A Sublethal Concentration of Chlorine Induces Antibiotic Resistance in Salmonella via Production of Reactive Oxygen Species
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsTo the authors:
Page 1, Abstract first you wrote: (small-sample statistics, the fold change revealed that Cl radicals were the) then few lines later you wrote (after chlorine treatment for obtaining resistant colonies).
Regarding chlorine (Cl) versus Hypochlorite (ClO-) and ROS (Cl.) radical, the chemists will not be happy if you are mixing these three species and calling then chlorine! In fact, Chlorine is (Cl2) gas. Please clarify. You have added another term (Chloride down in the conclusion) please clarify.
Pages 2 & 3, Material and Method:
All are good.
Pages 4 & 5, Results all are good.
Pages 6 & 7, Discussion
All are good
Page 8 Conclusion
You mentioned chloride however; you have been mentioning Chlorine throughout the manuscript. Please see the comment about the abstract. Please clarify the identity (if you know) of the ROS species. Is it (Chloride, Cl-) or (Chlorine Cl)?
The two figures are Okay.
The only table is good.
References looked good. However, references 8 and 9 have the year mentioned twice. One time un-bolded or between brackets and a second time in boldface.
8. O’Bryan, C.A.; Ricke, S.C.; Marcy, J.A. 2022. Public health impact of Salmonella spp. on raw poultry: Current concepts and future prospects in the United States. Food Control 2022, 132: 108539.doi.org/10.1016/j.foodcont.2021.108539.
9. Logue, C.; Sherwood, J.; Olah, P.; Elijah, L.; Dockter, M. (2003). The incidence of antimicrobial-resistant Salmonella spp.
Just be consistent with the citation, as the guide to authors requires it.
Author Response
See attached file.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsComment
A timely topic, as it sheds new light on the processes leading to multidrug resistance.
However, several main issues arise:
1) S. Typhimurium 14028s is not the only strain that conditions food infections
foodborne infections in humans. Why is only one strain being studied?
2) What supports the choice of antibiotics used in the study to determine resistance?
Why was e.g. azithromycin or cephalosporins not used?
3) It would be advisable to determine the confirmation of the mutation over time
4) 147-149 - the strain was treated for 12h. And the other times? Why was the MIC not
MIC was determined? What changes in the expression of resistance genes (e.g. to chloramphenicol)
observed? ROS may not only lead to mutations - they also cause environmental stress
environmental stress resulting in efflux production, which temporarily leads to increased
resistance, but this is not a permanent mechanism and returns to normal once the
stimulus.
5) Kanamycin uses oxygen-dependent receptors. Is there a link between ROS and
disruption of receptor transport that could explain resistance to
kanamycin after the action of ROS?
6) Section 2.2 - MSS method - no description, unclear, tab 2 - no description of analysis
7) Section 2.3 - why were the indicated concentrations of antibiotics used for the experiment?
8) 270-272 Kanamycin resistance is not an exhaustive definition of MDR
Future research directions are indicated in the discussion; however, the data in the article are
incomplete and the sample is too small.
Author Response
See attached file.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsIt’s weird that the authors did not cite any literature in the Methods. I assume the methods that the manuscript used were not created by the authors.
For example, Lines 128-130. Please cite in the ending of the sentence.
More importantly, the authors should explain why they chose Rifampicin as the way to calculate S.Tm mutation rate. Readers may feel very confused since rifampicin usually plays the role of the inhibitor of RNA pol to pause the transcription, while here obviously it’s not the case.
Starting from Line 160, It’s not comparable if the authors did not show the exact concentrations of different reagents (chlorine, Hydrogen Peroxide, and etc), although they said it in Methods. Also, the author did not explain why they chose sublethal concentration of chloride as the concentration.
Line 131, The authors did not clearly states why they chose 150mM Thiourea and 500uM H2O2. Are they also the sublethal concentration of S.Tm just as chlorine?
The phenotype that Chl treated S.Tm gained the resistance to Kan antibiotic seems very strong. If the authors can design an experiment that Chl concentration gradients is involved to Kan resistance ability, that would be more reliable and persuasive.
Author Response
See attached file.
Author Response File: Author Response.pdf
