From the journal RSC Chemical Biology Peer review history

01-Apr-2023

Dear Dr Coombes:

Manuscript ID: CB-ART-02-2023-000014
TITLE: Screening under infection-relevant conditions reveals chemical sensitivity in multidrug resistant non-typhoidal Salmonella (iNTS)

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary.

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The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

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Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

************

Reviewer 1

This work used two non-traditional screening strategies, e.g., by screening under the host-mimicking conditions or via a macrophage-based platform, to identify potential hits with anti-iNTS Salmonella activities. These two unconventional drug discovery approaches resulted in some positive hits. And the authors chose some of them to further explore their mode of actions. The whole study designed rationally, and may be a good example for scientists to discovery other new antimicrobial agents. Some of the suggestions or concerns were listed below for the authors to better improve their work.
1 Two positive hits (cetylbyridinium chloride and purpurogallin carboxylic acid) seems active in both screens, so I am wondering why the authors not follow them further, e.g., for their anti-iNTS Salmonella activities both in vitro and in animal infection models, as well as their toxicities on host cells.
2 The authors discovered AZT(3’azido-3’deoxythymidine) from the host-mimicking conditions screening, so how about its antimicrobial activity in normal nutrient replete media.
3 For Fig. 2d, I think there should be a growth control that without drugs.
4 “Similar results were observed by assessing viable bacterial counts of ST313 on solid media after treatment with AZT at 0, 2, 32, and 256 μg/mL.” Here, I think 0 μg/mL should be deleted, as obviously, it is a growth control.
5 “Concentrations at or above 32 μg/mL reduced bacterial viability of ST313 after 6 h of incubation, and after 22 h of incubation there was a～4.5-log decrease in ST313 viability relative to untreated bacterial cells (Figure 2e).” There is no statistical analysis here, but it seems that bacterial viability was reduced after 2h incubation. And from Fig. 2e, it is after 20 h, instead of 22 h incubation, there was a～4.5-log decrease in ST313 viability.
6 For Fig. 4a, to do a favor for the readers to compare the activities and toxicities, µg/ml instead of µM is appreciated to describe the concentration of the hits.
7 In the current host cell-directed screening strategy, macrophages were pretreated with the compounds, how about their effect when dosed at the beginning of infection or several hours after infection?

Reviewer 2

In “Screening under infection-relevant conditions reveals chemical sensitivity in multidrug resistant non-typhoidal Salmonella (iNTS)”, Tsai et al. develop and implement two small molecule screens that respecitvely: 1) reveal chemical sensitivities of ST313 iNTS in infection-relevant in vitro condition (screen 1); and 2) identify potential host-directed therapeutics that limit the proliferation of ST313 iNTS in macrophages (screen 2).

In Screen 1, the authors identify 92 compounds that inhibit STM proliferation in acidic, low-phosphate, low-magnesium host-mimicking media. Among these 92 compounds are several well characterised antibiotics (e.g. enrofloxacin, ciprofloxacin, other established antimicrobials). The authors further investigate one compound (3’azido-3’deoxythymidine (AZT)). This compound has been known to inhibit Salmonella Typhimurium proliferation in macrophages for ~30 years. The authors further show that: 1) AZT can also act synergistically with relevant antibiotics for treating iNTS (e.g. ciprofloxacin) to prevent ST313 proliferation in acidic, low-phosphate, low-magnesium host-mimicking media, which is of considerable interest; and 2) that mutations in the tdk gene of ST313 Salmonella confers resistance to AZT in acidic, low-phosphate, low-magnesium host-mimicking media.

In Screen 2, the authors identify 28 compounds where treatment of LPS-exposed RAW macrophages with these compounds prior to infection with ST313 Salmonella restricts Salmonella proliferation in the pre-treated LPS-exposed RAW macrophages following infection. The authors subsequently refine this list of 28 compounds to 3 compounds by excluding hits to only those that show both a clear dose-dependent effect on ST313 Salmonella proliferation and limited cytotoxicity for RAW macrophages: amodiaquine, berbamine and indatraline. Intriguingly, the authors show that pre-treatment with two of these compounds (amodiaquine, berbamine) also restrict ST313 proliferation in epithelial cells. Finally, the authors use qPCR to show amodiaquine and indatraline pre-treatment may increase transcription of the Nos2 gene in RAW macrophages.

Overall, the paper is well written and is potentially a useful contribution to the field. However, I have outlined below a few concerns/questions that I believe the authors should try to address prior to publication.

1) The authors identify 92 compounds that inhibit STM proliferation in acidic, low-phosphate, low-magnesium host-mimicking media (Screen 1). Although it is encouraging that many of these compounds are known to be bacteriostatic or bactericidal against STM in macrophages (e.g. fluoroquinolones, AZT), the authors should still directly investigate if all 92 compounds they identify significantly reduced STM proliferation in macrophages.

2) The authors should investigate directly in macrophages if AZT acts synergistically with ciprofloxacin and colistin, which their results in acidic, low-phosphate, low-magnesium host-mimicking media suggest (see Fig. 3a).

3) The authors should investigate if synergy between AZT and ciprofloxacin/colistin is abrogated in the case of treatment of a tdk ST313 mutant.

4) The authors should confirm that a tdk ST313 mutant that is resistant to AZT treatment in in acidic, low-phosphate, low-magnesium host-mimicking media is similarly resistant in macrophages. It may be the case that this mutant cannot proliferate in macrophages, and therefore that resistance via the identified tdk point mutations is unlikely to arise/be relevant in vivo.

5) The authors’ results with amodiaquine and berbamine pre-treatment of RAW macrophages and epithelial cells is very interesting. Do the authors know at this point if the effects of amodiaquine or berbamine pre-treatment result in bacteriostatic or bactericidal macrophages/epithelial cells?

6) I commend the authors for being careful in not overstating the results of Fig. 5d. Nonetheless, I still think that the final paragraph of the Results section (beginning “Each of the three host-dependent compounds have known physiological activities …”) is still a reach based on transcriptional up-regulation of a single gene by qPCR. Additionally, the effects of amodiaquine and berbamine are also seen in epithelial cells that inherently have a much-reduced innate immune response upon ST313 infection compared with macrophages. Can the dramatic effects of amodiaquine and berbamine in epithelial cells really be explained by transcriptional up-regulation of Nos2?

7) I noted in the methods that the authors have pre-treated RAW macrophages with LPS prior to infection. Although there are valid reasons why they would have done so, the authors have not explicitly justified this choice in the text. Can the authors please explain their rationale for this in the manuscript? Additionally, are the effects of amodiaquine, berbamine and indatraline pre-treatment observed in macrophages not pre-treated with LPS?

8) I noted in the methods that the authors opsonise STM313 prior to infection with RAW macrophages with human serum, even though RAW macrophages are a mouse cell line. Why have the authors chosen to do this? Can the authors confirm that their observations are the same if opsonisation is carried out with mouse serum? Relatedly, since RAW macropahges are a mouse cell line, Fig 5b and the related text and figure legends should read “Nos2” and “Nox2” instead of “NOS2” and “NOX2”.

Responses to Referee Comments

Referee: 1

Comments to the Author
This work used two non-traditional screening strategies, e.g., by screening under the host- mimicking conditions or via a macrophage-based platform, to identify potential hits with anti- iNTS Salmonella activities. These two unconventional drug discovery approaches resulted in some positive hits. And the authors chose some of them to further explore their mode of actions. The whole study designed rationally, and may be a good example for scientists to discovery other new antimicrobial agents. Some of the suggestions or concerns were listed below for the authors to better improve their work.

Response: We thank the reviewer for their support and positive comments on our manuscript.

1 Two positive hits (cetylbyridinium chloride and purpurogallin carboxylic acid) seems active in both screens, so I am wondering why the authors not follow them further, e.g., for their anti- iNTS Salmonella activities both in vitro and in animal infection models, as well as their toxicities on host cells.

Response: We agree with the reviewer that it would be interesting to assess the antimicrobial activity of cetylbyridinium chloride and purpurogallin carboxylic acid, given their potency in both chemical screens. However, in this study, we were specifically interested in the separation between compounds active in one or the other chemical screen, and not both. We previously reported (PMID: 30643129) that the host-mimicking conditions of LPM and macrophages illuminate distinct chemical target spaces and select for different active compounds against Salmonella. Thus, we chose to focus specifically on compounds with selective activity in either screen. We believe that further investigation of cetylbyridinium chloride and purpurogallin carboxylic acid would fall outside of the scope of this paper.

2 The authors discovered AZT(3’azido-3’deoxythymidine) from the host-mimicking conditions screening, so how about its antimicrobial activity in normal nutrient replete media.

Response: We thank the reviewer for this suggestion. To address this, we performed AZT MIC testing in the original LPM pH 5.8 media and with variations of LPM media that were supplemented to provide nutrient replenishment as suggested. The data shows a 4-fold increase in MIC in supplemented media that appears to be mainly driven by pH. The MIC table with these data are provided below. Given that our focus was on host-mimicking conditions and on macrophage infections where AZT was also found to be active, we include these data here for the purpose of review but have not incorporated them into the manuscript in order to keep the manuscript focused.
Condition (MIC (ug/ml))
LPM pH 5.8 (0.25)
LPM pH 5.8 + 10 mM MgCl2 (0.25)
LPM pH 5.8 + 10 mM PO43- (0.5)
LPMpH5.8+10mMMgCl2 +10mMPO43- (0.5)
LPM pH 7.4 (2)
]LPM pH 7.4 + 10 mM MgCl2 (2)
LPM pH 7.4 + 10 mM PO43- (2)
LPMpH7.4+10mMMgCl2 +10mMPO43- (2)

3 For Fig. 2d, I think there should be a growth control that without drugs.

Response: We agree with the reviewer and have now included growth control data in Figure 2d.

4 “Similar results were observed by assessing viable bacterial counts of ST313 on solid media after treatment with AZT at 0, 2, 32, and 256 μg/mL.” Here, I think 0 μg/mL should be deleted, as obviously, it is a growth control.

Response: We agree with the reviewer and have deleted the text referring to this growth control.

5 “Concentrations at or above 32 μg/mL reduced bacterial viability of ST313 after 6 h of incubation, and after 22 h of incubation there was a~4.5-log decrease in ST313 viability relative to untreated bacterial cells (Figure 2e).” There is no statistical analysis here, but it seems that bacterial viability was reduced after 2h incubation. And from Fig. 2e, it is after 20 h, instead of 22 h incubation, there was a~4.5-log decrease in ST313 viability.

Response: We have edited the corresponding text in the manuscript to incorporate these observations.

6 For Fig. 4a, to do a favor for the readers to compare the activities and toxicities, μg/ml instead of μM is appreciated to describe the concentration of the hits.

Response: We thank the reviewer for this suggestion. The graphs in Figure 4a all depict compound concentrations in μM, as per standard chemical screening and reconfirmation protocols (the screening stock concentrations of all compounds in library format are at 5 mM). For data presentation and consistency, changing the graphs shown in Figure 4a from μM to μg/mL would alter the axes considerably and the data would be more difficult to interpret (as the molecular weight of the compounds varies considerably).

7 In the current host cell-directed screening strategy, macrophages were pretreated with the compounds, how about their effect when dosed at the beginning of infection or several hours after infection?

Response: We also believe that this is an interesting experiment to consider. However, we designed the pretreatment methodology of this study to specifically minimize the exposure of bacteria to compound after internalization into macrophages to enrich for host-directed actives. This protocol allowed us to select more specifically for compounds likely to possess host- (and not bacterial-targeting) modes of activity. The specific timing of the pretreatment step in this study allowed for the macrophages to be washed before infection to eliminate residual compound. Thus, we believe that the suggested experiments would be most useful for future work in a separate study focused specifically on interrogating the mechanisms of action and prospective clinical utility of the lead compounds from the initial paper. In line with this view, we have now mentioned this option in the discussion of the paper as an area for future development.

Referee: 2

Comments to the Author
In “Screening under infection-relevant conditions reveals chemical sensitivity in multidrug resistant non-typhoidal Salmonella (iNTS)”, Tsai et al. develop and implement two small molecule screens that respecitvely: 1) reveal chemical sensitivities of ST313 iNTS in infection- relevant in vitro condition (screen 1); and 2) identify potential host-directed therapeutics that limit the proliferation of ST313 iNTS in macrophages (screen 2). In Screen 1, the authors identify 92 compounds that inhibit STM proliferation in acidic, low- phosphate, low-magnesium host-mimicking media. Among these 92 compounds are several well characterised antibiotics (e.g. enrofloxacin, ciprofloxacin, other established antimicrobials). The authors further investigate one compound (3’azido-3’deoxythymidine (AZT)). This compound has been known to inhibit Salmonella Typhimurium proliferation in macrophages for ~30 years. The authors further show that: 1) AZT can also act synergistically with relevant antibiotics for treating iNTS (e.g. ciprofloxacin) to prevent ST313 proliferation in acidic, low-phosphate, low- magnesium host-mimicking media, which is of considerable interest; and 2) that mutations in the tdk gene of ST313 Salmonella confers resistance to AZT in acidic, low-phosphate, low- magnesium host-mimicking media.
In Screen 2, the authors identify 28 compounds where treatment of LPS-exposed RAW macrophages with these compounds prior to infection with ST313 Salmonella restricts Salmonella proliferation in the pre-treated LPS-exposed RAW macrophages following infection. The authors subsequently refine this list of 28 compounds to 3 compounds by excluding hits to only those that show both a clear dose-dependent effect on ST313 Salmonella proliferation and limited cytotoxicity for RAW macrophages: amodiaquine, berbamine and indatraline. Intriguingly, the authors show that pre-treatment with two of these compounds (amodiaquine, berbamine) also restrict ST313 proliferation in epithelial cells. Finally, the authors use qPCR to show amodiaquine and indatraline pre-treatment may increase transcription of the Nos2 gene in RAW macrophages.
Overall, the paper is well written and is potentially a useful contribution to the field. However, I have outlined below a few concerns/questions that I believe the authors should try to address prior to publication.

Response: We thank the reviewer for their support and positive comments on our manuscript.

1) The authors identify 92 compounds that inhibit STM proliferation in acidic, low- phosphate, low-magnesium host-mimicking media (Screen 1). Although it is encouraging that many of these compounds are known to be bacteriostatic or bactericidal against STM in
macrophages (e.g. fluoroquinolones, AZT), the authors should still directly investigate if all 92 compounds they identify significantly reduced STM proliferation in macrophages.

Response: We agree that this would be an interesting set of experiments that would help to further interrogate the chemical sensitivity profile of ST313. However, we believe that such a comprehensive investigation would fall outside of the scope of this current paper, which as submitted contains two separate chemical screens designed to access two distinct target spaces (bacterial- and host-targeted mechanisms of action). For the purposes of this study, we selected AZT as a sole lead compound emerging from the LPM screen for its favorable properties and potency. Performing similar reconfirmation experiments for all 92 compounds in macrophages would require significant iterative experimentation that we believe would detract from the narrative of the paper as written by expanding into a target space that was not our specific focus.

2) The authors should investigate directly in macrophages if AZT acts synergistically with ciprofloxacin and colistin, which their results in acidic, low-phosphate, low-magnesium host- mimicking media suggest (see Fig. 3a).

Response: We thank the reviewer for this suggestion. We have now added new panels to Figure 3 (b,c) to address this point. Our updated results indicate that AZT does act synergistically with ciprofloxacin and colistin, aligning with our results from acidic, low-phosphate, low-magnesium host-mimicking media.

3) The authors should investigate if synergy between AZT and ciprofloxacin/colistin is abrogated in the case of treatment of a tdk ST313 mutant.

Response: We thank the reviewer for this suggestion. We have added these data to Figure 3 (e,f). As expected, we observed that synergy between AZT and both ciprofloxacin and colistin was abrogated in the case of both the ∆tdk and tdkE168* mutants.

4) The authors should confirm that a tdk ST313 mutant that is resistant to AZT treatment in in acidic, low-phosphate, low-magnesium host-mimicking media is similarly resistant in macrophages. It may be the case that this mutant cannot proliferate in macrophages, and therefore that resistance via the identified tdk point mutations is unlikely to arise/be relevant in vivo.

Response: We thank the reviewer for this suggestion. The suppression experiment was designed to help us identify the possible mechanism by which AZT was exerting function in Salmonella and not to understand the full spectrum of mutations that might give rise to biologically relevant/selective mutations in the context of host infection. It is also true that compensatory, unlinked mutations might also arise under host selection that would restore fitness to certain mutants. Again, this is not the focus of this first manuscript and is likely better suited to future investigations.

5) The authors’ results with amodiaquine and berbamine pre-treatment of RAW macrophages and epithelial cells is very interesting. Do the authors know at this point if the effects of amodiaquine or berbamine pre-treatment result in bacteriostatic or bactericidal macrophages/epithelial cells?

Response: We agree that this is an interesting question to consider. Based on the data presented in this study, we are unable to conclusively infer whether amodiaquine or berbamine are having a bacteriostatic or bactericidal impact on macrophages and/or epithelial cells and therefore chose not to speculate too heavily. Further experimentation to address this question will be an important step in the future, to continue characterizing the mechanisms of action of these drugs. We have added a description of these possible experiments to the discussion of the paper to capture the concept raised by this reviewer.

6) I commend the authors for being careful in not overstating the results of Fig. 5d. Nonetheless, I still think that the final paragraph of the Results section (beginning “Each of the three host-dependent compounds have known physiological activities ...”) is still a reach based on transcriptional up-regulation of a single gene by qPCR. Additionally, the effects of amodiaquine and berbamine are also seen in epithelial cells that inherently have a much-reduced innate immune response upon ST313 infection compared with macrophages. Can the dramatic effects of amodiaquine and berbamine in epithelial cells really be explained by transcriptional up-regulation of Nos2?

Response: We thank the reviewer for this suggestion and agree that our qPCR results are preliminary and cannot be used to conclusively infer a mechanism of action dependent upon nitric oxide host defenses. In line with this view, we have edited the referenced paragraph of the results section to more carefully describe our interpretation of the Nos2 upregulation that we observed.

7) I noted in the methods that the authors have pre-treated RAW macrophages with LPS prior to infection. Although there are valid reasons why they would have done so, the authors have not explicitly justified this choice in the text. Can the authors please explain their rationale for this in the manuscript? Additionally, are the effects of amodiaquine, berbamine and indatraline pre- treatment observed in macrophages not pre-treated with LPS?

Response: We agree with the reviewer’s suggestion and have now explained our rationale for LPS pretreatment in the text of our results section. We thank the reviewer for the additional question about LPS. We do not yet know whether the effects of host-directed compounds synergize with LPS pretreatment or not, but this is a screening condition that we are considering in future iterations with a broader panel of TLR agonists.

8) I noted in the methods that the authors opsonise STM313 prior to infection with RAW macrophages with human serum, even though RAW macrophages are a mouse cell line. Why have the authors chosen to do this? Can the authors confirm that their observations are the same if opsonisation is carried out with mouse serum? Relatedly, since RAW macropahges are a mouse cell line, Fig 5b and the related text and figure legends should read “Nos2” and “Nox2” instead of “NOS2” and “NOX2”.

Response: Opsonization of Salmonella using non-immune human serum for the purpose of phagocytic uptake into mouse cells is done routinely in the field. There are over 50 proteins in nonimmune serum that can act as opsonins for Gram-negative bacteria, including complement proteins C4b, C3b, iC3b, and C1q, and other molecules. Human IgGs cross react with mouse Fc- gamma-receptors (Dekkers, G., 2017) and so non-specific IgG opsonization is also possible. There is also cross reactivity between mouse CR3 (CD11b/CD18) and targets opsonized with human complement proteins and beta-glucans. Regarding gene nomenclature, as per the reviewer’s suggestion, we have edited the appropriate sections of the text and figures referring to Nos2 and Nox2.

21-Jun-2023

Dear Dr Coombes:

Manuscript ID: CB-ART-02-2023-000014.R1
TITLE: Screening under infection-relevant conditions reveals chemical sensitivity in multidrug resistant non-typhoidal Salmonella (iNTS)

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link :

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You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

All RSC Chemical Biology articles are published under an open access model, and the appropriate article processing charge (APC) will apply. Details of the APC and discounted rates can be found at https://www.rsc.org/journals-books-databases/about-journals/rsc-chemical-biology/#CB-charges.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy, https://credit.niso.org/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines https://www.rsc.org/journals-books-databases/author-and-reviewer-hub/authors-information/responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

************

Reviewer 1

Generally, the authors have addressed most of my concerns. I would be happy to recommend its publication if the following comments were payed attentions.
1.The authors mentioned figure S1 in the revised-manuscript, does the authors mean the table appeared in the rebuttal letter to show the retaining activity in nutrient-replete media? If it is the truth, please do not forget to supplement this result in the final version of the manuscript.
2. In Page 11, the authors suggested that “Concentrations at or above 2 μg/mL reduced bacterial viability of ST313 after 6 h of incubation, and after 20 h of incubation there was a ~4.5-log decrease in ST313 viability relative to untreated bacterial cells (Figure 2e).” Here should be clearly noticed that only the group with 256 μg/mL AZT treatment results in a ~4.5-log decrease in ST313 viability relative to untreated bacterial, the other two groups reduced the survive of ST313 in a much less degree.
3.From figure 3b and 3c, it seems that AZT combined with ciprofloxacin has synergistic activity towards ST313, but this is not the case for AZT-colistin combinations, as it did not further reduce the survival of ST3131 when compared with the single AZT group. So, the authors should be noticed not to be overstatement.

Responses to Referee Comments Revision 2
Referee: 1

Comments to the Author:

Generally, the authors have addressed most of my concerns. I would be happy to recommend its publication if the following comments were payed attentions.

Response: We are pleased that the reviewer was satisfied with how we addressed their original concerns and we have also addressed their additional comments fully in this second revision.

1. The authors mentioned figure S1 in the revised-manuscript, does the authors mean the table appeared in the rebuttal letter to show the retaining activity in nutrient-replete media? If it is the truth, please do not forget to supplement this result in the final version of the manuscript.

Response: We have included the table of MIC data as Figure 2f, as it relates to this portion of the results.

2. In Page 11, the authors suggested that “Concentrations at or above 2 μg/mL reduced bacterial viability of ST313 after 6 h of incubation, and after 20 h of incubation there was a ~4.5-log decrease in ST313 viability relative to untreated bacterial cells (Figure 2e).” Here should be clearly noticed that only the group with 256 μg/mL AZT treatment results in a ~4.5-log decrease in ST313 viability relative to untreated bacterial, the other two groups reduced the survive of ST313 in a much less degree.

Response: We have noted this in the results section as requested. This now reads:
“Concentrations at or above 2 μg/mL reduced bacterial viability of ST313 after 6 h of incubation in a dose-dependent manner, and after 20 h of incubation there was a ~4.5- log decrease in ST313 viability relative to untreated bacterial cells at the highest concentration tested (Figure 2e).”

3. From figure 3b and 3c, it seems that AZT combined with ciprofloxacin has synergistic activity towards ST313, but this is not the case for AZT-colistin combinations, as it did not further reduce the survival of ST3131 when compared with the single AZT group. So, the authors should be noticed not to be overstatement.

Response: We agree with the review. So as not to overstate our conclusions, this sentence has been revised as follows:
“Consistent with our in vitro results, we found that the combination of AZT and ciprofloxacin reduced the intracellular replication of ST313 more effectively than either AZT or ciprofloxacin alone at both 6h (Figure 3b) and 20 h post-infection (Figure 3c). Although the combination of AZT plus colistin was highly synergistic in vitro, this combination was not as effective as AZT and ciprofloxacin at reducing intracellular ST313 during infection of macrophages.”

30-Jun-2023

Dear Dr Coombes:

Manuscript ID: CB-ART-02-2023-000014.R2
TITLE: Screening under infection-relevant conditions reveals chemical sensitivity in multidrug resistant invasive non-typhoidal Salmonella (iNTS)

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Reviewer 1

I have not any other questions, and now I am happy to recommend its publication.