It’s not all about the MIC: Daptomycin synergy with β-lactams varies between enterococcal species where cardiolipin synthase mutations confer daptomycin resistance
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Introduction: Administering daptomycin with b-lactams may prevent resistance or achieve synergy in enterococcal infections. Daptomycin-non-susceptibility (DAP-NS) phenotype is commonly associated with mutations in the liaFSR and YycFGHIJ systems. Cardiolipin synthase (cls) mutations have been found in conjunction with liaFSR mutations, associated with higher MICs, suggesting they are stepwise mutations. In vitro, ampicillin restores daptomycin bactericidal activity in Enterococcus faecium isolates with liaFSR mutations, but not when they occur in the cls gene; cls-only Enterococcus faecalis mutants have not yet been reported. Aim: The aim was to explore daptomycin synergy with b-lactams for high-MIC DAP-NS E. faecalis and E. faeciumcls mutants. Methodology: Experimentally-induced DAP-NS E. faecalis (A7) and DAP-NS E. faecium (C_002) from a persistent infection on daptomycin-ampicillin therapy had whole-genome sequencing (AB SOLiD 5500, CLC Genomics Workbench 9). Time-kill studies were performed with DAP, ampicillin, and DAP-AMP for both isolates; and ceftriaxone and DAP-ceftriaxone for E. faecalis. Results: Both isolates’ only DAP-NS-associated mutation was in cls. DAP-ceftriaxone demonstrated bactericidal activity and synergy for E. faecalis A7, whereas E. faecium C_002 grew in the presence of DAP and DAP-AMP. Conclusion: High-level DAP-NS arose in association with a cls mutation alone in E. faecalis A7, and in the absence of other documented DAP-NS-associated genetic mutations in E. faecium C_002. DAP-ceftriaxone combination therapy may be a useful salvage option in DAP-NS E. faecalis infections carrying the cls mutation identified in this study. There was no in vitro DAP-ampicillin synergy for cls-associated DAP-NS E. faecium C_002.
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Thank you for taking the time to respond to the reviewers' comments. Unfortunately, the reviewers' comments have not been addressed sufficiently after revision, and with only a single biological replicate the work performed does not meet the criteria required for a scientific publication. Some of the findings are potentially interesting, but the study has not been carried out in a robust manner and therefore the data cannot support any conclusions. I hope that this study can be completed at some point in the future.
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Comments to Author
The author presented an interesting topic in this work. In general, the article is well written despite the few comments that are listed below. 1. Regarding the abstract, in the methodology section, the author mentioned that time-kill studies were performed for E. faecalis. E. faecium was missing and should be written. (lines 33-35). 2. As regards the introduction, it is very informative and to the point. In line 60, the author should write the reference number of Diaz et al. 3. The design and methodology section was clearly defined and appropriate. In line 83, the author stated that E. faecalis O7 was used to obtain the mutated strains. But in lines 91-92, the author wrote E. faecalis O. The author needs to revise if they are the same isolate or if it is a transcriptional error. In line 85, …
Comments to Author
The author presented an interesting topic in this work. In general, the article is well written despite the few comments that are listed below. 1. Regarding the abstract, in the methodology section, the author mentioned that time-kill studies were performed for E. faecalis. E. faecium was missing and should be written. (lines 33-35). 2. As regards the introduction, it is very informative and to the point. In line 60, the author should write the reference number of Diaz et al. 3. The design and methodology section was clearly defined and appropriate. In line 83, the author stated that E. faecalis O7 was used to obtain the mutated strains. But in lines 91-92, the author wrote E. faecalis O. The author needs to revise if they are the same isolate or if it is a transcriptional error. In line 85, there is a spelling mistake, '35C' was written instead of '35 °C'. In line 89, the author wrote, "Daptomycin MIC rose from 2 mg/L to 48 mg/L (E. faecalis A7) and 32 mg/L (B7)". And in lines 94-95, the author stated that "A vanB vancomycin-resistant E. faecium (C_002) isolated from peritoneal fluid during prolonged daptomycin-ampicillin combination therapy had daptomycin MIC of 32 mg/L.". However, in the time-kill studies, lines 121-123, the author wrote, "DAP MIC was determined by Etest on Mueller-Hinton agar from growth at log phase in BHI broth; this was 32 mg/L for E. faecalis A7, and 12 mg/L for E. faecium C_002.". The author needs to clarify this discrepancy regarding the DAP MIC for E. faecalis A7 and E. faecium (C_002). 4. As for the results section, I found it very simple and obvious. However, it is recommended to supply figures with a better resolution. It was noted that, in the methodology section, in lines 118-119, the author mentioned that time-kill studies were done for E. faecalis O, but in the result section, I could not find the result for E. faecalis O. Please the write the results of time-kill studies for E. faecalis O. 5. Regarding the discussion and conclusion sections, the references used are somehow old, as the most recent included is from 2014. I recommend the author compare this work to at least one or two recent researches. 6. The references must be thoroughly revised according to the journal style. For example, the abbreviated names of the journal should be written, the number of pages should be written in the form of (1738-1743) instead of (1738-43), and the references miss the doi. Reference number 13 should be written as "National Committee for Clinical Laboratory Standards" or "NCCLS", as mentioned in the manuscript, and not "Standards NCfCL".
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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I have now secured two peer review assessments of your manuscript which give differing reviews of the work presented. Taken with my own assessment I regret to inform that there are several significant concerns about the experimental design and scientific rigor of the work, and the structuring and presentation of the results, which mean that the data presented are not sufficient to support the conclusions. The manuscript requires a re-write to improve structure and legibility, but also requires further experimental work to bring it up to the standard required for publication. Specifically, the number of experimental repeats is not sufficient, and the evidence to support the role of cls mutations in DNS is also not sufficient. Please can you address the reviewers comments below, and revise your manuscript accordingly.
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Comments to Author
The manuscript by McKew aims to identify the effect of cls mutations on synergy between daptomycin and beta-lactams. Characterisation of two daptomycin non-susceptible enterococcal strains with different mutations in cls demonstrates that synergy between daptomycin and beta-lactams in these strains depends on the strain and the beta-lactam. However, whilst the topic is interesting, the data supporting this are fairly weak, with insufficient replicates and a lack of statistical analysis. Additionally, it is limited by the very small number of strains and beta-lactams studied. 1.Data in Fig 1 and 2 were done in duplicate but it's not clear if this is one replicate in duplicate, or two independent experiments. Further, there is no statistical analysis or error bars. Despite this, line 147 states "no …
Comments to Author
The manuscript by McKew aims to identify the effect of cls mutations on synergy between daptomycin and beta-lactams. Characterisation of two daptomycin non-susceptible enterococcal strains with different mutations in cls demonstrates that synergy between daptomycin and beta-lactams in these strains depends on the strain and the beta-lactam. However, whilst the topic is interesting, the data supporting this are fairly weak, with insufficient replicates and a lack of statistical analysis. Additionally, it is limited by the very small number of strains and beta-lactams studied. 1.Data in Fig 1 and 2 were done in duplicate but it's not clear if this is one replicate in duplicate, or two independent experiments. Further, there is no statistical analysis or error bars. Despite this, line 147 states "no significant change" and lines 151-152 claim "no significant difference". Data should consist of at least three independent biological replicates and should be analysed by an appropriate statistical test e.g. two-way ANOVA. 2. Different inocula are used in Fig 1 and Fig 2. Line 119 states the inoculum of these assays is 5x10^5 but in Fig 1 it looks more like 10^7? Due to the inoculum effect, the inocula should be the same to enable comparisons to be made between the strains. Lines 161-164 try to compare the survival of the two strains to daptomycin but this is not a valid comparison with such different inocula. 3. I have several concerns with the MIC data provided: There are no details on how many replicates were performed to generate the MIC data. Different daptomycin MICs for the same strain are stated throughout the manuscript e.g. strain A7 has an MIC of 48 mg/L (line 89) and 32 mg/L (line 122), E. faecium has an MIC of 12 mg/L (line 122) and 32 mg/L (line 95). Different methods for determining MICs are used throughout and details on these methods are lacking. Daptomycin MICs were determined by E-test - details on inoculum are needed and details of whether the MHA was supplemented with calcium should be provided e.g. on line 123 it is stated that '0.01 ml aliquot of log-phase bacteria…'. But it's not clear how many bacteria that corresponds to. Ampicillin MICs were determined by Vitek2 - more information is needed on how this was performed and justification of why this method was chosen as it is not the gold-standard. Exact MIC values should be determined for each strain as it can't be said that "Ampicillin MICs for E. faecalis A7 and B7 remained the same as for E. faecalis O, at ≤ 2 mg/L" (line 92) if the exact MIC wasn't determined. 4. The manuscript should be edited for clarity regarding the strains used e.g. lines 81/83 - not clear whether there is a difference between strain O and O7? Information about all the strains used could be presented as a strain table (which could contain the MIC data). 5. Similarly, it is unclear what reference genome was used to compare the sequences of A7/B7 to. It's important that the mutations in A7/B7 are compared to the parental strain O/O7. It's not clear whether this is the case as line 135 states "Compared to E. faecalis O, the DAP-NS E. faecalis A7 and B7 isolates had mutations" but this conflicts with lines 109-110 which says they were compared to "E. faecalis D32 (GenBank accession CP003726)". 6. Line 139. Is it reasonable to compare E. faecium C_002 to an unrelated strain? Can it be said to have mutations, rather than differences? 7. Lines 161-163. What evidence is there to link cls mutations to the DNS phenotype, especially since loss of cls does not decrease susceptibility to daptomycin (Woodall et al., 2021 Front Micro). The description of the genomic data implies loss of function of cardiolipin synthase (unclear which one), but there is no evidence that cardiolipin levels are affected in these strains. Therefore, text should be clarified to make this point. 8. 171-172. I don't think it reasonable to claim a single mutation in E. faecium C_002 cls leads to the DNS phenotype, since only a few genes were examined and the comparator strain is not directly related. To demonstrate this it would be necessary to make the mutation de novo in a daptomycin susceptible strain or repair the mutation in E. faecium C_002 and show loss of DNS phenotype. 9. Lines 212-213. How is it known that 'daptomycin resistance mediated by a cls mutation in E. faecium C_002 arose in vivo'? Since there is no directly related daptomycin susceptible pre-cursor isolate, I'm not clear how this can be shown. 10. Line 120 states "Isolate E. faecalis B7 was isogenic with A7 so was not used for time-kill studies." but these isolates have different daptomycin MICs. Reasons for this difference should be discussed and time-kills performed on both strains. 11. I think it would be beneficial to also perform the time-kill assay on the parental strain O/O7 to enable the impact of the mutated cls on synergy to be assessed 12. The materials and methods section contains considerable results data. This section should be revised to focus on methods only. The results section should include a section of the generation of non-susceptible strains and determination of the MICs of all strains. 13. The methods section should be re-written to contain one method per subheading. For example, the time-kill section currently contains details about MIC determination which should be under its own subheading. 14. The manuscript should contain justification of why these beta-lactams were chosen. Why was this concentration of ampicillin used? Information on the ceftriaxone MICs of the strains should be provided. How does the concentration of ceftriaxone used for the time-kill assays relate to the MIC? 15. Line 130 - needs a better definition of synergy. Is the decrease compared to each antibiotic separately? 16. Why use DAP at 5X MIC? Are these clinically-achievable concentrations? Same for the concentration of ampicillin - why this concentration and is it clinically relevant? Good clinical justification is provided for ceftriaxone but what multiple of the MIC is this and why? Additional points: It would be helpful if each statement in the introduction had a supporting reference. As it stands the first few sentences have no supporting citations. Line 52. Please swap 'develop' for 'acquire'. Line 57. Delete 'genetic'. Also, the text should say …genes encoding regulatory systems… Line 58. 'liaFSR' should be italicised and 'YycFGHIJ' be lower case and italicised. Line 60. E. faecalis has two cls genes [Nguyen et al., 2020 Open Forum Infect Dis; Woodall et al., 2021 Front Micro]. Are mutations seen in cls1 or cls2 or both? Lines 106-107: which cls gene was examined? Why restrict WGS analyses to only those genes previously described? Lines 221-227 need supporting citations. Line 189. 'degrading' would be better than 'decomposing'. Figures 1 and 2 are very low resolution. I know that journal pdf conversion processes often produce poor quality figures, but these are almost impossible to read.
Please rate the manuscript for methodological rigour
Very poor
Please rate the quality of the presentation and structure of the manuscript
Very poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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