Genomic analysis of Acinetobacter baumannii DUEMBL6 reveals diesel bioremediation potential and biosafety concerns

Nizam Uddin; Monjima Islam Prova; Muttasim Billaha; Tasnimul Arabi Anik; Rahat Uzzaman; Nadia Haider; Faruk Islam; Humaira Akhter; Anowara Begum

doi:10.1371/journal.pone.0339456

Peer Review History

Original SubmissionMay 2, 2025
30 May 2025 Decision Letter - Ranjit Gurav, Editor Dear Dr. Begum, Please submit your revised manuscript by Jul 14 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'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Ranjit Gurav, Ph.D Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following financial disclosure: “The research was funded by the Ministry of Science and Technology, and University Grant Commission, Bangladesh.” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 3. Thank you for stating the following in the Funding Section of your manuscript: “The research was funded by the Ministry of Science and Technology, and University Grant Commission, Bangladesh.” We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “The research was funded by the Ministry of Science and Technology, and University Grant Commission, Bangladesh.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments: Thank you for your submission. Upon reviewing the manuscript, we found that Figures 1 to 7 are not sufficiently clear in their current form. To proceed with the review process, we kindly request that you resubmit the manuscript with high-resolution versions of these figures to ensure their clarity and legibility for the reviewers. https://doi.org/10.1371/journal.pone.0339456.r001
Revision 1
10 Jun 2025 Author Response 1.Manuscript Formatting: We have revised our manuscript according to PLOS ONE’s style requirements, using the formatting templates provided for both the main body and the title/author/affiliations sections. 2.Role of the Funders: The funding statement has been updated to clarify the role of funders. Please amend the Funding Disclosure to read: “The research was funded by the Ministry of Science and Technology, and University Grant Commission, Bangladesh. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” 3.Funding Statement Location: We have removed any funding-related information from the Acknowledgments and other manuscript sections. The Funding Statement remains exclusively in the appropriate section of the submission form. 4.Supporting Information Captions: Captions for all Supporting Information files have now been included at the end of the manuscript, and corresponding in-text citations have been updated accordingly to meet the journal's guidelines. 5.High-Resolution Figures: The previous lower resolution may have resulted from the conversion to a combined PDF during initial submission. According to the journal, ‘The compiled submission PDF includes low-resolution preview images of the figures after the reference list. The function of these previews is to allow you to download the entire submission as quickly as possible. Click the link at the top of each preview page to download a high-resolution version of each figure.’ Yet, we have replaced some of the figures with high resolution versions. Attachments Attachment Submitted filename: Response_to_Reviwer.docx https://doi.org/10.1371/journal.pone.0339456.r002
1 Oct 2025 Decision Letter - Ranjit Gurav, Editor Dear Dr. Begum, 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. plosone@plos.org 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'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . Ranjit Gurav, Ph.D Academic Editor Journal Requirements: Reviewers' comments: Comments to the Author ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Partly Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: I Don't Know Reviewer #3: 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 Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** Reviewer #1: (No Response) Reviewer #2: The authors report the isolation of bacteria from oil-contaminated sites in Dhaka, Bangladesh using 1% diesel enrichment/selection media. The authors isolated a range of bacteria (including Gammaproteobacteria) from this environment, many of which would be predicted based on previous literature. The authors test biofilm formation and diesel utilization of the strains before choosing an isolate of Acinetobacter baumannii for genome sequencing and bioinformatic analysis. My specific comments are below: 1. The paper would benefit from further background discussion of the alkB and catE genes, their roles biodegradation, and previous uses in the field. 2. The authors mention comparison of % diesel degradation to other previously tested strains (lines 269-270), but these analyses were not done “side by side” as far as I can see. Were similar conditions used, so as to make this a valid comparison? Ideally, it would have been optimal to include other control/comparison strains in their analysis. 3. I did not see any figure legends in the material that was available for review. Please provide figure legends for the reader. 4. The authors discuss genes/relevant pathways that are present in the sequenced Acinetobacter isolate. The study would benefit from deletion/inactivation of some of these genes and then observing if this affects the biodegradation activity. 5. The authors discuss the presence of genes/systems for biosynthesis pathways in the sequenced isolate (as opposed to biodegradation). Is there any evidence that these pathways are functional? Could some assays be performed to test this? 6. The authors discuss the fact that virulence genes and antibiotic resistance genes were detected in the sequenced Acinetobacter isolate and that these genes could pose a problem for the use of the strain in biodegradation. The authors should suggest that these genes could possibly be deleted/inactivated in this strain, and then the resulting strain tested to see if this affects the biodegradation activity. In other words, the strain could possibly be engineered to eliminate virulence and antibiotic resistance for future use/applications. Experiments toward this end would improve the paper. 7. Was the sequenced isolate tested for relevant antibiotic resistance? This would improve the paper and give insight into whether those genes are expressed/functional. 8. Whenever the authors refer to the sequenced strain DUEMBL6, it would help the reader if they include the genus/species in key locations to help the reader (some examples: titles to Figures 4, 5, and 7; line 357, line 362, etc). I realize you mention this strain in the text, but it helps the reader overall when reading the whole paper to include the genus/species, especially since there are many strain numbers used in the paper (for example, write as “Acinetobacter baumannii DUEMBL6”). Reviewer #3: Dear Authors, Thank you for the opportunity to review your manuscript titled “Genomic Analysis of Acinetobacter baumannii DUEMBL6 Reveals Diesel Bioremediation Potential and Biosafety Concerns.” The study is methodologically sound, timely, and relevant, but I believe it can be strengthened by addressing the following points: 1. Comparative Performance of DUEMBL6 o Please consider including a comparative framework, such as a table summarizing the diesel degradation efficiencies of DUEMBL6 alongside those of other globally reported strains or consortia. This will help contextualize your findings. 2. Biosafety and Application Risks o The biosafety discussion requires more depth. I recommend elaborating on potential strategies such as the use of non-pathogenic microbial consortia, genetic attenuation of virulence factors, and immobilization or encapsulation techniques to prevent uncontrolled spread. 3. Functional Validation of Genomic Insights o The alkB mutation analysis (I138F, A334T) is interesting, but conclusions regarding functional advantage remain speculative. Please acknowledge this as a limitation and recommend future work, such as enzyme activity assays, transcriptomics, and proteomics, to validate functionality. 4. Figures and Visuals o A summary table of antibiotic resistance genes and virulence factors should be moved into the main text for ease of reference rather than being confined to supplementary materials. 5. Language and Formatting o The manuscript would benefit from polishing for clarity and consistency. In particular, I noted several typographical errors that should be corrected: a) Page 31, Line 306: “Idetification of biosynthetic gene clusters” → “Identification of biosynthetic gene clusters” b) Page 36, Line 366: “Antibitotic resistance genes and mobile genetic elements” → “Antibiotic resistance genes and mobile genetic elements” c) Page 20, Line 219 / Table 2: “Strenotrophomonas maltophila” → “Stenotrophomonas maltophilia” With improvements in comparative analysis, biosafety framing, data presentation, and language/formatting, the manuscript will present its findings with much greater clarity and impact. ****** what does this mean? ). 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Revision 2
8 Oct 2025 Author Response REVIEWER 1 1.The paper would benefit from further background discussion of the alkB and catE genes, their roles biodegradation, and previous uses in the field. Response to Comment 1: We thank the reviewer for this insightful suggestion. In response, we have expanded the discussion of the alkB and catE genes to include both their established roles in hydrocarbon biodegradation and their previous applications in the field. Specifically, we now highlight that alkB-encoded alkane hydroxylases are widely used as biomarkers for alkane degradation in marine and soil environments, while catE-encoded catechol-2,3-dioxygenases play a central role in aromatic compound degradation and have been applied in the bioremediation of oil-contaminated soils and groundwater (van Beilen & Funhoff, 2007; Harayama et al., 1999; Wang et al., 2011; Jiao et al., 2016). These additions are included in the Results and Discussion section (Lines 251-255). 2.The authors mention comparison of % diesel degradation to other previously tested strains (lines 269-270), but these analyses were not done “side by side” as far as I can see. Were similar conditions used, so as to make this a valid comparison? Ideally, it would have been optimal to include other control/comparison strains in their analysis. Response to Comment 2: We thank the reviewer for this valuable comment. Since different studies were conducted under varying experimental conditions, we have included the respective conditions in Table 3 for proper context. It was not possible to identify studies performed under exactly identical parameters, as a study may have preferential temperatures and conditions based on their region or specific goals. Therefore, the comparisons presented here are intended to provide contextual rather than experimental insights into the relative degradation efficiency of our isolates. This clarification has been added to the revised manuscript (Lines 260–266, Table 3). 3.I did not see any figure legends in the material that was available for review. Please provide figure legends for the reader. Response to comment 3 We thank the reviewer for this suggestion. Figure legends have now been added to accompany all figures to provide clarity for the reader. 4. The authors discuss genes/relevant pathways that are present in the sequenced Acinetobacter isolate. The study would benefit from deletion/inactivation of some of these genes and then observing if this affects the biodegradation activity. 5. The authors discuss the presence of genes/systems for biosynthesis pathways in the sequenced isolate (as opposed to biodegradation). Is there any evidence that these pathways are functional? Could some assays be performed to test this? 6. The authors discuss the fact that virulence genes and antibiotic resistance genes were detected in the sequenced Acinetobacter isolate and that these genes could pose a problem for the use of the strain in biodegradation. The authors should suggest that these genes could possibly be deleted/inactivated in this strain, and then the resulting strain tested to see if this affects the biodegradation activity. In other words, the strain could possibly be engineered to eliminate virulence and antibiotic resistance for future use/applications. Experiments toward this end would improve the paper. Response to comments 4, 5, and 6: We thank the reviewer for these insightful suggestions. The primary scope of the present study was to identify and characterize the isolate and to provide a genomic overview of its biodegradation potential. We agree that functional validation through gene deletion/inactivation studies (comment 4), biochemical assays to test the activity of biosynthetic pathways (comment 5), and engineering approaches to remove virulence and antibiotic resistance determinants (comment 6) would substantially strengthen the understanding and potential application of this strain. We have now acknowledged these points in the Conclusion (lines 503-508) as important directions for future work. At present, we are seeking funding support to pursue these experimental approaches, and we believe that the publication of this study will help us justify and build the foundation for such follow-up research. 7.Was the sequenced isolate tested for relevant antibiotic resistance? This would improve the paper and give insight into whether those genes are expressed/functional. Response to comment 7 We thank the reviewer for this valuable suggestion. We had previously performed phenotypic antibiotic resistance testing, but initially excluded these results to keep the focus on genomic characteristics and avoid excessive length. As suggested, we have now incorporated these data and linked the genomic resistance determinants to the corresponding phenotypic profiles (Lines 198-204, 419-421, 424-426, 434-436, and 442-443). 8.Whenever the authors refer to the sequenced strain DUEMBL6, it would help the reader if they include the genus/species in key locations to help the reader (some examples: titles to Figures 4, 5, and 7; line 357, line 362, etc). I realize you mention this strain in the text, but it helps the reader overall when reading the whole paper to include the genus/species, especially since there are many strain numbers used in the paper (for example, write as “Acinetobacter baumannii DUEMBL6”). Response to comment 8 We thank the reviewer for this valuable suggestion. We have revised the manuscript to include the genus and species name (e.g., Acinetobacter baumannii DUEMBL6) in the specified figure titles (Figures 4, 5, and 7) and key text locations (lines 357, 362, etc.). We believe this change improves clarity and consistency for readers, especially given the presence of multiple strain numbers in the manuscript. REVIEWER 3 1.Comparative Performance of DUEMBL6 o Please consider including a comparative framework, such as a table summarizing the diesel degradation efficiencies of DUEMBL6 alongside those of other globally reported strains or consortia. This will help contextualize your findings. Response to Comment 1: We thank the reviewer for this thoughtful suggestion. In response, we have included a comparative framework (Table 3) summarizing the diesel degradation efficiencies of A. baumannii DUEMBL6 alongside other globally reported strains and consortia. Since these studies were conducted under different environmental and experimental conditions, we have also included their respective parameters (e.g., temperature, incubation period) to ensure proper contextualization. The comparison is intended to provide perspective on the relative degradation efficiency of DUEMBL6 rather than a direct experimental equivalence. These additions can be found in the revised manuscript (Lines 260–266, Table 3). 2.Biosafety and Application Risks The biosafety discussion requires more depth. I recommend elaborating on potential strategies such as the use of non-pathogenic microbial consortia, genetic attenuation of virulence factors, and immobilization or encapsulation techniques to prevent uncontrolled spread. Response to comment 2 We thank the reviewer for this important suggestion. We agree that the biosafety aspects of applying A. baumannii DUEMBL6 in bioremediation require more depth. We have now expanded the Discussion to highlight potential strategies to mitigate these risks, including the use of non-pathogenic microbial consortia, genetic attenuation of virulence and resistance factors, and immobilization or encapsulation techniques to prevent uncontrolled environmental spread (Lines 490–495). We believe this addition strengthens the manuscript by addressing biosafety considerations and providing a framework for future applied research. 3. Functional Validation of Genomic Insights The alkB mutation analysis (I138F, A334T) is interesting, but conclusions regarding functional advantage remain speculative. Please acknowledge this as a limitation and recommend future work, such as enzyme activity assays, transcriptomics, and proteomics, to validate functionality. Response to comment 3 We thank the reviewer for this valuable suggestion. We have acknowledged the speculative nature of the alkB mutation analysis as a limitation and have recommended future validation through enzyme activity assays, transcriptomics, and proteomics in the Conclusion (Lines 501–505). 4. Figures and Visuals A summary table of antibiotic resistance genes and virulence factors should be moved into the main text for ease of reference rather than being confined to supplementary materials. Response to comment 4 We appreciate the reviewer’s suggestion. However, the summary tables of antibiotic resistance genes and virulence factors span more than one page, and per the journal’s formatting requirements, such large tables must remain in the supplementary materials. Therefore, we have kept these tables in the supplementary file. 5. Language and Formatting o The manuscript would benefit from polishing for clarity and consistency. In particular, I noted several typographical errors that should be corrected: a) Page 31, Line 306: “Idetification of biosynthetic gene clusters” → “Identification of biosynthetic gene clusters” b) Page 36, Line 366: “Antibitotic resistance genes and mobile genetic elements” → “Antibiotic resistance genes and mobile genetic elements” c) Page 20, Line 219 / Table 2: “Strenotrophomonas maltophila” → “Stenotrophomonas maltophilia” Response to comment 5 We thank the reviewer for pointing out these errors. The typographical and formatting issues noted (Pages 20, 31, and 36) have been corrected in the revised manuscript. Attachments Attachment Submitted filename: Response_to_Reviwer_auresp_2.docx https://doi.org/10.1371/journal.pone.0339456.r004
18 Nov 2025 Decision Letter - Ranjit Gurav, Editor Dear Dr. Begum, 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. ============================== As per Reviewer 2 comments, the manuscript discusses virulence and antibiotic resistance genes but lacks experimental validation of their functional roles. The authors were encouraged to consider gene deletion or inactivation studies targeting key virulence and antibiotic resistance determinants in future work. Such experiments would offer valuable insights into the genetic basis of resistance and virulence and could demonstrate the feasibility of engineering strains with reduced pathogenicity and resistance. However, it is understandable that these experiments may not be feasible within the current revision timeline. Reviewer 2 also noted that the authors had mentioned performing antibiotic resistance assays in the previous revision. Upon review, it was observed that while the assay has been described in the Methods section and discussed in the Results, the corresponding data (e.g., figures, tables, or supplementary material) are missing. Therefore, it is recommended to revise the manuscript and incorporate the missing antibiotic resistance assay data (figures or tables) to support their discussion in the manuscript. ============================== Please submit your revised manuscript by Dec 28 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'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. 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Revision 3
24 Nov 2025 Author Response As per Reviewer 2 comments, the manuscript discusses virulence and antibiotic resistance genes but lacks experimental validation of their functional roles. The authors were encouraged to consider gene deletion or inactivation studies targeting key virulence and antibiotic resistance determinants in future work. Such experiments would offer valuable insights into the genetic basis of resistance and virulence and could demonstrate the feasibility of engineering strains with reduced pathogenicity and resistance. However, it is understandable that these experiments may not be feasible within the current revision timeline. Response to Comment 1 We thank the reviewer for this constructive comment. We agree that functional validation through gene deletion or inactivation studies would provide deeper insights into the roles of key virulence and antibiotic resistance determinants. While these experiments are beyond the scope of the current revision, we previously added this point to the conclusion as a prospect for future work. We believe this addition not only acknowledges the limitation but also highlights a clear direction for extending the findings of this study. Reviewer 2 also noted that the authors had mentioned performing antibiotic resistance assays in the previous revision. Upon review, it was observed that while the assay has been described in the Methods section and discussed in the Results, the corresponding data (e.g., figures, tables, or supplementary material) are missing. Therefore, it is recommended to revise the manuscript and incorporate the missing antibiotic resistance assay data (figures or tables) to support their discussion in the manuscript. Response to Comment 2 We thank the reviewer for pointing this out. We have now incorporated the missing antibiotic resistance assay data in Table 5 (Line 428) to support the corresponding descriptions in the Methods and Results sections. This table summarizes the phenotypic susceptibility profile. Attachments Attachment Submitted filename: Response_to_Reviwer_auresp_3.docx https://doi.org/10.1371/journal.pone.0339456.r006
9 Dec 2025 Decision Letter - Ranjit Gurav, Editor Genomic Analysis of Acinetobacter baumannii DUEMBL6 Reveals Diesel Bioremediation Potential and Biosafety Concerns PONE-D-25-23823R3 Dear Professor 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. For questions related to billing, please contact billing support . 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, Ranjit Gurav, Ph.D Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0339456.r007
Formally Accepted
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