Review Reports - Development of Resazurin-Based Assay for Rapid Evaluation of Sodium Hypochlorite Tolerance in Salmonella

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

FOODS 4183757 v1

Title: Development of resazurin-based assay for rapid evaluation of sodium hypochlorite tolerance in Salmonella

Here are my suggestions:

Minor:

The resazurin method detects more resistant strains than the MIC method, so these strains are false resistant. It is a problem of sensitivity and specificity. In my opinion, the resazurin assay can be used as a screening test. I suggest adding a phrase endorsing its use as a screening method.

Authors added this phrase in response to my suggestion (Lines 245-245) “The results indicate that the resazurin method detects a greater number of resistant strains compared to the MIC method, with a false resistance rate of 1.6% (Table 1). The reason could be that there is an overgrowth at the surface of the well determining false-positive results when it partially covers the well and blocks the fluorescence emission”.

The explanation is not correct if there is turbidity in the wells means a true positive result. Please remove this phrase. The OD values of these false positives were not mentioned. The problem may be the threshold value.

Author Response

We agreed with the reviewer that the discrepancy between the Resazurin assay and the MIC method (the 1.6% "false resistance") likely stems from the higher sensitivity of the resazurin indicator to low-level metabolic activity, which may not yet have reached the threshold of visible turbidity required for a standard MIC reading. Following your suggestion, we have removed the sentence: "The reason could be that there is an overgrowth at the surface of the well determining false-positive results when it partially covers the well and blocks the fluorescence emission" (formerly lines 244-245). We have updated the Discussion section to reflect that while the Resazurin assay is an excellent screening tool, its threshold parameters need careful calibration to align with gold-standard MIC results (Lines 256-257).

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The authors have addressed some of the issues raised in the previous review although others persist. Of main concern is the reporting of the chlorine concentrations that did not take into account the effect of pH on the proportion of hypochlorous vs hypochlorite. The authors report 56 ppm free chlorine in the undiluted bleach but then refer to 512 ppm in the MIC studies. It should also be noted that Salmonella would be sensitive to alkali pH being a Gram negative bacteria. In the challenge trails the authors suggest Salmonella could grow in the presence of 64 ppm free-chlorine. Again, the authors need to confirm the pH of the media along with final free-chlorine concentration rather than estimated.

The authors should also cite support of using 0.1% hypochlorite for surface disinfection directly from WHO as none of the cited papers contain the information. Also, the authors need to differentiate between applying hypochlorite in domestic environments vs poultry processing. The latter would adjust the pH to near neutral to increase free-chlorine and minimize generation of disinfection byproducts.

Author Response

Item 1. The authors have addressed some of the issues raised in the previous review although others persist. Of main concern is the reporting of the chlorine concentrations that did not take into account the effect of pH on the proportion of hypochlorous vs hypochlorite.

The authors report 56 ppm free chlorine in the undiluted bleach but then refer to 512 ppm in the MIC studies.

It should also be noted that Salmonella would be sensitive to alkali pH being a Gram negative bacteria. In the challenge trails the authors suggest Salmonella could grow in the presence of 64 ppm free-chlorine. Again, the authors need to confirm the pH of the media along with final free-chlorine concentration rather than estimated.

Reply: We sincerely thank the reviewer for these insightful and critical comments regarding the chemical behavior of sodium hypochlorite (NaClO) and the influence of pH on its antimicrobial efficacy. We have addressed the concerns as follows:

Effect of pH on HClO/ClO⁻ Distribution：

We fully agree with the reviewer that the bactericidal activity of NaClO is pH-dependent, as the ratio of hypochlorous acid (HClO) to hypochlorite ion (ClO⁻) shifts with pH. At neutral pH, HClO is the dominant active species, whereas ClO⁻ becomes more prevalent and stable in alkaline conditions (typically pH > 10) (Fukuzaki, 2006).

Justification for Not Adjusting pH in MIC Testing

While we acknowledge that pH significantly impacts antimicrobial efficacy, we chose not to artificially adjust the pH of the media for the following reasons:

Methodological Consistency: A review of established protocols for chlorine MIC testing (Wang, 2025; Kampf, 2024; Gao et al., 2014) reveals that pH adjustment is generally not performed. To ensure our results are comparable with existing literature on Salmonella and Klebsiella pneumoniae tolerance (Gao et al., 2014; Wu et al.), we maintained consistency with these established methodologies. For example, according to the reported from Geo et al., bacterial suspension was added to a 96-well microplate containing 50 μL of chlorine disinfectant per well, resulting in final concentrations of 1–512 mg/L. Besides, in the study of Wu et al. found that the Klebsiella pneumoniae was more tolerant among Gram-negative strains to chlorine disinfectants, followed by Salmonella and Escherichia coli.

Standardization Challenges: We agree that the lack of standardized pH controls in chlorine susceptibility testing complicates the comparison of tolerance trends across studies. We have added a detailed discussion on this limitation and the potential impact of pH on our results in the revised manuscript (Lines 294–307). Future studies will prioritize the optimization of pH-controlled procedures.

Clarification on Chlorine Concentrations We apologize for the confusion regarding the concentrations. To clarify:

The commercial NaClO stock solution contained 56,800 ppm (56.8 mg/mL pH 12.2) of free chlorine, not 56 ppm. This stock was then diluted to the target working concentrations used in the MIC studies (up to 512 ppm). This clarification has been updated in Lines 137-139.

In response to the reviewer’s suggestion, we have quantified the actual pH and final free-chlorine concentrations within the test media. These results are now detailed in Table S1 (provided as a Supplementary File) (Lines 151-153).

Table S1 Information on final free-chlorine concentration and pH value

NaClO working concentrations	Final free chlorine concentration in the media	pH values in the media
128 mg/L	2.3 mg/L	7.37
256 mg/L	41.2 mg/L	7.41
512 mg/L	76.9 mg/L	7.43
1024 mg/L	219 mg/L	7.48

Fukuzaki, S. Mechanisms of Actions of Sodium Hypochlorite in Cleaning and Disinfection Processes. Biocontrol Sci. 2006, 11, 147–157, doi:10.4265/bio.11.147.

Wang, B.; Xiong, F.; Rao, W.; Lyu, C.; Chen, H.; Xu, X.; Wang, H. Characterizing variations in resistance of serratia species to sodium hypochlorite: Cell membrane and oxidative stress responses. Food Res. Int. 2025, 221, 117629. doi.org/10.1016/j.foodres.2025.117629.

Gao, H.; Liu, C. Biochemical and morphological alteration of Listeria monocytogenes under environmental stress caused by chloramine-T and sodium hypochlorite. Food Control 2014, 46, 455-461, doi.org/10.1016/j.foodcont.2014.05.016.

Kampf, G. Antiseptic Stewardship: Biocide Resistance and Clinical Implications; 2nd ed.; Springer International Publishing AG: Cham, 2024; ISBN 978-3-031-66074-0, doi.org/10.1007/978-3-031-66074-0.

Wu, G.; Yang, Q.; Long, M.; Guo, L.; Li, B.; Meng, Y.; Zhang, A.; Wang, H.; Liu, S.; Zou, L. Evaluation of Agar Dilution and Broth Microdilution Methods to Determine the Disinfectant Susceptibility. J. Antibiot. 2015, 68, 661–665, doi:10.1038/ja.2015.51.

Item 2. The authors should also cite support of using 0.1% hypochlorite for surface disinfection directly from WHO as none of the cited papers contain the information. Also, the authors need to differentiate between applying hypochlorite in domestic environments vs poultry processing. The latter would adjust the pH to near neutral to increase free-chlorine and minimize generation of disinfection byproducts.

Reply: We appreciate the reviewer’s guidance on clarifying the application of hypochlorite in different contexts and providing the appropriate citations.

WHO Citation: As requested, we have added references to support the use of 0.1% sodium hypochlorite for surface disinfection (Line 39).

World Health Organization. Home Care for Patients with COVID-19 Presenting with Mild Symptoms and Management of Their Contacts: Interim Guidance. 2020. Available online:https://apps.who.int/iris/bitstream/handle/10665/331473/WHO-nCovIPC-HomeCare-2020.3-eng.pdf?sequence=1 (accessed on 17 March 2020).

Xiao, S.; Yuan, Z.; Huang, Y. Disinfectants against SARS-CoV-2: A Review. Viruses 2022, 14, 1721, doi: 10.3390/v14081721.

Domestic vs. Poultry Processing Context: We have revised the manuscript to clearly differentiate between the general use of hypochlorite in domestic environments and its specialized application in poultry processing. In the latter, pH control is critical for both efficacy and safety. The revised text now reads: " During the spin-chill process in poultry processing, chicken carcasses are immersed in cold water (< 5 °C) with NaClO added to maintain a near-neutral pH. This serves to both maximize free chlorine concentration and minimize disinfection byproduct formation" in lines 41-43.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The authors have addressed some issues but the major weakness of the study persists. Specifically, when comparing the sensor vs microdilution MIC there would be a need to report if the chlorine is total or free. The foundation of the work is based on the sensor being comparable to the standard method and this can only be concluded if the active chlorine was the same.

Line 15: Does this refer to total or free chlorine?

Line 20: Is the short analysis time the only benefit of the sensor?

Line 53: Would 5 log CFU/ml be considered low?

Line 26: Is this free or total chlorine?

Line 30: Unclear if the authors are referring to the growth inhibition or inactivation?

Line 32: How does the sensor correlate to that of the microdilution method?

Line 42: What concentration of chlorine is typically applied in carcass chill tanks?

Line 63: By chlorine disinfectant do the authors mean alkali stabilized hypochlorite?

Line 137: The free chlorine concentration at pH 12 would be low so the authors should check they don’t mean total chlorine.

Line 203: What is the advantage of a rapid test time?

Line 218: Was 64 mg/L measured or predicted?

Line 243: The authors have to report on the free or total chlorine. The MIC determined using the resazurin-based assay cannot be compared to the microdilution method without considering pH.

Line 298: The cited references did not refer to pH but did measure free- and total chlorine. This should have been done in the current study.

Author Response

Responses to Reviewers’ Comments

Reply: We sincerely appreciate the reviewer's insightful comments. Firstly, we clarify that all reported concentrations of the NaClO working solution refer to free chlorine, verified using a calibrated Palintest ChlorSense meter (DPD standard method) (lines 150-152). To provide full transparency, we have included Supplementary Table S1, which details the free chlorine concentrations and pH values for both the initial working solutions and the final reaction mixtures.

Table S1 Characterization of free chlorine concentration and pH values in experimental systems

Free Chlorine of the initial NaClO working Concentration (mg/L)	pH of NaClO working solutions	Free Chlorine in Reaction Mixture (mg/L) *	pH of Reaction Mixture
16	7.39	<0.1	7.31
32	7.44	0.18	7.33
64	7.55	0.37	7.35
128	7.62	2.3	7.37
256	7.88	41.2	7.41
512	8.14	76.9	7.43
1024	8.53	219	7.48

*Note: Measured immediately after mixing the NaClO working solution with the bacterial suspension in the culture media, representing the actual initial exposure concentration after accounting for instantaneous chlorine demand.

Crucially, the initial 30-minute chlorine exposure phase was conducted identically for both the resazurin-based sensor assay and the standard broth microdilution method (as shown in Figure 1). During this period, both systems shared the same free chlorine concentrations and pH values (as shown in Table S1), maintaining consistent chlorine speciation across both methodologies.

Following the exposure, the antimicrobial action in both systems was simultaneously terminated by adding sodium thiosulfate (Na₂S₂O₃) to quench residual free chlorine and stabilize the pH at near-neutral levels. Then, the methodologies diverged only during the subsequent detection phase: the sensor assay quantified viability via fluorescence signaling from resazurin, whereas the microdilution method assessed the growth of survival bacteria via optical density. Since the resazurin dye was introduced only after this neutralization (Lines 152-154, 170-172), the transient alkalinity of the initial NaClO did not interfere with the metabolic readout.

In summary, because all fundamental treatment parameters, including chlorine species, concentration, pH, and contact time, were held constant, the results from these two approaches are inherently comparable.

Item 1. Line 15: Does this refer to total or free chlorine?

Reply: It is refer to the free chlorine of the initial NaClO working concentration.

Item 2. Line 20: Is the short analysis time the only benefit of the sensor?

Reply: The short analysis time is the primary advantage and not the only benefit of the approach. Beyond speed, this method provides higher sensitivity to subtle phenotypic shifts among bacteria isolates with the same MIC value that turbidity-based methods might overlook (lines 279-281). To clarify the benefit of the sensor, the sentence has been revised as “This methodology is recognized as a rapid and high-throughput, and quantitative screening approach for assessing bacterial chlorine resistance” in lines 31-32.

Item 3. Line 23: Would 5 log CFU/ml be considered low?

Reply: The sentence has been revised as “Even with an inoculum of 10⁵ CFU/mL, the resazurin-based method determines NaClO tolerance in just 6 hours” in lines 22-23.

Item 4. Line 26: Is this free or total chlorine?

Reply: As we mentioned in Item 1, it’s referred to the free chlorine of the initial NaClO working concentration.

Item 5. Line 30: Unclear if the authors are referring to the growth inhibition or inactivation?

Reply: It’s referred to the inactivation effects, and the sentence was revised as “When the strains exhibiting an MIC value of 256 mg/L, the fluorescence intensity varied from around 1.2×10⁵ to 4×10⁵RFU, reflecting inactivation effects at practical chlorine concentrations to make it clarification (lines 29-31)”.

Item 6. Line 32: How does the sensor correlate to that of the microdilution method?

Reply: Both the resazurin-based assay and the broth microdilution method serve to evaluate bacterial tolerance under chlorine treatment by measuring population responses to specific concentrations. They differ in the type of response signal monitored. The broth microdilution method relies on turbidity as a signal for the survival bacteria growth. The resazurin-based assay leverages a colorimetric change driven by metabolic activity of the survival bacteria, offering a more sensitive and potentially quantitative readout of bacterial viability.

Item 7. Line 42: What concentration of chlorine is typically applied in carcass chill tanks?

Reply: The concentration of chlorine typically applied in poultry carcass chill tanks is 50 mg/L free chlorine. To clarify this, the information has been added in lines 41-42 as “During the spin-chill process in poultry processing, chicken carcasses are immersed in cold water with 50 mg/L free chlorine added to maintain a near-neutral pH (6.5~7.0)”.

FAO/WHO. Benefits and risks of the use of chlorine-containing disinfectants in food production and food processing: report of a joint FAO/WHO expert meeting, Ann Arbor, MI, USA, 27-30 May 2008. Geneva World Health Organization. 2009. https://www.fao.org/4/i1357e/i1357e00.htm.

Item 8. Line 63: By chlorine disinfectant do the authors mean alkali stabilized hypochlorite?

Reply: According to the joint FAO/WHO report, chlorine-containing disinfectants encompass a broad range of reactive species, including hypochlorous acid (HClO), hypochlorite ion (OCl^-), chlorine gas (Cl²), and other formulations like chloramines and chlorine dioxide (FAO/WHO, 2008). The chlorine disinfectant used in this study refers to free chlorine derived from sodium hypochlorite (NaClO), rather than alkali-stabilized hypochlorite.

FAO/WHO. Benefits and risks of the use of chlorine-containing disinfectants in food production and food pro-cessing: report of a joint FAO/WHO expert meeting, Ann Arbor, MI, USA, 27-30 May 2008. Geneva World Health Organization. 2009. https://www.fao.org/4/i1357e/i1357e00.htm.

Item 9. Line 137: The free chlorine concentration at pH 12 would be low so the authors should check they don’t mean total chlorine.

Reply: We agree with the reviewer that the speciation of chlorine is highly pH-dependent. However, we would like to clarify that the pH=12 mentioned refers exclusively to the undiluted commercial NaClO stock solution (Sangon Biotech), where high alkalinity is required to stabilize the concentrated available chlorine (5.68%). We have revised the manuscript (lines 137-140) to clearly distinguish between the stock solution properties and the experimental working conditions: "The commercial NaClO stock solution (Sangon Biotech, Shanghai, China) was used in this study. The available chlorine content of the original NaClO solution was 5.68% (56800 mg/L, pH 12), which was subsequently diluted to target working concentrations for all assays."

Item 10. Line 203: What is the advantage of a rapid test time?

Reply: Current protocols, particularly the broth microdilution method, rely on visible turbidity which necessitates an 18–24 hour incubation period. This turnaround time is often insufficient for rapid decision-making in food processing or clinical settings. Our study addresses this gap by developing a 6-hour resazurin-based framework that enables rapid quantification of chlorine tolerance. Such a time-critical tool is essential for implementing precision disinfection strategies, minimizing chemical waste, and reducing the selective pressure for antimicrobial resistance (lines 325-327).

Item 11. Line 218: Was 64 mg/L measured or predicted?

Reply: The value of 64 mg/L (and other values in the series) represents the theoretically calculated NaClO working concentration based on the standard two-fold serial dilution method. Consistent with established protocols for chlorine MIC testing (Wang, 2025; Wu et al., 2015; Gao et al., 2014), we expressed the MIC results using the conventional 2-fold dilution series (e.g., 32, 64, 128 mg/L). Moreover, to ensure the scientific rigor of these theoretical values, we performed experimental verification as presented in the newly added Supplementary Table 1. By adhering to these standard numerical representations, we ensure that our results are directly comparable with existing literature and providing the measured data in the supplement for full transparency (lines 145-159).

Item 12. Line 243: The authors have to report on the free or total chlorine. The MIC determined using the resazurin-based assay cannot be compared to the microdilution method without considering pH.

Reply: We fully agree with the reviewer that the comparability of these two methods hinges on the precise control of free chlorine concentration and pH. To address this, we have quantified both the free chlorine and pH in working solution and reaction mixtures, which are now detailed in Supplementary Table S1.

To ensure a rigorous comparison, the initial stages of both the resazurin-based sensor assay and the broth microdilution method were conducted under identical conditions. Specifically, during the first 30 minutes of the chlorine exposure phase, both systems shared the same free chlorine concentrations and pH values, as detailed in Table S1. Following this exposure, the antimicrobial action in both systems was simultaneously terminated by adding sodium thiosulfate (Na₂S₂O₃) to quench all residual free chlorine.

The methodologies diverged only during the subsequent detection phase: the sensor assay quantified bacterial viability via fluorescence signaling, whereas the broth microdilution method assessed growth of survival bacteria via optical density measurements. Given that the fundamental treatment parameters (chlorine species, concentration, and contact time) were held constant, the results from these two approaches are inherently comparable and reflect the same antimicrobial efficacy.

Item 13. Line 298: The cited references did not refer to pH but did measure free- and total chlorine. This should have been done in the current study.

Reply: The cited references, Gao and Liu (2014) and Wang et al., (2025) measured the active chlorine in MIC testing. As suggested, we have included Supplementary Table S1, which details the free chlorine concentrations and pH values for both the initial working solutions and the final reaction mixtures. The MIC represents the theoretically calculated NaClO working concentration based on the standard two-fold serial dilution method. To maintain consistency with numerical representations in previous literature and ensure the continuity of the dilution series, the free chlorine concentrations of the NaClO working solutions were utilized to report the MIC values. The methodology was modified from the protocol established by previous studies to better achieve the aims of the current investigation (Gao & Liu, 2014; Wang et al., (2025). The sentence has been added in lines 155-159.

Gao, H.; Liu, C. Biochemical and morphological alteration of Listeria monocytogenes under environmental stress caused by chloramine-T and sodium hypochlorite. Food Control 2014, 46, 455-461, doi.org/10.1016/j.foodcont.2014.05.016

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper reported on a resazurin-based assay to measure the metabolic activity of Salmonella. In the current application, the assay was to determine the tolerance of different Salmonella to chlorine. Although the research could be of interest, there are concerns relating to the foundation of the assay, structure of the script, methods and interpretation of results. Specifically, it is unclear whether there is a need for such an assay, as the need to prepare suspensions and set up the assay is time-consuming. Moreover, the assay would not detect VBNC or sub-lethally injured cells, whereas the more standard Dilution assay would.

The introduction should provide more background especially in relation to the MBC of Salmonella to chlorine. To suggest that sensitivity to 128 ppm chlorine is not in line with a typical value of 10 -20 ppm. The assay does not appear to include a carbon and electron source for the Salmonella to have reducing power to react with the dye. Addition points are provided below.

Title: Is the sensor specific for Salmonella? Hypochlorite tolerance in any specific matrix?

Line 14: Sub-lethal concentration of chlorine?

Line 18: What is the principle of the assay?

Line 24: Was the culture growing or stationary phase?

Line 29: What was considered tolerant? When reporting chlorine concentration free or total chlorine?

Line 31: Is it MIC or MBC that the authors are referring to?

Line 33: How does the technique compare to the Dilution test that verifies a 5 log CFU reduction with a 30 second contact time?

Keywords: MIC/MBC, sanitizer, dilution-test, hypochlorite

Line 38: Do the authors mean bacterial or total foodborne illness cases?

Line 40: Chlorine is applied as a sanitizer.

Line 45: Chlorine is mainly used to prevent cross-contamination in wash tanks. This should be considered in the revised script. The authors should also describe the different forms of chlorine as a function of pH and organic matter. Can also refer to chlorine demand?

Line 51: Is chlorine resistance the issue or more the instability of the santitizer in the presence of organics or inorganics.

Line 54: Dilution assay is the standard method.

Line 58: By turbidity test, do the authors mean the increase in optical density if the pathogen?

Line 61: Is there a demand for a rapid test given that the culture needs to be prepared and also post-treatment recovery requires to be determined?

Line 66: By digestion do the authors mean reduction by active metabolizing cells?

Line 73: How does the assay determine sub-lethally injured cells?

Line 80: Need to define what is considered tolerance? Bacteriocidial concentration is more significant.

Line 88: 128 mg/l is equivalent to 128 ppm, which would support a 5-log reduction of Salmonella. Did the authors measure the free-chlorine concentration and pH?

Line 101: Was the pH adjusted? Why not use buffered solutions of chlorine?

Line 120: To confirm, the assay was performed in MH broth as this is not clear from reading the text.

Line 122: Presumably, the broth had a chlorine demand that would reduce the concentration of the sanitizer in the assay. Were chlorine measurements made before the addition of Salmonella?

Line 134: How many times were the experiments repeated?

Line 141: It would have been informative to determine detection of stationary cells vs actively growing to determine if the inoculating suspension had to be performed in a controlled manner.

Line 152: Do the authors mean when cell lysis occurs?

Line 200: The authors should report on the MBC of Salmonella to chlorine reported by other researchers.

Line 204: Would this be the case of a global oxidizer such as chlorine?

Line 201-225: The section has speculative statements with no supporting evidence. Therefore, the paragraph should be removed or made more concise.

Table 1: The Microdiultion method reports on the growth of Salmonella while the Reszurin assay measures metabolic flux. In this regard, it is unclear why the assays would be considered equivalent.

Comments on the Quality of English Language

could be improved

Author Response

Reviewers 1:

The paper reported on a resazurin-based assay to measure the metabolic activity of Salmonella. In the current application, the assay was to determine the tolerance of different Salmonella to chlorine. Although the research could be of interest, there are concerns relating to the foundation of the assay, structure of the script, methods and interpretation of results. Specifically, it is unclear whether there is a need for such an assay, as the need to prepare suspensions and set up the assay is time-consuming. Moreover, the assay would not detect VBNC or sub-lethally injured cells, whereas the more standard Dilution assay would.

Reply: In this study, we emphasize the planktonic bacteria and recommend using MIC to assess resistance at the species or strain levels. The broth microdilution method established by the Clinical Laboratory Standards Institute (CLSI) is a widely utilized technique for evaluating bacterial resistance to antibiotics or disinfectant. But this method requires overnight culturing for 18 to 24 hours to monitoring the turbidity changes. To optimize this method, this work employed a resazurin-based assay to evaluate the NaClO tolerance of Salmonella, which results compared to broth microdilution method for assessing bacterial tolerance. Besides, the suspensions preparation work would be considered to optimize the in future as suggested. The necessary of this study was revised to make it clarified (lines 17-18).

Item 1. The introduction should provide more background especially in relation to the MBC of Salmonella to chlorine. To suggest that sensitivity to 128 ppm chlorine is not in line with a typical value of 10 -20 ppm. The assay does not appear to include a carbon and electron source for the Salmonella to have reducing power to react with the dye. Addition points are provided below.

Reply: As suggested, the sentence on MBC has been added in the Introduction as “Kampf proposed isolates that are inadequately killed by below-use concentrations of a disinfectant solution in suspension tests could be considered tolerant and potentially resistant; moreover, regardless of changes in the MIC value, any isolate or strain should be considered resistant if it demonstrates a significantly elevated MBC, provided the concentration used in the MBC test complies with standard practice and achieves the commonly accepted minimum log₁₀ reduction”(lines 67-73). Many studies showed that the chlorine resistance of bacteria was different among species or strains. In a study on chlorine resistance of E. coli, the MIC range of free chlorine from the most sensitive strain to the most resistant strain was 0.04–0.2 mg/L. In the study of Wu et al., six additional Salmonella serotypes were used as quality control strains in determining the chlorine disinfectant tolerance and the range of MIC values was 8-256 mg/L. In our study, Salmonella Enteritidis CVCC 1806 was used as quality control strain (MIC = 128 mg/L) (lines 100-101). Besides, as suggested the sentence on “The dye acts as an intermediate electron acceptor in the electron transport chain without interference of the normal transfer of electrons. As the indicator dye accepts electrons, it changes from the oxidized, non-fluorescent, blue state to the reduced, fluorescent, pink state. Resazurin reduction may signify an impairment of cellular metabolism” were added in lines 174-178.

Wang, W.; Liu, F.; Huang, K.; Yu, X.; Xiao, X. Chlorine Tolerance in Foodborne Pathogens: Definitions, Identification Methods, and Mechanisms. Current Opinion in Food Science. 2025, 62, 101271, doi:10.1016/j.cofs.2025.101271.

Luo, L., Wu, Y., Yu, T., Wang, Y., Chen, G., Tong, X., Bai, Y., Xu, C., Wang, H., Ikuno, N., & Hu, H. Evaluating method and potential risks of chlorine-resistant bacteria (CRB): A review. Water Research. 2021, 188, 116474, doi.org/10.1016/j.watres.2020.116474.

Wu, G., Yang, Q., Long, M., Guo, L., Li, B., Meng, Y., Zhang, A., Wang, H., Liu, S., & Zou, L. Evaluation of agar dilution and broth microdilution methods to determine the disinfectant susceptibility. Journal of Antibiotics. 2015, 68, 661-665, doi.org/10.1038/ja.2015.51

Calvo, L.G.; Villarino, R.-A.; Rama, J.L.R.; Abril, A.G.; De Miguel, T. A Modification of the Resazurin Cell Viability Assay, Suitable for the Quantification of Lactic Acid Producing Bacteria. LWT 2025, 215, 117259, doi:10.1016/j.lwt.2024.117259.

Petiti, J.; Revel, L.; Divieto, C. Standard Operating Procedure to Optimize Resazurin-Based Viability Assays. Biosensors 2024, 14, 156, doi:10.3390/bios14040156.

Lavogina, D.; Lust, H.; Tahk, M.-J.; Laasfeld, T.; Vellama, H.; Nasirova, N.; Vardja, M.; Eskla, K.-L.; Salumets, A.; Rinken, A.; et al. Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon. Biosensors 2022, 12, 196, doi:10.3390/bios12040196.

Item 2. Title: Is the sensor specific for Salmonella? Hypochlorite tolerance in any specific matrix?

Reply: The sensor was specific for Salmonella and can be applied in the Salmonella isolates from samples.

Figure 1. Fluorescence intensity changes of sodium hypochlorite tolerance in Salmonella, E.coli and S.aureus

Item 3. Line 14: Sub-lethal concentration of chlorine?

Reply: Concentration of chlorine was established by the Clinical Laboratory Standards Institute (CLSI) and our previous studies.

Xiao, X.; Bai, L.; Wang, S.; Liu, L.; Qu, X.; Zhang, J.; Xiao, Y.; Tang, B.; Li, Y.; Yang, H.; et al. Chlorine Tolerance and Cross-Resistance to Antibiotics in Poultry-Associated Salmonella Isolates in China. Front. Microbiol. 2022, 12, 833743, doi:10.3389/fmicb.2021.833743.

Item 4. Line 18: What is the principle of the assay?

Reply: On entering the cells, this blue colored non-fluorescent oxidized form of the redox indicator dye is reduced by the intracellular reducing environment created by the acceptance of electrons from nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), nicotinamide adenine dinucleotide (reduced form) (NADH), flavin adenine dinucleotide (semiquinone form) (FADH), flavin mononucleotide (semiquinone form) (FMNH) and cytochromes by the enzymes (cytosolic and mitochondrial dehydrogenases in particular) to a red or pink fluorescent compound (resorufin).

Figure 2 principle of the assay

O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Investigation of the Alamar Blue (Resazurin) Fluorescent Dye for the Assessment of Mammalian Cell Cytotoxicity. European Journal of Biochemistry. 2000, 267, 5421–5426, doi:10.1046/j.1432-1327.2000.01606.x.

Rampersad, S.N. Multiple Applications of Alamar Blue as an Indicator of Metabolic Function and Cellular Health in Cell Viability Bioassays. Sensors 2012, 12, 12347–12360, doi:10.3390/s120912347.

Munshi, S.; Twining, R.C.; Dahl, R. Alamar Blue Reagent Interacts with Cell-Culture Media Giving Different Fluorescence over Time: Potential for False Positives. Journal of Pharmacological and Toxicological Methods. 2014, 70, 195–198, doi:10.1016/j.vascn.2014.06.005.

Item 5. Line 24: Was the culture growing or stationary phase?

Reply: The bacterial was cultured to the stationary phase before to use.

Item 6. Line 29: What was considered tolerant? When reporting chlorine concentration free or total chlorine?

Reply: The information on “Tolerance refers to a microorganism’s ability to survive after a short time exposure to chlorine at typically lethal concentrations (lines 56-57)” and “Chlorine concentration is referring to the total chlorine (line 121)” has been added in the manuscript.

Item 7. Line 31: Is it MIC or MBC that the authors are referring to?

Reply: The minimal inhibitory concentration (MIC), which is a key indicator of an antimicrobial agent’s potency, is defined as the concentration (mg L^-1) at which visible growth of bacteria is prevented under defined growth conditions. Minimum bactericidal concentration (MBC) is the lowest concentration of antibiotics that kills 99.9% of the inoculum. The MBC of an antibacterial agent is determined by subculturing the last clear MIC tube onto the growth medium and examining for bacterial growth.

Kadeřábková, N.; Mahmood, A.J.S.; Mavridou, D.A.I. Antibiotic Susceptibility Testing Using Minimum Inhibitory Concentration (MIC) Assays. npj Antimicrob Resist. 2024, 2, 37, doi:10.1038/s44259-024-00051-6.

Cerf, O.; Carpentier, B.; Sanders, P. Tests for Determining In-Use Concentrations of Antibiotics and Disinfectants Are Based on Entirely Different Concepts: “Resistance” Has Different Meanings. International Journal of Food Microbiology. 2010, 136, 247–254, doi:10.1016/j.ijfoodmicro.2009.10.002.

Item 8. Line 33: How does the technique compare to the Dilution test that verifies a 5 log CFU reduction with a 30 second contact time?

Reply: MIC is referring to the minimum disinfectant concentration that inhibits the growth of bacteria in the medium. MIC unifies the reflection endpoint and is suitable for comparison of chlorine resistance among strains, which provides a direct measure of intrinsic resistance. Logarithmic inactivation through Dilution test can reflects disinfection efficacy. Monitoring bacterial populations over time, which can be labor intensive and slow compared to single-point measurements like MIC.

Luo, L.-W.; Wu, Y.-H.; Yu, T.; Wang, Y.-H.; Chen, G.-Q.; Tong, X.; Bai, Y.; Xu, C.; Wang, H.-B.; Ikuno, N.; et al. Evaluating Method and Potential Risks of Chlorine-Resistant Bacteria (CRB): A Review. Water Research. 2021, 188, 116474, doi:10.1016/j.watres.2020.116474.

Item 9. Keywords: MIC/MBC, sanitizer, dilution-test, hypochlorite

Reply: The keywords were revied as suggested (line 35).

Item 10. Line 38: Do the authors mean bacterial or total foodborne illness cases?

Reply: The sentence has been revised as “Salmonella is responsible for approximately 70-80% of foodborne disease outbreaks in China” in lines 38-39.

Item 11. Line 40: Chlorine is applied as a sanitizer.

Reply: As suggested, “Chlorine disinfectants” was revised as “chlorine sanitizer” (line 39).

Item 12. Line 45: Chlorine is mainly used to prevent cross-contamination in wash tanks. This should be considered in the revised script. The authors should also describe the different forms of chlorine as a function of pH and organic matter. Can also refer to chlorine demand?

Reply: Chlorine is mainly used to prevent cross-contamination in wash tanks. The effectiveness of chlorine is dependent not only on the concentration of chlorine but also on the chemical composition of the chill water. In aqueous solution chlorine exists in three different forms, i.e., C1₂, HOCl, and -OC1, which are called free available chlorine or free residual chlorine. The solubility of Cl₂ in water is very low and therefore Cl₂ readily escapes from the solution. Also, in the neutral pH region (6.5 to 7.5), undissociated HOCl tends to be decomposed at a relatively high rate and its concentration decreases gradually during the storage period. On the other hand, dissociated -OCl is more stable in alkaline water, especially at pHs above 10. Chlorine

effectiveness was diminished by the inorganic and organic components present in the chill water, because chlorine reacted not only with microorganisms but also with the inorganic and organic materials. Previous study has been reported that chlorination of chill water at concentrations lower than 30 ppm total residual chlorine did not prevent bacterial cross–contamination, while total residual chlorine higher than 30 ppm greatly reduced post–chill bacterial incidences. The sentences were added in manuscript in lines 46-51.

Figure 3 Distribution of HOCl in aqueous solution as function of pH.

Fukuzaki, S. Mechanisms of Actions of Sodium Hypochlorite in Cleaning and Disinfection Processes. Biocon-trol Sci. 2006, 11, 147–157, doi:10.4265/bio.11.147.

Yang, H.; Li, Y.; Griffis, C.L.; Waldroup, A.L. A probability model for cross-contamination by Campylobacter jejuni and Salmonella Typhimurium in poultry chilling process. Appl. Eng. Agric. 2002, 18, 717–724, doi:10.13031/2013.11313.

Item 13. Line 51: Is chlorine resistance the issue or more the instability of the santitizer in the presence of organics or inorganics.

Reply: With the regular use of surface disinfectants (sodium hypochlorite, sodium dichloroisocyanurate), many microorganisms gradually adapt to these conditions and increase their resistance to subsequent stresses. This resistance can amplify the severity and frequency of bacterial infections. If not addressed, this can lead to incomplete food sterilization and the persistence of resistant bacteria. Consequently, there has been a surge in studies focusing on the interactions between sterilization methods and microbial resistance. Some researchers have linked increased chlorine tolerance with the presence of antimicrobial resistance determinants in bacteria, and have described chlorine and antibiotic cross-resistance. The chlorine tolerance of foodborne bacteria has emerged as a critical concern in food safety prevention. There is an urgent necessity for research on novel intervention strategies on chlorine-tolerant pathogen, such as the screening or synthesis of broad-spectrum, highly effective bacteriophages, and the development of revolutionary antibacterial materials driven by light, heat, or friction. Photocatalysis rely on potent oxidants, such as hydroxyl radicals and superoxide anions, to break down organic contaminants.

Xiao, X.; He, M.; Ma, L.; Lv, W.; Huang, K.; Yang, H.; Li, Y.; Zou, L.; Xiao, Y.; Wang, W. Insights into Microbial Contamination and Antibiotic Resistome Traits in Pork Wholesale Market: An Evaluation of the Disinfection Effect of Sodium Hypochlorite. Journal of Hazardous Materials. 2024, 468, 133811, doi:10.1016/j.jhazmat.2024.133811.

Wang, W.; Liu, F.; Huang, K.; Yu, X.; Xiao, X. Chlorine Tolerance in Foodborne Pathogens: Definitions, Identification Methods, and Mechanisms. Current Opinion in Food Science. 2025, 62, 101271, doi:10.1016/j.cofs.2025.101271.

Item 14. Line 58: By turbidity test, do the authors mean the increase in optical density if the pathogen?

Reply: Cell growth in the plates was also quantified by measuring the optical density

at 600 nm in the turbidity test (lines 127-128).

Item 15. Line 61: Is there a demand for a rapid test given that the culture needs to be prepared and also post-treatment recovery requires to be determined?

Reply: The broth microdilution method established by the Clinical Laboratory Standards Institute (CLSI) is a widely utilized technique for evaluating bacterial resistance to antibiotics or disinfectant. But this method requires overnight culturing for 18 to 24 hours to monitoring the turbidity changes. The principle of this test is based on the visual and fluorescence intensity changes after adding resazurin and the results of NaClO tolerance can be finalized within one hour.

Item 16. Line 66: By digestion do the authors mean reduction by active metabolizing cells?

Reply: Active cells and sub-lethally injured cells.

Item 17. Line 73: How does the assay determine sub-lethally injured cells?

Reply: Resazurin is a small-molecular-weight phenoxazin-3-one dye that can be reduced to resorufin by NADH, NADPH, FADH, or other biologically abundant reductive species in the presence of mitochondrial or cytoplasmic reductases. Resazurin reduction may signify an impairment of cellular metabolism. In our previous studies, we found that VBNC state Salmonella Enteritidis has a significant increase in NAD+/NADH ratios.

Wang, W., Wang, W., Yi, Z., Ma, J., Hou, W., Xiao, Y., Yang, H., Wang, H., & Xiao, X. An integrated transcriptome and proteomic investigation of the stress responses of viable but non-culturable (VBNC) state Salmonella enteritidis triggered by sodium hypochlorite. Food Science and Human Wellness. 2025, 14, null-null, doi.org/10.26599/FSHW.2024.9250213

Item 18. Line 80: Need to define what is considered tolerance? Bacteriocidial concentration is more significant.

Reply: Tolerance refers to a microorganism’s ability to survive after a short time exposure to chlorine at typically lethal concentrations (lines 56-57).

Kampf, G. Proposal for a Definition to Describe Resistance to Biocidal Active Substances and Disinfectants. Journal of Hospital Infection. 2023, 139, 251–252, doi:10.1016/j.jhin.2023.05.009.

Wang, W.; Liu, F.; Huang, K.; Yu, X.; Xiao, X. Chlorine Tolerance in Foodborne Pathogens: Definitions, Identification Methods, and Mechanisms. Current Opinion in Food Science. 2025, 62, 101271. doi:10.1016/j.cofs.2025.101271.

Item 19. Line 101: Was the pH adjusted? Why not use buffered solutions of chlorine?

Reply: Chlorine solutions was prepared according to our previous studies.

Item 20. Line 120: To confirm, the assay was performed in MH broth as this is not clear from reading the text.

Reply: The information was added in line 134 to make it clarification.

Item 21. Line 122: Presumably, the broth had a chlorine demand that would reduce the concentration of the sanitizer in the assay. Were chlorine measurements made before the addition of Salmonella?

Reply: The methods was established by the Clinical Laboratory Standards Institute (CLSI) and our previous studies.

Item 22. Line 134: How many times were the experiments repeated?

Reply: All experiments were performed in triplicate on separate days (lines 147-148).

Item 23. Line 141: It would have been informative to determine detection of stationary cells vs actively growing to determine if the inoculating suspension had to be performed in a controlled manner.

Reply: The bacterial was cultured to the stationary phase before to use.

Item 24. Line 152: Do the authors mean when cell lysis occurs?

Reply: As suggested, the sentence was revised in line 172.

Item 25. Line 200: The authors should report on the MBC of Salmonella to chlorine reported by other researchers.

Reply: The sentence on “The majority of S.Typhimurium (65.6%) isolated from carcass washing exhibited MIC at 80 ppm. 68.95% of isolates exhibited MBC at 80 ppm for chlorine. S.Typhimurium isolated from the environmental samples exhibited similar MIC and MBC values for chlorine, while the highest proportion was exhibited at 40 ppm and it was 41.1% and 54.5% respectively” has been added in lines 219-223.

Zhang, L.; Yin, D.; Li, L.; Gao, Y. Effects of Cross-Resistance of Salmonella Enterica Serovar Enteritidis Induced by Sodium Hypochlorite to Environmental Stress. Indian J Microbiol. 2024, 64, 1558–1569, doi:10.1007/s12088-023-01170-w.

Weersooriya, G.; Dulakshi, H.M.T.; De Alwis, P.S.; Priyantha, M.A.R. Chlorine Resistance in Salmonella Typhimurium and Campylobacter Jejuni Isolated from Poultry Processing Line. Preprints 2025, 2025010734, doi.org/10.20944/preprints202501.0734.v1.

Item 26. Line 204: Would this be the case of a global oxidizer such as chlorine?

Reply: A total of 1722 publications were available on the Web of Science under the topic ‘chlorine resistant bacteria’, with a significant increase in the past five years. Prolonged exposure to disinfectants or excessive use of chlorine disinfectants poses a global risk of antibiotic-resistant bacteria proliferation, endangering human health.

Wang, W.; Liu, F.; Huang, K.; Yu, X.; Xiao, X. Chlorine Tolerance in Foodborne Pathogens: Definitions, Identification Methods, and Mechanisms. Current Opinion in Food Science. 2025, 62, 101271, doi:10.1016/j.cofs.2025.101271.

Item 27. Line 201-225: The section has speculative statements with no supporting evidence. Therefore, the paragraph should be removed or made more concise.

Reply: As suggested, the paragraph was made more concise (lines 224-236).

Item 28. Table 1: The Microdiultion method reports on the growth of Salmonella while the Reszurin assay measures metabolic flux. In this regard, it is unclear why the assays would be considered equivalent.

Reply: The Microdiultion method is the reference method to determine hypochlorite tolerance. But this method requires overnight culturing for 18 to 24 hours to monitoring the turbidity changes. The resazurin assay can be used as a rapid screening test. The Table 1 was revised to make it clarified.

Other revision: The Figures in the manuscript were improved to clearly communicate the research.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript number: Foods 4088474

Title: Development of resazurin-based assay for rapid evaluation of sodium hypochlorite tolerance in Salmonella

Here are my suggestions:

Major:

Figure 1 is a scheme of the experimental protocol and is not a result. I suggest moving to the materials and methods section.
Figure 2: The turbidity of the wells is not observable in a photograph. I suggest changing the photo to a graph or a table.
The MIC is the reference method to determine hypochlorite tolerance. The resazurin method detects more resistant strains than the MIC method, so these strains are false resistant. It is a problem of sensitivity and specificity. In my opinion, the resazurin assay can be used as a screening test.
The MICs values of hypochlorite from resistant and sensitive strains are not clear. Please add them.
Table 1 could be summarized in percentages or numbers of sensitive, resistant, false sensitive, and false resistant strains by each method.
It would be useful to summarize the best conditions for the resazurin method (temperature, inoculum, time).
Which are the reference RFUs that differentiate between resistant and sensitive strains?

Author Response

Item 1. Figure 1 is a scheme of the experimental protocol and is not a result. I suggest moving to the materials and methods section.

Reply: As suggested, the Figure 1 was moved to the materials and methods section 2.5.

Item 2. Figure 2: The turbidity of the wells is not observable in a photograph. I suggest changing the photo to a graph or a table.

Reply: As suggested, the photo was revised as a graph.

Figure 4. Visual observation time of sodium hypochlorite tolerance in S. Enteritidis CVCC 1806 at a range of bacterial concentrations between 10¹ to 10⁷ CFU/mL. The final concentrations of the NaClO solution were 64 mg/L. (A) NaClO tolerance in colorimetric; (B) NaClO tolerance by turbidity changes.

Item 3. The MIC is the reference method to determine hypochlorite tolerance. The resazurin method detects more resistant strains than the MIC method, so these strains are false resistant. It is a problem of sensitivity and specificity. In my opinion, the resazurin assay can be used as a screening test.

Reply: The manuscript was revised in Table 1 to make it clarification as suggested (line 237).

Item 4. The MICs values of hypochlorite from resistant and sensitive strains are not clear. Please add them.

Reply: The sentence on “In the broth microdilution method, tolerance to NaClO at a minimum inhibitory concentration (MIC) above 128 mg/L was observed in 98.4% (63 out of 64) of our Salmonella isolates. Sensitive to NaClO at MIC = 128 mg/L was observed in 1.6% (1 out of 64). In the resazurin-based experiment, tolerance to NaClO at a MIC above 128 mg/L was observed in 100%” were added in lines 211-214 as suggested.

Item 5. Table 1 could be summarized in percentages or numbers of sensitive, resistant, false sensitive, and false resistant strains by each method.

Reply: The Table 1 has been revised as suggested.

Item 6. It would be useful to summarize the best conditions for the resazurin method (temperature, inoculum, time).

Reply: As suggested, the best conditions for the resazurin method were summarized in the Conclusion part (lines 245-246).

Item 7. Which are the reference RFUs that differentiate between resistant and sensitive strains?

Reply: The reference that differentiate between resistant and sensitive strains was 3.2´10⁵RFU. The information was added in lines 214-215.

Other revision: The Figures in the manuscript were improved to clearly communicate the research.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed some of the issues but others remain. The major concerns relate the the need for a rapid assay. In addition, total rather than free chlorine concentrations were reported with the consequence that differences in pH could explain the difference between the assay and microdilution approach. To be useful the authors need to report the free-chlorine concentration. Additional points are provided below

Line 14: Is there a correlation between chlorine tolerance and antibiotic resistance? Moreover, would exposing a culture to chlorine cause selection of tolerant cells?

Line 21: Unclear what point the authors are trying to say so the sentence should be re-written.

Line 26: What does identifiable mean within the context of the paper?

Line 29: How was tolerance defined and verified?

Line 42: What pH?

Line 45: Increase?

Line 53: What evidence has been published for microbes growing in chlorine solutions? What is considered excessive exposure? What is the link to AMR?

Line 56: What is considered a short time?

Line 79: Reduced rather then digested?

Line 83: Not synthesis but reduction. What enzyme would reduce resazurin?

Line 84: Would this include dormant and VBNC cells?

Line 89: Why is there a need for a rapid method? What actions could be taken based in the results.

Line 100: What was the original isolation source of the SE strain?

Line 113: What pH? Is the ppm total or free-chlorine?

Line 121: why not measure free-chlorine as this would be the active form?

Line 129: By no visible growth do the authors mean there was an Optical Density threshold?

Line 138: By visually recorded do the authors mean a subjective assessment of color change?

Line 163: The low sensitivity of the assay would suggest that 5 log CFU/ml is the lower level of detection. If 7 log CFU/ml was the initial level then only a 2 log reduction would be needed to achieve to designate the test as negative. The microdilution test would be more sensitive.

Line 172: Would cell lysis occur with chlorine? The growth of Salmonella would be inhibited in any event.

Line 194: Would 1h make a difference?

Line 211: Should mention that the concentration is total chlorine not free-chlorine. Was the pH of the dye assay the same as the microdilution assay? This would impact on the free-chlorine concentration.

Line 224: This part would be better placed in the Introduction.

Table 1: The intensity had high SD values. Were statistics performed on the data?

Include a paragraph on the limitations of the study.

Comments on the Quality of English Language

could be improved

Author Response

Responses to Reviewers’ Comments

Dear editors,

Thank you for your letter with the reviewers’ comments on our manuscript entitled “Development of resazurin-based assay for rapid evaluation of sodium hypochlorite tolerance in Salmonella (foods-4088474)”. The authors have carefully reviewed the comments and revised the manuscript. The authors’ responses to the reviewers’ comments are listed below.

Xingning Xiao, Ph.D.

Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;

Tel: +86 13735593914

Qingli Dong, Ph.D.

University of Shanghai for Science and Technology, Shanghai, 200093, China;

Tel: +86 13585971239

Comments on reviewers:

The authors have addressed some of the issues but others remain. The major concerns relate the need for a rapid assay. In addition, total rather than free chlorine concentrations were reported with the consequence that differences in pH could explain the difference between the assay and microdilution approach. To be useful the authors need to report the free-chlorine concentration. Additional points are provided below

Item 1. Line 14: Is there a correlation between chlorine tolerance and antibiotic resistance? Moreover, would exposing a culture to chlorine cause selection of tolerant cells?

Reply: Previous studies reported that exposure to chlorine resulted in cross-resistance to ceftiofur, tetracycline, ciprofloxacin, and florfenicol. Expression of multidrug efflux pumps is a major mechanism mediating such cross-resistance. Similarly, pretreatment of S. Enteritidis with chlorine, sodium nitrite, sodium benzoate or acetic acid induced the over-expression of the marRAB operon, a global antibiotic resistance regulator involved in the production of AcrAB efflux pumps that extrude antibiotics. Therefore, using chlorine disinfectant could provide selection pressure for strains that acquire antibiotic resistance.

Item 2. Line 21: Unclear what point the authors are trying to say so the sentence should be re-written.

Reply: The sentence was revised as “The colorimetric method could clearly detect bacterial tolerance to NaClO during 7 hours of incubation. In contrast, the broth microdilution method, which relies on measuring turbidity, failed to detect the tolerance” in lines 20-22.

Item 3. Line 26: What does identifiable mean within the context of the paper?

Reply: The word “identifiable” was deleted and the sentence was revised as “Our research showed that the resazurin-based method, which uses fluorescence intensity as an indicator of NaClO tolerance, could detect Salmonella concentrations ranging from 10⁴ to 10⁷ CFU/mL” to make it clarification (lines 25-27).

Item 4. Line 29: How was tolerance defined and verified?

Reply: Tolerance refers to a microorganism’s ability to survive within one hour exposure to chlorine at typically lethal concentrations (lines 56-57). Salmonella Enteritidis CVCC 1806 (S. Enteritidis CVCC 1806) was used as quality control strain (MIC = 128 mg/L) (lines 113-114). Preliminary tests indicated the MIC of S. Enteritidis CVCC 1806 to NaClO was 128 mg/L and tolerance to NaClO was defined as > 128 mg/L.

Wang, W.; Xiao, X.; Wang, H.; Wang, S.; Xiao, Y.; Yang, H.; Hou, W.; Wang, W. Characterization and Comparative Transcriptome Analyses of Salmonella Enterica Enteritidis Strains Possessing Different Chlorine Tolerance Profiles. LWT 2022, 169, 113945, doi:10.1016/j.lwt.2022.113945.

Item 5. Line 42: What pH?

Reply: The pH usually ranges from 10 to 12.

Item 6. Line 45: Increase?

Reply: The sentence was revised as “With increasing NaClO concentration (0–200 mg/L), the viable count of pathogenic bacteria decreased significantly after a 5-minute treatment” in lines 44-46.

Item 7. Line 53: What evidence has been published for microbes growing in chlorine solutions? What is considered excessive exposure? What is the link to AMR?

Reply: During the sanitizing procedure, bacteria can endure and proliferate despite the presence of residual disinfectants, thereby earning the designation of being tolerant. In our previous study reported that the associations between NaClO and aminoglycosides, quinolone, polymyxin resistance. Besides, the observations from metagenome sequencing were found between disinfectant resistance genes and ARGs related to aminoglycosides, quinolone, polymyxin.

Xiao, X.; He, M.; Ma, L.; Lv, W.; Huang, K.; Yang, H.; Li, Y.; Zou, L.; Xiao, Y.; Wang, W. Insights into Microbial Contamination and Antibiotic Resistome Traits in Pork Wholesale Market: An Evaluation of the Disinfection Effect of Sodium Hypochlorite. Journal of Hazardous Materials. 2024, 468, 133811, doi:10.1016/j.jhazmat.2024.133811.

Item 8. Line 56: What is considered a short time?

Reply: Processed within 1 hour (lines 56-57).

Item 9. Line 79: Reduced rather then digested?

Reply: It was revised in line 79.

Item 10. Line 83: Not synthesis but reduction. What enzyme would reduce resazurin?

Reply: The word “synthesis” was revised as “reduction” in line 83. The enzymes, cytosolic and mitochondrial dehydrogenases, would reduce resazurin.

Item 11. Line 84: Would this include dormant and VBNC cells?

Reply: The resazurin reduction assay primarily detects metabolically active cells and may not respond to dormant or viable but non-culturable (VBNC) cells.

Item 12. Line 89: Why is there a need for a rapid method? What actions could be taken based in the results.

Reply: The broth microdilution method is the reference method to determine hypochlorite tolerance. The resazurin assay can be used as a screening test (line 256). The chlorine tolerance of foodborne bacteria has emerged as a critical concern in food safety prevention. There is an urgent necessity for research on novel intervention strategies on chlorine-tolerant pathogen, such as the screening or synthesis of broad-spectrum, highly effective bacteriophages, and the development of revolutionary antibacterial materials driven by light, heat, or friction (lines 260-264).

Item 13. Line 100: What was the original isolation source of the SE strain?

Reply: S. Enteritidis CVCC 1806 was isolated from poultry meat.

Item 14. Line 113: What pH? Is the ppm total or free-chlorine?

Reply: The pH was 12. It refers total (available) chlorine (line 126).

Item 15. Line 121: why not measure free-chlorine as this would be the active form?

Reply: The effectiveness of sodium hypochlorite in the cleaning and disinfection processes depends on the concentration of total (available) and the pH of the solution. The free and combined available chlorine in water are collectively described as total residual (available) chlorine. In the study of Yang et al., (2002), A hypochlorite stock solution (containing 5.88 mg/mL hypochlorite) was used to obtain 50 ppm of total chlorine in chill water to study cross-contamination by Campylobacter jejuni and Salmonella Typhimurium.

Item 16. Line 129: By no visible growth do the authors mean there was an Optical Density threshold?

Reply: The Optical Density threshold was 0.2 and the information was added in line 143.

Item 17. Line 138: By visually recorded do the authors mean a subjective assessment of color change?

Reply: The sentence on “The color of the wells was recorded through a subjective assessment of color change” was revised in lines 150-151.

Item 18. Line 163: The low sensitivity of the assay would suggest that 5 log CFU/mL is the lower level of detection. If 7 log CFU/mL was the initial level then only a 2 log reduction would be needed to achieve to designate the test as negative. The microdilution test would be more sensitive.

Reply: The broth microdilution method is the reference method to determine hypochlorite tolerance. The resazurin assay can be used as a screening test.

Item 19. Line 172: Would cell lysis occur with chlorine? The growth of Salmonella would be inhibited in any event.

Reply: According to our previous study, ultrastructural analyses indicated the presence of severe cell ruptures for S. Enteritidis CVCC 1806 following NaClO treatment and cytoplasmic contents were displaced.

Item 20. Line 194: Would 1h make a difference?

Reply: Our research showed that the resazurin-based method, which uses fluorescence intensity as an indicator of NaClO tolerance, could observe the results in a 1 h incubation period. When utilizing the changes of colorimetric (subjective assessment of color change) as the indicator of NaClO tolerance, the changes need to observe after 2 h. The sentence was added in lines 207-208.

Item 21. Line 211: Should mention that the concentration is total chlorine not free-chlorine. Was the pH of the dye assay the same as the microdilution assay? This would impact on the free-chlorine concentration.

Reply: The response to the comments on “total chlorine” was explained in Item 15. In the present study, the effect of pH was not considered. Future work will involve measuring the pH to address this factor (lines 243-244).

Item 22. Line 224: This part would be better placed in the Introduction.

Reply: As suggested, it was moved to the Introduction (lines 85-97).

Item 23. Table 1: The intensity had high SD values. Were statistics performed on the data?

Reply: The Coefficient of Variation (CV%) of the data was calculated. The results showed that the CV value is less than 10%, indicating the data are relatively stable. Besides, the other reviewer had the comment in Table 1 “I suggested summarizing Table 1 in percentages or numbers of sensitive, resistant, false sensitive, and false resistant strains by each method and the individual samples need to remove from the table. Therefore, the Table 1 has been revised in lines 248-250.

Item 24. Include a paragraph on the limitations of the study.

Reply: Several limitations became apparent during our study. The limited sample size of our collection may be seen as a constraint, necessitating more investigations with a more extensive array of Salmonella isolates to further substantiate the test's accuracy. The effectiveness of sodium hypochlorite in the cleaning and disinfection processes depends on the pH of the solution. In the present study, the effect of pH was not considered. Future work will involve measuring the pH to address this factor. The resazurin reduction assay primarily detects metabolically active cells and may not respond to dormant or viable but non-culturable (VBNC) cells. The limitations of the study were added in lines 239-246 as suggested.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript number: Foods 4088474v2

Title: Development of resazurin-based assay for rapid evaluation of sodium hypochlorite tolerance in Salmonella

Here are my suggestions:

Major:

In my previous revision, I suggested changing Figure 2 because the turbidity of the wells is not observable in a photograph. The authors replace it with a scheme analogous to the photo, but it is still not clear. Please replace with a graph of OD 544 nm vs. time.
The resazurin method detects more resistant strains than the MIC method, so these strains are false resistant. It is a problem of sensitivity and specificity. In my opinion, the resazurin assay can be used as a screening test. I suggest adding a phrase endorsing its use as a screening method.
In my previous revision, I suggested summarizing Table 1 in percentages or numbers of sensitive, resistant, false sensitive, and false resistant strains by each method. The authors added this information; the individual samples are still in the table. Please remove them.
The strains tested in the manuscript are the 96% resistant to hypochlorite. To evaluate the suitability of the resazurin method vs the MIC method, it would be more accurate to have a larger sample and a greater number of sensitive ones. If it is not possible to increase the total number of samples and the sensitive ones, a phrase mentioning that should be added.

Author Response

Responses to Reviewers’ Comments

Dear editors,

Xingning Xiao, Ph.D.

Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;

Tel: +86 13735593914

Qingli Dong, Ph.D.

University of Shanghai for Science and Technology, Shanghai, 200093, China;

Tel: +86 13585971239

Comments on reviewers:

Item 1. In my previous revision, I suggested changing Figure 2 because the turbidity of the wells is not observable in a photograph. The authors replace it with a scheme analogous to the photo, but it is still not clear. Please replace with a graph of OD 544 nm vs. time.

Reply: A graph of OD 544 nm vs. time was added in the revised manuscript as suggested (line 141, lines 198-201).

Item 2. The resazurin method detects more resistant strains than the MIC method, so these strains are false resistant. It is a problem of sensitivity and specificity. In my opinion, the resazurin assay can be used as a screening test. I suggest adding a phrase endorsing its use as a screening method.

Reply: As suggested, the sentence on “Evaluation method of sodium hypochlorite tolerance in Salmonella using resazurin was developed and can be used as a screening test.” has been added in lines 255-256.

Item 3. In my previous revision, I suggested summarizing Table 1 in percentages or numbers of sensitive, resistant, false sensitive, and false resistant strains by each method. The authors added this information; the individual samples are still in the table. Please remove them.

Reply: As suggested, the individual samples were removed from Table 1 (line 248).

Item 4. The strains tested in the manuscript are the 96% resistant to hypochlorite. To evaluate the suitability of the resazurin method vs the MIC method, it would be more accurate to have a larger sample and a greater number of sensitive ones. If it is not possible to increase the total number of samples and the sensitive ones, a phrase mentioning that should be added.

Reply: The sentence on “The limited sample size of our collection may be seen as a constraint, necessitating more investigations with a more extensive array of Salmonella isolates to further substantiate the test's accuracy” were added in lines 239-241.

Author Response File: Author Response.pdf