AMR prevalence in the oral microbiome is an important factor to consider. In the revised manuscript, we have added a section in the Discussion addressing how the use of chlorhexidine and other antimicrobial agents may alter the prevalence of AMR genes in the oral cavity. We reference the study by Bartsch et al. (2024), highlighting its findings on how chlorhexidine affects the composition of the oral microbiome and contributes to the selection of resistant strains. This contextualizes our findings and provides an additional perspective on how external factors may influence AMR prevalence in the dental student population. We are grateful for your comments, which have helped improve the quality of our manuscript. We believe that the revisions made in response to your feedback enhance the robustness and relevance of the study. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0315450.r002
26 Nov 2024 Decision Letter - Farah Al-Marzooq, Editor Comparative Analysis of Salivary Antimicrobial Resistance Genes in Dental Students: A PCR and Questionnaire Study PONE-D-24-33161R1 Dear Dr. Mohammed, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Farah Al-Marzooq, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0315450.r003
Formally Accepted
6 Jan 2025 Acceptance Letter - Farah Al-Marzooq, Editor PONE-D-24-33161R1 PLOS ONE Dear Dr. Mohammed, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Farah Al-Marzooq Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0315450.r004

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