Resistance trend in bacteria isolated from corneal ulcers: A retrospective analysis from Pakistan

Naila Obaid; Ayesha Saeed; Sidra Abbas; Shaghufta Perveen; Hajira Younas

doi:10.1371/journal.pone.0325157

Peer Review History

Original SubmissionNovember 18, 2024
18 Nov 2024 Author Response https://doi.org/10.1371/journal.pone.0325157.r001
19 Jan 2025 Decision Letter - Mengistu Hailemariam Zenebe, Editor Dear Dr. Abbas, Please submit your revised manuscript by Mar 05 2025 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 . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. 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Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The article presents significant findings on antibiotic resistance patterns from corneal ulcers. Globally, antibiotic resistance is recognized as a major public health threat. These infections can lead to visual impairment and even blindness if not treated. Pseudomonas aeruginosa shows high resistance to commonly used antibiotics such as ciprofloxacin and gentamicin, which are frequently prescribed for corneal infections. This trend is alarming, necessitating that the protocols may need to be reevaluated to manage these infections. In the context of Pakistan, the study's implications are particularly pronounced. The country faces a significant burden of infectious diseases, compounded by inadequate healthcare infrastructure and the overuse of antibiotics. Even though the sample size is small, I have not seen such a study reported in the Pakistani population. There is a lack of data on antibiotic resistance patterns in Pakistan concerning corneal ulcers. The current study fills this gap by providing valuable insights. The appealing visual illustrations of resistance patterns enhance the understanding of the relationships between different antibiotics and bacterial strains. This study not only informs clinical practice but also underscores the urgent need for public health initiatives to address antibiotic misuse. Overall, I suggest minor revisions to improve the quality of the manuscript further. Introduction Explain antibiotics use and the prevalence of resistance in Pakistan, especially in ophthalmic infections? Highlight how local practices may contribute to resistance patterns? Methodology Please elaborate on the disc diffusion method. Discuss how the results were interpreted, including the criteria for determining resistance and susceptibility. This is crucial for readers to understand the significance of your findings. Discussion It is important to acknowledge the role of non-bacterial agents in corneal infections. Discuss the prevalence of fungal pathogens such as Aspergillus and Candida, as well as viral and parasitic causes. Highlight how the resistance patterns observed in the current study reflect or diverge from global trends. Reviewer #2: The study's measurement of healing rates in corneal ulcer patients, alongside assessing antibiotic resistance levels across various bacterial strains and their specific treatment responses, is highly relevant in the context of rising antibiotic resistance. Understanding the healing rates allows for the evaluation of treatment efficacy while analyzing resistance patterns, which provides critical insights into the challenges faced in managing corneal infections as well as informs more personalized treatment plans towards optimizing patient outcomes. However, here are some comments and recommendations. • The timeline of the study should be reviewed and consistently stated throughout the document to ensure clarity. • The sample size utilized in this study is insufficient. • Currently, relying solely on disc diffusion test results is not widely accepted for detecting antibiotic resistance. Therefore, it is recommended that the authors validate their disc diffusion results, particularly for resistant strains, using additional approved methods such as the microdilution method or E-test. Employing these techniques will provide accurate minimum inhibitory concentration (MIC) values for each resistant antibiotic strain identified. • The detection of vancomycin resistance through disc diffusion is not considered valid according to CLSI and EUCAST guidelines. It is essential to perform E-tests alongside microdilution methods to assess vancomycin resistance and investigate the presence of VanA, VanB, and VanC genes in the isolated strains. • Although phenotypic approaches are valuable, integrating high-throughput molecular methods would yield data, leading to stronger conclusions. Notwithstanding, the authors did impressive work regarding their statistical analysis. The highlights of some of the statistical analysis, especially from the frequency of interactions between each antibiotic and bacterial strain, can be stated in the abstract. ****** 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: Yes: ALEXANDER KWARTENG ******** [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 Attachments Attachment Submitted filename: Reviewer comments and recommendation.docx https://doi.org/10.1371/journal.pone.0325157.r002
Revision 1
17 Feb 2025 Author Response Response to Reviewers’ Comments The authors are thankful to the reviewers for their valuable insights and constructive feedback which has significantly contributed to enhance the quality of the manuscript. All the revisions are highlighted in yellow. Reviewer # 1 General comment The article presents significant findings on antibiotic resistance patterns from corneal ulcers. Globally, antibiotic resistance is recognized as a major public health threat. These infections can lead to visual impairment and even blindness if not treated. Pseudomonas aeruginosa shows high resistance to commonly used antibiotics such as ciprofloxacin and gentamicin, which are frequently prescribed for corneal infections. This trend is alarming, necessitating that the protocols may need to be reevaluated to manage these infections. In the context of Pakistan, the study's implications are particularly pronounced. The country faces a significant burden of infectious diseases, compounded by inadequate healthcare infrastructure and the overuse of antibiotics. Even though the sample size is small, I have not seen such a study reported in the Pakistani population. There is a lack of data on antibiotic resistance patterns in Pakistan concerning corneal ulcers. The current study fills this gap by providing valuable insights. The appealing visual illustrations of resistance patterns enhance the understanding of the relationships between different antibiotics and bacterial strains. This study not only informs clinical practice but also underscores the urgent need for public health initiatives to address antibiotic misuse. Overall, I suggest minor revisions to improve the quality of the manuscript further. Comment 1 Explain antibiotics use and the prevalence of resistance in Pakistan, especially in ophthalmic infections? Response The required information regarding antibiotics use and the prevalence of resistance in Pakistan, particularly in ophthalmic infections, has been added in lines 78-80. Comment 2 Highlight how local practices may contribute to resistance patterns? Response The required information on how local practices contribute to resistance patterns has been incorporated from line 76. This section now includes details on antibiotic overuse, self-medication, and other key factors influencing resistance trends. Comment 3 Please elaborate on the disc diffusion method. Discuss how the results were interpreted, including the criteria for determining resistance and susceptibility. This is crucial for readers to understand the significance of your findings. Response 3 The disc diffusion method, also known as the Kirby-Bauer method, is a widely used technique for assessing the antibiotic susceptibility of bacterial isolates. This method follows the guidelines set by the Clinical and Laboratory Standards Institute (CLSI). The description is added from line 126. Comment 4 It is important to acknowledge the role of non-bacterial agents in corneal infections. Discuss the prevalence of fungal pathogens such as Aspergillus and Candida, as well as viral and parasitic causes. Response The introduction has been expanded from line 61 to include a discussion on the prevalence of fungal pathogens, such as Aspergillus and Candida, as well as viral and parasitic causes of corneal infections. Relevant sources have been cited to highlight the significance of these pathogens in corneal infections. Reviewer # 2 General comment Highlight how the resistance patterns observed in the current study reflect or diverge from global trends. The study's measurement of healing rates in corneal ulcer patients, alongside assessing antibiotic resistance levels across various bacterial strains and their specific treatment responses, is highly relevant in the context of rising antibiotic resistance. Understanding the healing rates allows for the evaluation of treatment efficacy while analysing resistance patterns, which provides critical insights into the challenges faced in managing corneal infections as well as informs more personalized treatment plans towards optimizing patient outcomes. However, here are some comments and recommendations. Comment 1 The timeline of the study should be reviewed and consistently stated throughout the document to ensure clarity. Response The timeline has been carefully reviewed and ensure consistency throughout the document to improve clarity. Comment 2 The sample size utilized in this study is insufficient. Response The incidence of corneal infections is relatively low, and patient recruitment was inherently limited by case availability during the study period. Notably, corneal ulcer cases are more prevalent during the summer and monsoon seasons, which may be attributed to increased exposure to environmental pathogens and humid conditions favouring microbial growth. Nevertheless, the findings provide valuable insights into corneal ulcer pathology and microbial patterns. Future studies with larger cohorts will further substantiate these observations. Comment 3 Currently, relying solely on disc diffusion test results is not widely accepted for detecting antibiotic resistance. Therefore, it is recommended that the authors validate their disc diffusion results, particularly for resistant strains, using additional approved methods such as the microdilution method or E-test. Employing these techniques will provide accurate minimum inhibitory concentration (MIC) values for each resistant antibiotic strain identified. Response Due to economic constraints in Pakistan, the disc diffusion method remains the most widely used and feasible approach in clinical settings. Despite its limitations, it provides reliable and reproducible results under local laboratory conditions. In the current study, E-test is typically performed for vancomycin-resistant strains; however, as no vancomycin resistance was observed, further MIC determination was not required. Comment 4 The detection of vancomycin resistance through disc diffusion is not considered valid according to CLSI and EUCAST guidelines. It is essential to perform E-tests alongside microdilution methods to assess vancomycin resistance and investigate the presence of VanA, VanB, and VanC genes in the isolated strains. Response No vancomycin-resistant strains were identified in this study. The authors acknowledges that as per CLSI and EUCAST guidelines, disc diffusion alone is not a reliable method for detecting vancomycin resistance. However, all isolates showed susceptibility to vancomycin based on the disc diffusion method, and no resistance patterns were observed that would justify further confirmation by E-test, broth microdilution, or PCR for VanA, VanB, and VanC genes. Future studies will consider molecular confirmation where necessary. Comment 5 Although phenotypic approaches are valuable, integrating high-throughput molecular methods would yield data, leading to stronger conclusions. Notwithstanding, the authors did impressive work regarding their statistical analysis. The highlights of some of the statistical analysis, especially from the frequency of interactions between each antibiotic and bacterial strain, can be stated in the abstract. Response Abstract has been modified accordingly. I hope that the responses have satisfied the queries/ comments of the reviewers. Once again I am thankful to the reviewers whose input has raised the quality of the manuscript. Dr. Sidra Abbas Attachments Attachment Submitted filename: Comments_Eye.docx https://doi.org/10.1371/journal.pone.0325157.r003
16 Mar 2025 Decision Letter - Mengistu Hailemariam Zenebe, Editor 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 submit your revised manuscript by Apr 30 2025 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 . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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We look forward to receiving your revised manuscript. Kind regards, Mengistu Hailemariam Zenebe, PhD Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: (No Response) Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Partly Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The study addresses a critical public health issue of antibiotic resistance in corneal ulcer pathogens in Pakistani region with limited data. The topic is timely and clinically relevant as I stated in my previous review comments also. But I have noticed many discrepancies in the revised manuscript which are as following; In response to the comment “Explain antibiotics use and the prevalence of resistance in Pakistan, especially in ophthalmic infections”, The authors addressed the comment partially which is not satisfactory. The authors acknowledged empirical antibiotic use (fluoroquinolones, aminoglycosides) and drivers of resistance (self-medication, over the counter availability of drugs). Quantitative data on resistance prevalence, such as national and regional resistance from Pakistan, is missing. The text lacks data and citations on local ophthalmic infections related antibiotic resistance. The authors response to address the comment “Please elaborate on the disc diffusion method. Discuss how the results were interpreted, including the criteria for determining resistance and susceptibility. This is crucial for readers to understand the significance of your findings.” is incomplete. The CLSI reference is noted, but critical details are missing. Specific CLSI breakpoints for each antibiotic have not been enlisted. No quality control strains have been used to validate the method. I cannot find the addressal of the comment “Highlight how the resistance patterns observed in the current study reflect or diverge from global trends.” In the discussion section of the article. The authors fail to compare findings to regional studies. For example, there is no mention of P. aeruginosa resistance rates to ciprofloxacin in South Asia/Pakistan/India and global data. This comment has not been addressed. The authors need to justify why fungal/viral testing was not performed despite Sabouraud agar use to confirm any co-infection in patients. Discuss the implications of missing fungal/viral co-infection data. Kirby-Bauer disc diffusion alone for vancomycin susceptibility is invalid as per CLSI guidelines. Disc diffusion lacks reliability for glycopeptides and carbapenems. Biochemical tests for bacterial identification lack specificity. Biochemical characterization is rudimentary. Misidentification of Staphylococcus aureus , like coagulase-negative isolates labeled as MRSA raises concerns. No secondary confirmation test was performed, such as broth microdilution and molecular assays. It is better to confirm species using 16S rRNA sequencing. Mixed infections are common in corneal ulcers and influence resistance profiles. Perform fungal cultures or PCR and viral testing to rule out co-infections. The retrospective design lacks a control group, reducing the ability to infer causality or resistance trends. The choice of 26 antibiotics is broad but lacks justification. Why Nitrofurantoin antibiotic also checked as it is used in urinary tract infections? The conclusion is generic and lacks specificity. It overstates the study’s impact given the small sample size. Some citations (Woodland et al., 1992, Title: Causes of conjunctivitis and keratoconjunctivitis in Karachi, Pakistan) are outdated. Include recent studies. The authors did not mention inclusion and exclusion criteria. Decision Major Revisions Suggested. Most of the comments have been addressed superficially. The manuscript suffers from inconsistencies. Reviewer #2: (No Response) ****** 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 Attachments Attachment Submitted filename: Reviewe Comments 2nd R.docx https://doi.org/10.1371/journal.pone.0325157.r004
Revision 2
18 Apr 2025 Author Response Response to Reviewers’ Comments The authors are thankful to the reviewers for their valuable insights and constructive feedback which has significantly contributed to enhance the quality of the manuscript. All the revisions are highlighted in yellow. Reviewer Comments The study addresses a critical public health issue of antibiotic resistance in corneal ulcer pathogens in Pakistani region with limited data. The topic is timely and clinically relevant as I stated in my previous review comments also. But I have noticed many discrepancies in the revised manuscript which are as following. Comment 1 In response to the comment “Explain antibiotics use and the prevalence of resistance in Pakistan, especially in ophthalmic infections”, The authors addressed the comment partially which is not satisfactory. The authors acknowledged empirical antibiotic use (fluoroquinolones, aminoglycosides) and drivers of resistance (self-medication, over the counter availability of drugs). Quantitative data on resistance prevalence, such as national and regional resistance from Pakistan, is missing. The text lacks data and citations on local ophthalmic infections related antibiotic resistance. Response Quantitative data on resistance prevalence at the regional level have been added in the introduction page 3. Comment 2 The authors response to address the comment “Please elaborate on the disc diffusion method. Discuss how the results were interpreted, including the criteria for determining resistance and susceptibility. This is crucial for readers to understand the significance of your findings.” is incomplete. The CLSI reference is noted, but critical details are missing. Specific CLSI breakpoints for each antibiotic have not been enlisted. No quality control strains have been used to validate the method. Response The disc diffusion method has been revised on page 5. Specific CLSI breakpoints for each antibiotic have been included results and discussion in the under the heading antibiotic resistance profile of bacterial species. However, in clinical pathology laboratory, antibiotic susceptibility testing for different samples from different departments in the hospitals including the corneal ulcers are performed as part of routine diagnostic workflow, where hundreds of samples are processed daily under standardized operating procedures. While ideal for research studies, running concurrent QC strains with every routine clinical specimen is not feasible due to: Operational Considerations: As a high-volume diagnostic laboratory processing hundreds of specimens daily, we prioritize, timely reporting for patient care, Batch-level QC validation, Strict adherence to standardized operating procedures Standardized Identification Methods: All isolates underwent biochemical confirmation using API test kits (bioMérieux), consistent with established methodologies given below as well (Khor et al., 2024; Naik et al., 2021). Comment 3 I cannot find the addressal of the comment “Highlight how the resistance patterns observed in the current study reflect or diverge from global trends.” In the discussion section of the article. The authors fail to compare findings to regional studies. For example, there is no mention of P. aeruginosa resistance rates to ciprofloxacin in South Asia/Pakistan/India and global data. This comment has not been addressed. Response Resistance patterns of the tested antibiotics to regional/global data, emphasizing critical disparities have been added in the text on page 10 and 11. Comment 4 The authors need to justify why fungal/viral testing was not performed despite Sabouraud agar use to confirm any co-infection in patients. Discuss the implications of missing fungal/viral co-infection data. Response Fungal and viral keratitis cases were excluded from this study, as the focus was on admitted patients with bacterial keratitis. As part of our standard microbiological protocol, corneal scrapings from all patients were cultured on Sabouraud agar to screen for potential fungal co-infections. However, patients diagnosed with fungal or viral keratitis were managed separately and not included in the final analysis to maintain a focused and uniform study population. Comment 5 Kirby-Bauer disc diffusion alone for vancomycin susceptibility is invalid as per CLSI guidelines. Disc diffusion lacks reliability for glycopeptides and carbapenems. Response Broth microdilution (BMD) MIC testing has now been incorporated for vancomycin susceptibility, as required by CLSI guidelines for glycopeptides. The revised manuscript includes these updated methods in the Methodology section. Comment 6 Biochemical tests for bacterial identification lack specificity. Biochemical characterization is rudimentary. Misidentification of Staphylococcus aureus , like coagulase-negative isolates labeled as MRSA raises concerns. No secondary confirmation test was performed, such as broth microdilution and molecular assays. Response We acknowledge that referring to coagulase-negative isolates as MRSA was a typographical error. In our hospital setting, biochemical identification remains a standard and practical approach for timely diagnosis and reporting, particularly given the high volume of samples and the need to initiate prompt treatment. While we recognize the superior specificity of molecular assays such as PCR, resource limitations and financial constraints precluded their use in this study. Nonetheless, the biochemical methods employed are routinely used in clinical microbiology and are considered sufficiently reliable for initial identification and management. Comment 7 It is better to confirm species using 16S rRNA sequencing. Response Thank you for the suggestion. We agree that 16S rRNA sequencing is a highly reliable and accurate method for bacterial species identification. However, due to the high volume of samples and limited financial and laboratory resources, it was not feasible to incorporate molecular sequencing methods in our study. Our identification protocol relied on conventional biochemical testing, which is routinely used in clinical microbiology laboratories for timely and cost-effective diagnosis. While we acknowledge that 16S rRNA sequencing would enhance the precision of species confirmation, the focus of our study was on clinical outcomes and management, for which conventional identification methods were considered sufficient within the given constraints. Below are the few studies which used biochemical and the sensitivity assays for the identification of the bacterial strains. Khor, W.B., Lakshminarayanan, R., Periayah, M.H., Prajna, V.N., Garg, P., Sharma, N., Mehta, J.S., Young, A., Goseyarakwong, P., Puangsricharern, V. and Tan, A.L., 2024. The antibiotic resistance profiles of Pseudomonas aeruginosa in the Asia Cornea Society Infectious Keratitis Study. International Ophthalmology, 44(1), p.361. Fernandes, M., Vira, D., Medikonda, R. and Kumar, N., 2016. Extensively and pan-drug resistant Pseudomonas aeruginosa keratitis: clinical features, risk factors, and outcome. Graefe's Archive for Clinical and Experimental Ophthalmology, 254, pp.315-322. Naik, P., Pandey, S., Gagan, S., Biswas, S. and Joseph, J., 2021. Virulence factors in multidrug (MDR) and Pan-drug resistant (XDR) Pseudomonas aeruginosa: a cross-sectional study of isolates recovered from ocular infections in a high-incidence setting in southern India. Journal of Ophthalmic Inflammation and Infection, 11, pp.1-11. Cabrera‐Aguas, M., Chidi‐Egboka, N., Kandel, H. and Watson, S.L., 2024. Antimicrobial resistance in ocular infection: A review. Clinical & Experimental Ophthalmology, 52(3), pp.258-275. AlBahrani, S., Alqazih, T.Q., Aseeri, A.A., Al Argan, R., Alkhafaji, D., Alrqyai, N.A., Alanazi, S.M., Aldakheel, D.S., Ghazwani, Q.H., Jalalah, S.S. and Alshuaibi, A.K., 2024. Pattern of cephalosporin and carbapenem-resistant Pseudomonas aeruginosa: a retrospective analysis. IJID regions, 10, pp.31-34. Comment 8 Mixed infections are common in corneal ulcers and influence resistance profiles. Perform fungal cultures or PCR and viral testing to rule out co-infections. Response Thank you for the valuable comment. For all admitted patients with corneal ulcers, corneal scrapings were collected and processed for microbiological evaluation, including staining and culture on blood agar, chocolate agar, and Sabouraud dextrose agar. Smears were also prepared for microscopic examination. Patients who showed a positive culture and sensitivity to a specific antibiotic were started on targeted therapy and included in the study. Cases of viral keratitis were excluded, as these patients were managed on an outpatient basis and not part of the admitted cohort. Similarly, patients with fungal keratitis who developed complications such as non-healing ulcers or corneal perforation were excluded from the final analysis. However, patients presenting with mixed infections involving both bacterial and fungal pathogens, who responded to treatment, were included in the study. Comment 9 The retrospective design lacks a control group, reducing the ability to infer causality or resistance trends. Response Thank you for the comment. However, we respectfully disagree with the suggestion regarding the need for a control group in this context. The retrospective design of our study was intentionally chosen to allow for the efficient analysis of resistance patterns in clinically confirmed cases of infectious keratitis. The primary objective was to characterize antimicrobial resistance in real-world clinical isolates rather than to assess treatment efficacy or outcomes between different groups. Given the ethical considerations, it would not be appropriate to withhold diagnostic procedures, such as corneal scrapings, from symptomatic patients solely to establish an untreated control group. Additionally, control groups are more commonly utilized in prospective clinical trials or interventional studies, whereas retrospective observational studies, particularly those focused on microbiological profiling, do not typically include control arms. Therefore, the absence of a control group does not compromise the validity of our findings within the scope of this study's objectives. Comment 10 The choice of 26 antibiotics is broad but lacks justification. Why Nitrofurantoin antibiotic also checked as it is used in urinary tract infections? Response We acknowledge the reviewer’s concern regarding the inclusion of nitrofurantoin in our antibiotic panel. This was inadvertently added due to an administrative error during table compilation and was originally intended for a separate study on urinary tract infections. We sincerely apologize for this oversight and confirm that nitrofurantoin testing was not performed in our corneal ulcer study. The updated manuscript which now reflects only clinically relevant antibiotics used for ocular infections, with detailed justifications provided for each agent based on CLSI guidelines. Comment 11 The conclusion is generic and lacks specificity. It overstates the study’s impact given the small sample size. Response Conclusion have been revised Comment 12 Some citations (Woodland et al., 1992, Title: Causes of conjunctivitis and keratoconjunctivitis in Karachi, Pakistan) are outdated. Include recent studies. Response Recent references have been included, and the reference are updated accordingly. Comment 13 The authors did not mention inclusion and exclusion criteria. Response Inclusion and exclusion criteria have now been added in the manuscript. Decision Major Revisions Suggested. Most of the comments have been addressed superficially. The manuscript suffers from inconsistencies. I hope that the responses have satisfied the queries/ comments of the reviewers. Once again I am thankful to the reviewers whose input has raised the quality of the manuscript. Dr. Sidra Abbas Attachments Attachment Submitted filename: Eye_C2_18April.docx https://doi.org/10.1371/journal.pone.0325157.r005
8 May 2025 Decision Letter - Mengistu Hailemariam Zenebe, Editor Resistance trend in bacteria isolated from corneal ulcers: A retrospective analysis from Pakistan PONE-D-24-48906R2 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, Mengistu Hailemariam Zenebe, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: I Don't Know ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: The comments have been addressed to somehow satisfactory level. The editor may proceed for acceptance or forward to another reviewer for further evaluation of the manuscript to improve its scientific rigour. ****** 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 ******** https://doi.org/10.1371/journal.pone.0325157.r006
Formally Accepted
Acceptance Letter - Mengistu Hailemariam Zenebe, Editor PONE-D-24-48906R2 PLOS ONE Dear Dr. Abbas, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. Mengistu Hailemariam Zenebe Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0325157.r007

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