Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Could the authors more clearly define the primary objective of the study and specify whether the aim was to identify completely inert vehicle concentrations or merely concentrations that do not significantly reduce B. burgdorferi growth and metabolic activity?
2. What was the rationale for selecting DMSO, ethanol, and particularly sodium hydroxide as the three vehicles evaluated in this study, and how representative are these vehicles of those commonly used in B. burgdorferi antimicrobial screening assays?
3. Could the authors provide a stronger justification for the selected concentration range of 0.15–2.4% for all three vehicles, considering their substantially different physicochemical properties?
4. Why was only one B. burgdorferi strain, B31 clone 5A1, included, and how confident are the authors that the observed vehicle tolerances can be generalized to other laboratory strains or clinical isolates?
5. Could the authors clarify whether the three biological replicates represent completely independent cultures prepared on separate occasions or parallel cultures originating from the same inoculum?
6. Were technical replicates averaged before statistical analysis so that the biological replicate, rather than the individual technical measurement, represented the experimental unit, thereby avoiding potential pseudoreplication?
7. Given the relatively small number of biological replicates, did the authors perform or consider a statistical power analysis to determine whether the study was sufficiently powered to detect biologically relevant vehicle effects?
8. Could the authors justify the use of ordinary one-way ANOVA for these data rather than a model that explicitly accounts for vehicle type, concentration, and their interaction?
9. What is the justification for replacing zero values with 1 before logarithmic transformation, and could this approach introduce bias, particularly in groups showing complete loss of detectable cells or luminescence?
10. Could the authors provide the complete underlying biological-replicate-level data, rather than only normalized values and technical replicate measurements, to facilitate assessment of reproducibility?
11. Why were cultures evaluated only at a single time point, six days after inoculation, and could earlier measurements have provided important information on transient metabolic stress or growth inhibition?
12. Could the authors clarify whether the cultures were confirmed to be in comparable growth phases at the time of measurement, particularly given that different vehicles may have altered growth kinetics?
13. Since ATP-dependent luminescence is interpreted as a proxy for viability and metabolically active biomass, could the authors discuss more explicitly the limitations of equating intracellular ATP levels with bacterial viability?
14. How do the authors explain the marked increase in ATP-dependent luminescence observed with 0.6% and 1.2% ethanol despite the simultaneous reduction in cell counts, and could alternative assay-related mechanisms have contributed to this phenomenon?
15. Did the authors test whether ethanol itself, or ethanol-induced changes in membrane permeability or cell lysis efficiency, directly affected the BacTiter-Glo reaction independently of intracellular ATP concentration?
16. Could the observed ethanol-associated spirochaete aggregation have interfered with the accuracy of dark-field microscopy cell counting, and was any method used to disperse aggregates before counting?
17. Were morphological changes, including circular or pleomorphic forms, quantified systematically, or were these observations qualitative only?
18. Given that the measured pH values after NaOH addition ranged from 10.12 to 13.35, can the authors distinguish between a specific effect of NaOH as a vehicle and the more general biological effect of exposure to extreme alkaline conditions?
19. Would it be more informative to report the final NaOH concentration in molarity in addition to percentage volume/volume, particularly because a 1 M NaOH stock solution was used?
20. Did the authors measure the pH of the cultures after the six-day incubation period, and if not, how can they determine whether the initial alkaline conditions were maintained throughout the experiment?
21. Since ethanol is volatile, could the authors provide experimental evidence that ethanol concentrations remained reasonably stable during the six-day incubation rather than relying solely on the assumption that sealed tubes limited evaporation?
22. The manuscript concludes that DMSO was well tolerated up to 2.4%, but given that the study assessed primarily gross growth and ATP-dependent metabolic activity, would it be more appropriate to state that no detectable effect was observed using these specific endpoints rather than describing DMSO as biologically inert?
23. Could the authors more clearly define a recommended maximum working concentration of DMSO for future B. burgdorferi antimicrobial screening assays based on the present results?
24. Does the extensive discussion of PTLDS, persister forms, biofilms, rodent-targeted antimicrobial strategies, and Lyme disease treatment extend beyond the experimental scope of this vehicle-tolerance study, and could this section be shortened to maintain a stronger focus on the actual findings?
25. Could the authors conclude the manuscript with more specific practical recommendations regarding which vehicle and concentration ranges should be used or avoided in future B. burgdorferi drug-screening experiments, while clearly distinguishing conclusions directly supported by the present data from hypotheses requiring further molecular validation?

Author Response

The authors thank the reviewers for their thoughtful comments and have revised the manuscript to address all comments in full. The authors have attached the document including the responses. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a relevant methodological question regarding the use of different solvent vehicles in Borrelia burgdorferi in vitro assays. The comparison between direct cell counts and ATP-dependent luminescence is useful, and the different response observed with ethanol is particularly interesting. However, several points should be clarified before publication.

1. Please clarify the NaOH experiment. The methods indicate the use of a 1 M NaOH stock solution, while very high pH values (up to 13.35) are reported. The stock concentration, final concentrations, and pH measurements should be carefully checked.
2. Please explain more clearly how biological and technical replicates were handled in the statistical analysis. Technical replicates should not be considered independent biological observations.
3. The statement that DMSO was “well tolerated” may be somewhat strong. The results show no statistically significant reduction under the tested conditions, but this does not necessarily indicate complete biological inertness.
4. The increase in ATP-dependent luminescence in the presence of ethanol requires more cautious interpretation. It should be discussed whether ethanol may influence ATP release, membrane permeability, cell lysis, or the assay itself, rather than only reflecting increased metabolic activity.
5. The authors report increased spirochaete aggregation in the presence of ethanol. This may affect the accuracy of microscopic cell counting and could partly contribute to the difference between cell counts and luminescence.
6. Since measurements were performed only at day 6, the study mainly provides an endpoint assessment rather than replication kinetics. The use of terms such as “replication” and “mid-log phase” should therefore be justified or moderated.
7. The discussion is somewhat broad in the sections concerning PTLDS, persister forms, and rodent-targeted strategies. I suggest shortening these parts and focusing more on vehicle effects, assay reliability, and the implications for antimicrobial screening.

Overall, the study is useful and has practical methodological value. Addressing the points above would improve the clarity and reliability of the manuscript.

 

Author Response

The authors thank the reviewers for their thoughtful comments and have revised the manuscript to address all comments in full. The authors have attached the document including the responses. 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I am happy with the changes made

Author Response

The authors thank the reviewer for taking the time to review the revised manuscript and for the prompt response. 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed the majority of my previous comments, and the revised manuscript has been substantially improved. The methodological clarifications, revised statistical analysis, and more cautious interpretation of the findings have strengthened the manuscript.

I have only two minor points that should be addressed:

1. The response regarding NaOH states that the reported pH values were maintained “through the duration of the experiment,” whereas the revised Methods indicate that pH was measured at day 6. Please clarify that these values represent endpoint pH measurements, unless longitudinal measurements were performed.
2. In the Conclusion, the statement that “DMSO evidently does not affect cell counts and viability up to 2.4% v/v” remains somewhat stronger than supported by the measured endpoints. I suggest revising this to: “No statistically detectable reduction in cell counts or ATP-dependent luminescence was observed with DMSO up to 2.4% v/v under the conditions tested.” This wording would better reflect the limitations of the ATP-based assay and the experimental design.

Apart from these minor issues, the authors have adequately addressed my previous concerns.

Author Response

The authors thank the reviewer for reviewing the revised manuscript and for the prompt response. The manuscript has been revised in response to the two points below. The minor changes are accessible with tracked changes. 

Minor points: 

1. The response regarding NaOH states that the reported pH values were maintained “through the duration of the experiment,” whereas the revised Methods indicate that pH was measured at day 6. Please clarify that these values represent endpoint pH measurements, unless longitudinal measurements were performed.

The authors acknowledge the confusion regarding the wording in the response. The manuscript has also been revised in brief to confirm the endpoint measure, line 107. 

2. In the Conclusion, the statement that “DMSO evidently does not affect cell counts and viability up to 2.4% v/v” remains somewhat stronger than supported by the measured endpoints. I suggest revising this to: “No statistically detectable reduction in cell counts or ATP-dependent luminescence was observed with DMSO up to 2.4% v/v under the conditions tested.” This wording would better reflect the limitations of the ATP-based assay and the experimental design.

The authors thank the reviewer for suggesting revisions to the conclusion section. This text has been revised according to the recommendation.