Identification and detection of genetic markers associated with antimicrobial susceptibility and evaluation of efflux pump mechanisms in Mycoplasma iowae

Dominika Buni; Áron Botond Kovács; Enikő Wehmann; Dénes Grózner; Krisztián Bányai; Eszter Zsófia Nagy; Janet Bradbury; Marco Bottinelli; Elisabetta Stefani; Salvatore Catania; Inna Lysnyansky; László Kovács; Miklós Gyuranecz; Zsuzsa Kreizinger

doi:10.1371/journal.pone.0347345

Peer Review History

Original SubmissionNovember 24, 2025
2 Jan 2026 Decision Letter - Chih-Horng Kuo, Editor Dear Dr. Kreizinger, 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 Feb 16 2026 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.. 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.. 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Additional Editor Comments: Based on the expert reviewer’s detailed evaluation and my editorial assessment, I invite you to submit a major revision of this manuscript. The reviewer raised substantive concerns regarding the completeness and interpretation of resistance mechanisms, the lack of statistical support for genotype–phenotype associations, and the strength of conclusions drawn about efflux pump activity. As the handling editor with expertise in Mycoplasma genomics, I agree with these concerns and consider them important to address before further consideration. Due to the timing of the review process, I am proceeding with a decision based on the review comments received to date. Additional expert input may be sought after revision, depending on the extent of the changes. In particular, please ensure that the revised manuscript: - Clarifies which resistance mechanisms were examined and explicitly discusses unexplained resistant phenotypes as limitations; - Provides appropriate statistical support for SNP–MIC associations or clearly frames these analyses as descriptive; - Moderates and qualifies conclusions regarding efflux pump involvement, taking into account the limitations of inhibitor-based assays; - Aligns claims of novelty and diagnostic applicability with the scope and strength of the data. The reviewer’s additional technical and minor comments should also be addressed in your revision. [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: No ****** 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.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: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: This manuscript investigated mechanisms of antimicrobial resistance in Mycoplasma iowae using genomic and phenotypic approaches and developed molecular diagnostic tools for resistance detection. The authors analyzed genome sequences of 99 strains to identify SNPs associated with elevated MICs to fluoroquinolones, tetracyclines and macrolides; developed mismatch amplification mutation assays (MAMAs) for rapid detection of fluoroquinolone resistance markers; and evaluated the role of efflux pumps using efflux inhibitor assay, as well as explored potential regulatory regions of efflux pump genes using in silico analysis. This work provides molecular tools for fluoroquinolone resistance detection in M. iowae and demonstrates the involvement of active efflux pump in antimicrobial susceptibility in M. iowae, which fills an important knowledge gap. Below are my comments/suggestions: Major Concerns: 1. Incomplete results of resistance mechanisms The genetic mechanisms associated with elevated MICs to tetracyclines are not shown, although searching for SNPs in 16S rRNA gene was mentioned in Materials and Methods. It would also be interesting to check other tetracycline resistance mechanisms, such as mobile genetic elements carrying tetracycline resistance genes (e.g. tetM, tetO) and mutations in ribosomal proteins in the 99 isolates. For macrolide resistance, only SNPs in 23S rRNA are presented while no results for ribosomal proteins L22 and L23 (should be L4?). There are 4 isolates with high MICs for macrolides/ lincomycin without alteration in position 2059 in 23S rRNA. This needs to be investigated. 2. Lack of statistical support of genotype–phenotype associations The manuscript lacks statistical analysis (e.g., Fisher’s exact test or logistic regression) comparing MIC distributions between wild-type and mutant genotypes for gyrA, parC, and 23S rRNA. 3. Limited efflux pump functional validation Efflux pump activity validation is based on the MIC reductions by inhibitors, particularly with orthovanadate. Reserpine was excluded from the evaluation due to no effects on growth with high dose. However, experiences from other organisms, including Mycoplasma pneumoniae, even with a very high lethal dose, low level of reserpine still could reduce the MICs of certain drugs like macrolides. It would be a good completion to have this done in M. iowae. Another concern is that there is no transcriptional (e.g., RT-qPCR or RNA-seq) or protein-level validation. Minor Comments: 1. Abstract is long. Consider reducing the length and clarifying that MAMAs were developed only for fluoroquinolone resistance-associated SNPs. 2. Line 245: “and were aligned” should be deleted. 3. Clarify that the MAMA primers are allelic specific. The text contents in line 279-280 do not match the gyrA primers in Table 2: primer gyrA248-L is for genotype L (wild type, cytosine at 248, not adenine) and gyrA248-H is only for mutant strain carrying a thymine at 248 (not cytosine or adenine). There is no allelic specific primer for 248A. 4. According to Table 3, parC genotype L and H in a 1:1 mixed ratio (103:103) would be interpreted as L genotype. This will lead to false negative results for H genotype in application. This limitation should be discussed. 5. In Figure 2, include all six antimicrobials to make a whole view. 6. In Table S2, the 12 efflux pump gene names should be shown with their positions. ****** what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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? 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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0347345.r001
Revision 1
27 Feb 2026 Author Response Zsuzsa Kreizinger HUN-REN Veterinary Medical Research Institute Hungária krt. 21. H-1143 Budapest, Hungary Phone: +36 1 467 4060 Fax: +36 1 467 4076 E-mail: kreizinger.zsuzsa@vmri.hun-ren.hu Chih-Horng Kuo Academic Editor PLOS One 27th February, 2026 Dear Chih-Horng Kuo, We thank You and the Reviewer for Your e-mail on 3rd of January, 2026 and for Your careful evaluation of our manuscript” Identification and detection of genetic markers associated with antimicrobial susceptibility and evaluation of efflux pump mechanisms in Mycoplasma iowae” and for the constructive comments provided. The suggestions have significantly improved the clarity, and overall quality of the manuscript. I. Response to Journal Requirements 1. Compliance with style requirements and file naming We have thoroughly reviewed the manuscript to ensure full compliance with the journal’s style and formatting requirements, including correct file naming conventions. Hopefully, all structural, formatting, and stylistic requirements have now been met. 2. Code sharing requirements We fully agree with the journal’s policy regarding code transparency and reproducibility. In the revised version of the manuscript programs (Geneious, Promotech) were used according to the best practice that their developers recommend for sequence analyses, and online tools (Statistics Kingdom, https://www.socscistatistics.com/) were applied to perform the statistical analyses (logistic regression, Fisher’s exact test, Wilcoxon Signed-Rank test), hence the codes are available at the corresponding references. Mutations were further subjected to manual review and categorization, without the use of any codes. The text has been modified (Lines 140-157, 240-245, 276-281, 284-291, 294-297, 386), and data used for the statistical analyses and the corresponding results are now available in Tables S3-S6. 3. Funding statement clarification Thank you for this important clarification. We have now explicitly included the following statement in our Funding statement and cover letter: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” 4. Funding information placement We acknowledge the requirement that funding information must appear exclusively in the Funding Statement section of the online submission form. Accordingly, all funding-related text has been removed from the Acknowledgments. The amended Funding Statement has been included in the cover letter as requested. We respectfully request that the online submission form be updated accordingly. 5. “Data not shown” statement We acknowledge that the phrase “data not shown” does not meet data sharing standards. All relevant data previously referred to as “data not shown” have now been included in the manuscript (Lines 357-358, 386-389, 508-509). 6. Citation of reviewer-recommended publications We have carefully evaluated all suggested references. Relevant publications have been incorporated into the revised manuscript where appropriate (statistical analysis: citations No. 36-38; effect of reserpine in Mycoplasma pneumoniae: No. 54). In order to clarify nucleotide positions, the numbering has been given not only according to Escherichia coli, but according to the type strain of Mycoplasma iowae too, as recommended in the review of molecular assays for AMR detection by the MyMIC Consortium (citation No. 39; Lines 135-137, 159-160; Tables S1). II. Additional Editor Comments 1. Clarification of resistance mechanisms and discussion of unexplained phenotypes We have clarified in the Materials and Methods and Results sections which resistance mechanisms were examined, including SNP analyses and gene screening. Additionally, we have expanded the Discussion section to explicitly address resistant phenotypes that remain unexplained and have framed these as study limitations. We hope, that the need for further investigation of alternative mechanisms is now clearly acknowledged. 2. Statistical support for SNP–MIC associations We agree that statistical support strengthens genotype–phenotype interpretation, even if the study first evaluated the nucleotide positions based on literature data about their previous association to resistance. The recommended statistical analyses (including Fisher’s exact test and logistic regression) have now been performed to evaluate SNP–MIC associations. The Results and Materials and Methods sections have been updated accordingly. 3. Moderation of conclusions regarding efflux pump involvement The conclusions regarding efflux pump involvement have been carefully moderated. We now clearly state the limitations of inhibitor-based assays and avoid overinterpretation of efflux activity based solely on MIC reduction. The Discussion section has been revised to reflect a more cautious and evidence-based interpretation. 4. Alignment of novelty and diagnostic applicability claims We have carefully revised statements regarding novelty and diagnostic applicability to ensure that they accurately reflect the scope and strength of the data. Claims have been moderated and better aligned with the evidence presented. III. Reviewer’s Concerns Major Concerns 1: Incomplete results of resistance mechanisms We agree that a broader investigation of resistance mechanisms would strengthen the manuscript. However, as - to our knowledge – a first comprehensive investigation in this specific field, our intentions were to assess the presence of known mutations for target modification and develop diagnostic assays for their differentiation. However, Mycoplasma iowae proved to have very unique characteristics among the avian pathogen mycoplasmas, and in the absence of official standards in MIC testing in mycoplasmas, and the detected difference in MIC values in the different media, the description of new associations was beyond the scope of our aims. Thus, we completed our study with the inhibitor-based assay to assess the possibility of involvement of an efflux pump activity in the development of resistance. Thank You for pointing out the search for tetracycline resistance-associated mutations without reported results. Since the examined strains showed only slightly elevated MIC values - equal or below 4 µg/ml in Oxoid media (the MIC values which were used for the association study) - they were all considered susceptible to oxytetracycline, therefore genetic markers were not searched in the corresponding regions, and accordingly the sentence has been deleted from the Materials and Methods (Lines 128-129, 139-140). For macrolide resistance, results of the analysis of ribosomal proteins (L4 and L22, corrected as appropriate) have now been included (Lines 298-300). We agree that the findings of four isolates with elevated MICs lacking 23S rRNA position 2059 alterations warrant further investigations, which we now highlight in the discussion (Lines 454-458). As two of these strains were involved in the inhibitor-based assays in case of tilmicosin, we also discuss their phenotype changes caused by orthovanadate in Lines 522-526. 2: Lack of statistical support for genotype–phenotype associations Following the Reviewer’s recommendations, statistical analyses (Fisher’s exact test and logistic regression) comparing MIC distributions between wild-type and mutant genotypes for gyrA, parC, and 23S rRNA have now been conducted. Statistical methods and results are described in the Materials and Methods and Results sections (Lines 140-157, 276-281, 284-291, 294-297), and data used for the statistical analyses and the corresponding results in details are now available in Tables S3-S6. 3: Limited efflux pump functional validation We appreciate the valuable suggestions regarding the functional validation of efflux pump activity. Although we are not in the position to perform further experiments, such as the continuation of the studies with reserpine, and transcriptional or protein-level validation was beyond the scope of the current study, we now clearly acknowledge these limitations in the Discussion, and indicate them as important directions for future research. We have also explicitly discussed the limitations of inhibitor-based validation. (Lines 512-518, 528-530) Minor Comments Ad 1. The Abstract has been shortened and streamlined (Lines 35-55). It now clearly states that MAMAs were developed specifically for fluoroquinolone resistance-associated SNPs (Lines 42-44, 53-54). Ad 2. The phrase “and were aligned” has been deleted as suggested (Line 262). Ad 3. In accordance with the Reviewer’s correction, the discrepancy between the text and Table 2 has been arranged. Primer specificities (including clarification that no allele-specific primer was designed for 248A) are now accurately described and results for the non-specific alleles are presented (Lines 320-329). Ad 4. We agree that the interpretation of mixed genotypes is an important practical limitation. The potential for false-negative detection of parC H genotype in mixed populations has now been explicitly discussed in the Discussion section (Lines 488-495). Ad 5. Figure 2 has been revised to include all six antimicrobials to provide a comprehensive overview. The figure now shows the log2 transformed MIC values of the antibiotics in Oxoid media, MolliScience, and MolliScience supplemented with sodium orthovanadate. The log2 transformed MIC values of the two-fold dilution series of antimicrobial concentrations are shown to normalize data distribution. Ad 6. Table S2 has been updated to include the genomic positions of the efflux pump genes according to the type strain of Mycoplasma iowae, and the text has been completed regarding how regions were analyzed and positions were identified (Lines 263-265). In addition, in order to clarify nucleotide positions in the whole manuscript, the numbering has been given not only according to Escherichia coli, but according to the type strain of Mycoplasma iowae too, as recommended in the review of molecular assays for AMR detection by the MyMIC Consortium (citation No. 39; Lines 135-137, 159-160, Tables S1). We sincerely thank the Editor and Reviewer for their constructive and thoughtful evaluation. We believe that the revisions have significantly strengthened the scientific accuracy, clarity, and transparency of the manuscript. Sincerely, Zsuzsa Kreizinger On behalf of all co-authors Attachments Attachment Submitted filename: Response_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0347345.r002
16 Mar 2026 Decision Letter - Chih-Horng Kuo, Editor Dear Dr. Kreizinger, 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 2026 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.. 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.. 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.. 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 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 . 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 . 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 . 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, Chih-Horng Kuo, Ph.D. Academic Editor PLOS One Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. 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.] Reviewer's Responses to Questions Comments to the Author Reviewer #1: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 4. 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.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: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: The revised version has been significantly improved. There are only a few minor comments to be addressed: 1. Figure 2. Showing the MIC value bars in the negative direction for those less than 1 is somewhat strange. Maybe try to use a broken/split axis bar chart instead of using the log2 transformation. 2. One strain with A2059G mutation in 23S rRNA is susceptible to erythromycin. If this is not due to variant calling error, it would be interesting to provide an explanation. 3. The MAMA primer gyrA248-H specificity has been clarified to be only for C248T. The C248A information in M. iowae reference should be excluded also (lines 322, 468, 480, and table 3). ****** what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..--> Reviewer #1: 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". 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Revision 2
30 Mar 2026 Author Response Zsuzsa Kreizinger HUN-REN Veterinary Medical Research Institute Hungária krt. 21. H-1143 Budapest, Hungary Phone: +36 1 467 4060 Fax: +36 1 467 4076 E-mail: kreizinger.zsuzsa@vmri.hun-ren.hu Chih-Horng Kuo Academic Editor PLOS One 30th March, 2026 Dear Chih-Horng Kuo, We thank You and the Reviewer for Your e-mail on 17th of March, 2026 and for Your careful evaluation of our manuscript” Identification and detection of genetic markers associated with antimicrobial susceptibility and evaluation of efflux pump mechanisms in Mycoplasma iowae” and for the constructive comments provided. I. Response to Journal Requirements Citations We have carefully evaluated all suggested references during the previous revision, no new recommendations were added. Relevant publications have been incorporated into the revised manuscript where appropriate (statistical analysis: citations No. 36-38; effect of reserpine in Mycoplasma pneumoniae: No. 54). In order to clarify nucleotide positions, the numbering has been given not only according to Escherichia coli, but according to the type strain of Mycoplasma iowae too, as recommended in the review of molecular assays for AMR detection by the MyMIC Consortium (citation No. 39). All statistical analyses were repeated or performed according to the first revision of the manuscript using the cited tools, therefore the previously cited software (R 4.3.3) has been eliminated from the citation list. A full audit with publisher-level verification was conducted using ChatGPT to assess the reference list for retractions. This included checking direct sources (PubMed and official publisher pages) for retraction status, reviewing PubMed retraction flags, examining publisher platforms (Elsevier, Taylor & Francis, Springer, Frontiers, PLoS), and performing Retraction Watch–type searches using title and author combinations. No retracted publications or linked retraction notices were identified in our reference list. II. Reviewer’s Comments Ad 1. Figure 2 has been adjusted and now split axis is used instead of log2MIC bars to better visualize MIC values, the description of the figure has been corrected accordingly (Fig2, Lines 392, 396-397). Ad 2. In accordance with the Reviewer’s comment, we completed our discussion about unexplained resistant phenotypes as limitations with the unexplained susceptible phenotype in case of macrolides and lincomycin (Lines 455-457). Ad 3. According to the clarified MAMA specificity, the text has been corrected when the MAMA-targeted mutation was mentioned (Lines 322, Table 3). The discussion details examinations on the sequences and MIC values, not on the MAMA results, therefore in these cases, the “C248T/A” form was considered appropriate (Lines 469, 481). Nevertheless, the specificity of the gyrA-MAMA has been addressed in the discussion also (Lines 484-485). We thank the Editor and Reviewer for their evaluation. We consider that the revisions have improved the scientific accuracy, clarity, and transparency of the manuscript. Sincerely, Zsuzsa Kreizinger On behalf of all co-authors Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_2.docx https://doi.org/10.1371/journal.pone.0347345.r004
31 Mar 2026 Decision Letter - Chih-Horng Kuo, Editor Identification and detection of genetic markers associated with antimicrobial susceptibility and evaluation of efflux pump mechanisms in Mycoplasma iowae PONE-D-25-63087R2 Dear Dr. Kreizinger, 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 and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact 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, Chih-Horng Kuo, Ph.D. Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0347345.r005
Formally Accepted
Acceptance Letter - Chih-Horng Kuo, Editor PONE-D-25-63087R2 PLOS One Dear Dr. Kreizinger, 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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. Chih-Horng Kuo Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0347345.r006

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