A new pharmacodynamic approach to study antibiotic combinations against enterococci in vivo: Application to ampicillin plus ceftriaxone

Ivone Jimenez-Toro; Carlos A. Rodriguez; Andres F. Zuluaga; Julian D. Otalvaro; Omar Vesga

doi:10.1371/journal.pone.0243365

Peer Review History

Original SubmissionJuly 1, 2020
21 Sep 2020 Decision Letter - Yung-Fu Chang, Editor PONE-D-20-19379 A new pharmacodynamic approach to study antibiotic combinations against Enterococci in vivo: application to Ampicillin plus Ceftriaxone PLOS ONE Dear Dr. Rodriguez, 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. Your manuscript has been reviewed by two experts in your field. A minor revision is suggested before a decision can be made. Please submit your revised manuscript by two weeks. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: 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 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 Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: interesting pkpd study of combination therapy for Enterococcus. The authors applied proven pkpd concepts and the study is well done. My only comment to the authors is that I agree that it is clear that combination effect is most pronounced in the middle of the exposure curve, and perhaps not surprisingly as they note that at the extremes of exposures, the addition of a second agent is unlikely a priori to make too much of a difference. so with that in mind, it begs a clear question here of where on the ampicillin exposure curve are the mice relative to human exposures, so that one can put this all in clinical context. For example, are the ampicillin exposures, given it is given IV at high doses and frequent administration, already maxing out at T>MIC >50%, and if so then this model does not explain why ceftriaxone may be helpful as when ampicillin exposures are >50% you max out the effect. The only other comment I have is that another factor that is not taken into account in these studies is time. These are 24 h studies, you can only have so much antibacterial effect in 24h of time, and thus you might see potentiating or synergistic activities outside this 24h window. Many studies have shown that when you compare short and longer duration studies, you have a very similar ED50 but significantly change the Emax and slope as you get much deeper kill with longer duration. This is a good example that it does significantly depend where on the exposure curve you are located when translating over the animal model hill curves to clinical medicine (and again why elucidating where on the curve the human exposures are for ceftriazone and ampicillin would be expected is important to place this study into context). Reviewer #2: This study reveals very interesting antibiotic combinations against Enterococci in vivo for medical field. The manuscript is well written and the material and methods are sounds. I suggest the authors to use the latest version of CLSI. ****** 6. 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Revision 1
3 Nov 2020 Author Response Reviewer #1: interesting pkpd study of combination therapy for Enterococcus. The authors applied proven pkpd concepts and the study is well done. My only comment to the authors is that I agree that it is clear that combination effect is most pronounced in the middle of the exposure curve, and perhaps not surprisingly as they note that at the extremes of exposures, the addition of a second agent is unlikely a priori to make too much of a difference. so with that in mind, it begs a clear question here of where on the ampicillin exposure curve are the mice relative to human exposures, so that one can put this all in clinical context. For example, are the ampicillin exposures, given it is given IV at high doses and frequent administration, already maxing out at T>MIC >50%, and if so then this model does not explain why ceftriaxone may be helpful as when ampicillin exposures are >50% you max out the effect. We agree with the reviewer that it is necessary to determine where in the ampicillin exposure curve are the mice relative to humans to be able to put the results in clinical context. For this end, we simulated the human pharmacokinetics of two doses of ampicillin, one for soft-tissue infections (500 mg every 6 hours) and one for endocarditis (2000 mg every 4 hours), using the population model published by Soto et al. (Clinical Pharmacol 2014; 77(3):509-21). This model was developed for ampicillin-sulbactam, but it is useful because sulbactam does alter ampicillin pharmacokinetics. New sections of Human AMP PK/PD simulations were added to Methods (lines 197-205 of the revised version), Results (lines 279-285), and Supporting information (Table S2, Figures S4 and S5). After obtaining the PK profile we estimated the AMP fT>MIC for a range of minimal inhibitory concentrations from 0.5 to 256 mg/L (new S2 Table) to be able to compare murine and human exposures. With the lower dose, we found that for strains with MIC up to 2 mg/L (the majority of isolates: 94% of 13,858 reported in EUCAST international MIC distribution, https://mic.eucast.org) the fT>MIC is at least 61.8%, an exposure leading to maximal efficacy and therefore without benefit from CRO combination. Notwithstanding, for more resistant strains (MIC up to 8 mg/L), this dose is located in middle to left region of the curve, where the addition of CRO would turn an ineffective dose into a bactericidal one, without increasing the amount of ampicillin. With the higher dose, the simulation showed that the fT>MIC was �63% for strains with MICs up to 16 mg/L, suggesting that this dose is located in the right region of the curve, reaching maximal efficacy and not sensitive to CRO potentiation. However, endocarditis is a difficult to treat infection due to factors like poor penetration of some antibiotics into the vegetations and altered metabolic bacterial state, and even a fT>MIC of 100% is not sufficient to achieve cure (see below Carbon C. 1993). Then, another PK/PD index is required, but unfortunately it has not been determined yet. Notwithstanding, the data from the rabbit endocarditis model suggest that for �-lactams, plasma concentration needs to be all the time above 10 times the minimal bactericidal concentration (MBC). Due to the high MBC of ampicillin among Enterococci (>128 mg/L), even the maximal ampicillin dose (12 g/day) will fail to reach those concentrations, indicating that this dose is actually located in the left region of the curve (yet to be determined experimentally), where CRO potentiation would be feasible (a hypothesis worth testing). Two new paragraphs were included in the Discussion (lines 313-334) with two additional supporting references: • Carbon C. Experimental endocarditis: a review of its relevance to human endocarditis. J Antimicrob Chemother. 1993;31 • Pericas JM, Garcia-de-la-Maria C, Brunet M, Armero Y, Garcia-Gonzalez J, Casals G, et al. Early in vitro development of daptomycin non-susceptibility in high-level aminoglycoside-resistant Enterococcus faecalis predicts the efficacy of the combination of high-dose daptomycin plus ampicillin in an in vivo model of experimental endocarditis. J Antimicrob Chemother. 2017;72. The only other comment I have is that another factor that is not taken into account in these studies is time. These are 24 h studies, you can only have so much antibacterial effect in 24h of time, and thus you might see potentiating or synergistic activities outside this 24h window. Many studies have shown that when you compare short and longer duration studies, you have a very similar ED50 but significantly change the Emax and slope as you get much deeper kill with longer duration. This is a good example that it does significantly depend where on the exposure curve you are located when translating over the animal model hill curves to clinical medicine (and again why elucidating where on the curve the human exposures are for ceftriaxone and ampicillin would be expected is important to place this study into context). We agree that a longer treatment may lead to higher bactericidal effect. With the highest doses tested in combination, the mean bacterial count after 24 hours was around 3 log10 CFU/g, indicating that there is room for an additional 1 log10 kill (the model’s limit of detection is 2 log10 CFU/g). However, even with this increased killing, the combination would not reach the 2 log10 CFU/g threshold necessary for synergism and still be considered potentiation. Further studies in other models would be required to expand the understanding of the in vivo exposure-response relationship, specially the impact of treatment duration. Additionally, we chose the standard murine thigh infection model with 24 hours treatment (see Zak and Sande (Ed.). Handbook of animal models of infection, chapter 15, Academic Press 1999), because it has been thoroughly validated and the PK/PD indices derived from it have been shown to accurately predict findings in humans (see Ambrose PG et al. Pharmacokinetics-Pharmacodynamics of antimicrobial therapy: it’s just not for mice anymore. Clinical Infectious Diseases 2007; 44(1):79-86). Reviewer #2: This study reveals very interesting antibiotic combinations against Enterococci in vivo for medical field. The manuscript is well written and the material and methods are sounds. I suggest the authors to use the latest version of CLSI. Thank you for the observation. The latest version of CLSI (2020) was used and the reference was updated: Performance standards for antimicrobial susceptibility testing. 30th edition. CLSI supplement M100.: Wayne, PA: Clinical and Laboratory Standards Institute; 2020. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0243365.r002
20 Nov 2020 Decision Letter - Yung-Fu Chang, Editor A new pharmacodynamic approach to study antibiotic combinations against Enterococci in vivo: application to Ampicillin plus Ceftriaxone PONE-D-20-19379R1 Dear Dr. Rodriguez, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Yung-Fu Chang Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: 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. Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: Yes:** Jiabin Li https://doi.org/10.1371/journal.pone.0243365.r003
Formally Accepted
25 Nov 2020 Acceptance Letter - Yung-Fu Chang, Editor PONE-D-20-19379R1 A new pharmacodynamic approach to study antibiotic combinations against Enterococci in vivo: application to Ampicillin plus Ceftriaxone Dear Dr. Rodriguez: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Yung-Fu Chang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0243365.r004

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