In Vitro synergy of Farnesyltransferase inhibitors in combination with colistin against ESKAPE bacteria

Marian Klose; Lea Weber; Hagen Sjard Bachmann

doi:10.1371/journal.pone.0331440

Peer Review History

Original SubmissionApril 2, 2025
16 Jun 2025 Decision Letter - Shengwei Sun, Editor In Vitro Synergy of Farnesyltransferase Inhibitors in Combination with Colistin against ESKAPE Bacteria PLOS ONE Dear Dr. Bachmann, 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 Jul 31 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). 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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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: The manuscript investigates the effects of FTIs and additional compounds on key pathogenic bacteria when co-administered with colistin. The potential synergistic interactions were illustrated by a modified checkerboard assay. The approach enables FTIs to exhibit antimicrobial activity against several gram-negative bacteria when co-administered with sub-inhibitory concentrations of colistin. The authors found FTIs were able to reduce colistin MICs, even in a colistin-resistant gram-negative strain, thus offering a novel strategy to combat antimicrobial resistance. However, several key issues are not clarified clearly, some modification should be made according to the following questions: Comments: 1. Line 216, why does S. aureus MRSA have antibiotic resistance to the quantities of ampicillin, chloramphenicol, kanamycin and tetracycline, while S. aureus doesn’t? 2. Line 223, the author should provide the specific value of the sub-inhibitory concentration of colistin. 3. In the “Growth kinetics” section, the author only conducted the growth kinetics for S. aureus. Why did the authors not monitor the dynamic changes in the combined inhibitory effects of FTIs and colistin on E. cloacae growth? Time-kill assays should be included to evaluate the antibacterial effect. 4. Line 382, the author should discuss the lack of activity of the peptidomimetic FTIs, B581 and FTI-277 against gram-positive bacteria. Why the antimicrobial effects of peptidomimetic FTIs lower than non-peptidomimetic FTIs? 5. Line 438-440 and figure 8, the author only speculated the mechanism of the synergistic effects on gram-negative bacteria with the combined use of colistin and FTIs by disrupting the OM’s integrity and increasing permeability. The conclusion is not supported by any data. To further strengthen the conclusions, the authors may consider performing outer membrane permeability assay to confirm. 6. Line 476-479, “…colistin-resistant strains, including those harboring mobile colistin resistance genes “, “this approach could target bacteria with a strong propensity for heteroresistance to colistin and other polymyxins”, please give examples to illustrate which bacteria are included respectively. 7. Line 481-483, could you give more examples/details about how the FTIs affect the heteroresistance in gram-negative pathogens bacteria with or without colistin? ****** what does this mean? ). If published, this will include your full peer review and any attached files. 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Revision 1
30 Jul 2025 Author Response Dear Editor, dear Reviewer, We would like to sincerely thank you for your thorough evaluation of our manuscript and the opportunity to contribute to PLOS ONE. We greatly appreciate the constructive feedback and the chance to further improve our work in response to your valuable comments. We have carefully reviewed all submitted files and, to the best of our knowledge, ensured that they now fully comply with PLOS ONE's style and file naming requirements. Concerning the use of the image in the S1_File, we contacted the Tecan Deutschland GmbH, which provides both the device and the associated software shown in the screenshot. We received written confirmation that the reported screenshot does not constitute a copyright issue and can be used within the scope of this publication without permission. The relevant correspondence is provided as a supporting document. We now respond point-by-point to the reviewer’s comments below. 1. Line 216, why does S. aureus MRSA have antibiotic resistance to the quantities of ampicillin, chloramphenicol, kanamycin and tetracycline, while S. aureus doesn’t? We agree that this aspect needed clarification. The MRSA strain used in our study, Staphylococcus aureus ATCC 33592, is mecA-positive (PMID: 19752281), whereas S. aureus ATCC 25923 is mecA-negative and widely used as a quality control strain (PMID: 25377701). The methicillin-sensitive strain is markedly more susceptible to the antibiotics tested compared to the methicillin-resistant one. It is well established that methicillin resistance in S. aureus is frequently associated with cross-resistances, and thus often coincides with multidrug resistance (PMID: 32257966). To demonstrate that established antibiotics can still be effective against this MRSA strain, we additionally included gentamicin and vancomycin in our control experiments, as shown in Fig 3. 2. Line 223, the author should provide the specific value of the sub-inhibitory concentration of colistin. The exact concentrations of colistin used in the combination experiments are already detailed in the Materials and Methods section under the description of the checkerboard assay, and are also indicated in the respective figures. Nevertheless, in accordance with the reviewer’s recommendation, we have now included the concentration range directly in the Results section for clarity (Line 248-250). 3. In the “Growth kinetics” section, the author only conducted the growth kinetics for S. aureus. Why did the authors not monitor the dynamic changes in the combined inhibitory effects of FTIs and colistin on E. cloacae growth? Time-kill assays should be included to evaluate the antibacterial effect. This is a valid point, and we have now extended our analysis by conducting additional growth kinetics for E. cloacae, specifically focusing on the combined application of colistin and FTIs to illustrate potential synergistic effects. Furthermore, we have implemented time-kill assays for both S. aureus and E. cloacae to more thoroughly assess the bactericidal activity and the dynamic interplay between colistin and FTIs over time. These new experiments have been incorporated into the revised manuscript (Line 33, 185-192, 210-213, 340, and 351-390). 4. Line 382, the author should discuss the lack of activity of the peptidomimetic FTIs, B581 and FTI-277 against gram-positive bacteria. Why the antimicrobial effects of peptidomimetic FTIs lower than non-peptidomimetic FTIs? The differences in antimicrobial activity between the tested FTIs likely result from distinct chemical properties. Lonafarnib and tipifarnib, which affect gram-positive and gram-negative bacteria, both carry halogen substituents (bromine and chlorine, respectively). Halogens are known to be important for the antibiotic activity of different classes of antibiotics, like chloramphenicol, tetracyclines, and fluoroquinolones. Faleye et al. (2023; PMID: 37845080) emphasized that halogenated compounds often show increased affinity for gram-positive bacteria. Similarly, Sanabria-Rios et al. (2022; PMID: 36118101) demonstrated brominated fatty acids to be effective against MRSA strains. These findings may explain the higher activity of lonafarnib and tipifarnib. In contrast, the peptidomimetic FTIs B581 and FTI-277 contain polar thiol groups, which may impair membrane permeability. We have added an explanation of the chemical structures and properties of the molecules to the manuscript (Line 471-475). Further investigations into membrane integrity and compound uptake will help clarify the role of these functional groups in antimicrobial activity. 5. Line 438-440 and figure 8, the author only speculated the mechanism of the synergistic effects on gram-negative bacteria with the combined use of colistin and FTIs by disrupting the OM’s integrity and increasing permeability. The conclusion is not supported by any data. To further strengthen the conclusions, the authors may consider performing outer membrane permeability assay to confirm. To address this concern, we conducted outer membrane permeability assays using the fluorescent probe N-Phenyl-1-naphthylamine (NPN) for E. coli as a model strain and E. cloacae, a colistin-resistant isolate, as representative gram-negative bacteria. The results confirmed that sub-inhibitory concentrations of colistin were sufficient to increase outer membrane permeability without affecting viability. These new data have been included in the revised manuscript (Line 194-204, 392-415). 6. Line 476-479, “…colistin-resistant strains, including those harboring mobile colistin resistance genes “, “this approach could target bacteria with a strong propensity for heteroresistance to colistin and other polymyxins”, please give examples to illustrate which bacteria are included respectively. As we were able to demonstrate a significant antimicrobial effect of FTIs against gram-negative bacteria when co-administered with sub-inhibitory concentrations of colistin, the required colistin concentration was notably reduced. This finding suggests that even colistin-resistant bacteria might be susceptible to such combination treatments. Notably, one of the tested strains, E. cloacae ATCC 13047, is known to be highly resistant to colistin (PMID: 20207761). Members of the Enterobacter cloacae complex (ECC) are not only important nosocomial pathogens but are also well documented for their high potential to exhibit colistin heteroresistance (PMID: 36627272). Furthermore, plasmid-mediated colistin resistance genes (mcr) have been identified across several gram-negative ESKAPE organisms, including E. coli (PMID: 30305321), K. pneumoniae (PMID: 34946038), and A. baumannii (PMID: 30936095). While we did not include mcr-positive isolates in our study, future work will investigate whether FTIs may help restore susceptibility in strains carrying mcr-mediated colistin resistance. Notably, previous studies have shown that mcr-mediated colistin resistance can be overcome by using rationally designed combination therapies (PMID: 29386620). These findings underscore the potential of similar approaches – such as combining FTIs with sub-inhibitory colistin – to restore susceptibility in resistant gram-negative pathogens. 7. Line 481-483, could you give more examples/details about how the FTIs affect the heteroresistance in gram-negative pathogens bacteria with or without colistin? This point raises an important aspect of our findings. When used alone, FTIs showed no impact on most gram-negative bacteria, clearly indicating resistance rather than heteroresistance. In our study, we mentioned the possibility that FTIs in combination with colistin potentially influence the formation of heteroresistant subcolonies. For instance, lonafarnib, when combined with sub-inhibitory colistin, did not consistently suppress bacterial growth across the triplicate near the MIC threshold in the K. pneumoniae checkerboard assay, which supports the hypothesis of heteroresistant escape events. However, this remains a hypothesis. We have therefore deleted the word 'heteroresistant' from the Results section and now describe the formation of subcolonies only. However, we assume that the observed inconsistencies in growth inhibition patterns, particularly near the MIC threshold, are characteristic of heteroresistance, where subpopulations can transiently survive otherwise inhibitory concentrations (PMID: 25567227). We therefore proceed to discuss this hypothesis in greater detail in the Discussion (Line 574-576). Attachments Attachment Submitted filename: Response_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0331440.r002
18 Aug 2025 Decision Letter - Shengwei Sun, Editor In Vitro Synergy of Farnesyltransferase Inhibitors in Combination with Colistin against ESKAPE Bacteria PONE-D-25-17781R1 Dear Dr. Bachmann, 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, Shengwei Sun, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0331440.r003
Formally Accepted
Acceptance Letter - Shengwei Sun, Editor PONE-D-25-17781R1 PLOS ONE Dear Dr. Bachmann, 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. Shengwei Sun Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0331440.r004

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