Bactericidal effect of chlorous acid water against enterohaemorrhagic Escherichia coli
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Background. Enterohaemorrhagic Escherichia coli (EHEC) is an important aetiologic agent of foodborne illness. Since the main source of EHEC is contaminated meat and fresh produce, these ingredients should be sanitized using methods that achieve efficient mitigation of bacterial load under organic-matter-rich conditions.
Materials and Methods. We examined the effect of chlorous acid water (CAW), which is a chlorous acid (HClO 2 )-based disinfectant, on seven major serotypes of EHEC, including O157, O111, O26, O91, O103, O121 and O145, and compared its bactericidal activity with that of sodium hypochlorite (NaClO).
Results. Both sanitizers rapidly inactivated all EHEC strains to below the detection limit at low free available chlorine (FAC) concentrations (5–10 mg l −1 ). However, organic matter significantly hampered the bactericidal activity of NaClO. In the presence of simulated blood or excretion contamination, CAW demonstrated superior stability and sustained bactericidal efficacy compared with NaClO. While NaClO lost its activity rapidly due to the consumption of FAC by organic matter, CAW maintained an effective concentration and achieved significant bacterial reduction. Furthermore, in EHEC biofilm assays, CAW exhibited potent bactericidal activity, achieving a >7-log reduction of viable cells within the biofilm, whereas NaClO showed limited efficacy under the same conditions.
Discussion. The results suggest that CAW is a robust alternative to NaClO, particularly in environments with high organic burdens and biofilm contamination, such as food processing facilities and healthcare settings.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature.
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Comments to Author
I thank the authors for their careful and extensive revision of the manuscript and for the detailed responses provided. The revised version represents a clear and substantial improvement over the original submission. Several of the major concerns raised previously have been addressed in a meaningful way. The inclusion of additional non-O157 EHEC serotypes significantly strengthens the study and allows the conclusions to be framed with greater confidence within the diversity of clinically relevant EHEC. In particular, the addition of biofilm experiments is a major improvement. The demonstration of substantially greater efficacy of chlorous acid water compared with sodium hypochlorite against biofilm-associated EHEC addresses a key limitation of the original manuscript and adds important practical …
Comments to Author
I thank the authors for their careful and extensive revision of the manuscript and for the detailed responses provided. The revised version represents a clear and substantial improvement over the original submission. Several of the major concerns raised previously have been addressed in a meaningful way. The inclusion of additional non-O157 EHEC serotypes significantly strengthens the study and allows the conclusions to be framed with greater confidence within the diversity of clinically relevant EHEC. In particular, the addition of biofilm experiments is a major improvement. The demonstration of substantially greater efficacy of chlorous acid water compared with sodium hypochlorite against biofilm-associated EHEC addresses a key limitation of the original manuscript and adds important practical relevance, given the central role of biofilms in both food processing and healthcare environments. The tone and balance of the Discussion have also improved considerably. Claims regarding applicability are now more cautious and scientifically defensible, with CAW appropriately positioned as a promising agent for risk reduction rather than a standalone sterilant. I welcome the clearer acknowledgment of regulatory constraints, experimental limitations, and environmental context, as well as the more transparent discussion of industry funding. Together, these changes reduce the promotional tone noted previously and improve the overall credibility of the work. Concerns regarding environmental realism and novelty have been reasonably addressed. While the work remains laboratory-based and incremental, the authors now clearly articulate the specific contributions of the study, particularly the expanded serotype profiling, comparative behaviour under different organic load conditions, and superior activity against biofilm-embedded cells. The mechanistic discussion has also been appropriately tempered and framed as hypothesis-generating rather than definitive. Overall, the manuscript has been substantially strengthened and now presents a coherent, balanced, and methodologically sound evaluation of chlorous acid water against EHEC under defined conditions. I am satisfied that the major scientific concerns have been adequately addressed and that the conclusions are appropriately supported by the data.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
The authors present a laboratory study comparing the bactericidal effect of chlorous acid water (CAW) and sodium hypochlorite (NaClO) against three EHEC serogroups (O157, O26, O111). They conclude that CAW is more effective than NaClO under organic-matter-rich conditions and suggest applications in food sanitation and healthcare. While the experiments are competently executed, the scope is narrow, the novelty limited, and the interpretation overstates the significance of the findings. The paper would benefit from substantial revision before being considered for publication. Major Concerns Limited Strain Selection Only three EHEC strains were tested. This is insufficient to make broad claims about CAW's superiority, particularly since illness is caused by a wider range of serogroups (O103, O121, O145, …
Comments to Author
The authors present a laboratory study comparing the bactericidal effect of chlorous acid water (CAW) and sodium hypochlorite (NaClO) against three EHEC serogroups (O157, O26, O111). They conclude that CAW is more effective than NaClO under organic-matter-rich conditions and suggest applications in food sanitation and healthcare. While the experiments are competently executed, the scope is narrow, the novelty limited, and the interpretation overstates the significance of the findings. The paper would benefit from substantial revision before being considered for publication. Major Concerns Limited Strain Selection Only three EHEC strains were tested. This is insufficient to make broad claims about CAW's superiority, particularly since illness is caused by a wider range of serogroups (O103, O121, O145, O91, etc.). Without a broader panel or cross-species comparison, the conclusions are overstated. Artificial Experimental Models The bean sprout assay is highly artificial: autoclaved sprouts inoculated with E. coli in pure culture do not mimic real-world produce surfaces where complex microbiota and biofilms play a major role. The absence of any biofilm work is a serious omission given its central relevance in food safety and healthcare. Regulatory and Practical Relevance The maximum reported reduction (>3 log10 on sprouts after 30 minutes) falls short of regulatory standards for food safety, which typically require greater log reductions. This should not be described as "suitable for food processing." Claims of applicability are premature. Conflict of Interest and Balance The study is funded by the manufacturer of CAW, yet the discussion reads as promotional, emphasising strengths while downplaying limitations. Greater caution and balance are required; otherwise, the paper risks being perceived as biased. Environmental Realism All testing was performed at 25 °C in controlled laboratory conditions. This does not approximate food production environments (cold chain, variable contamination) or healthcare settings. Without testing in more realistic conditions, claims of utility outside the lab are unconvincing. Novelty and Contribution Comparative studies of CAW versus NaClO are already available in the literature, including reports of stability under organic load. The novelty here is incremental at best. The manuscript would need to clearly define what new knowledge it contributes beyond confirming known properties of CAW. Minor Concerns The mechanistic discussion of chloroperoxy radicals is speculative. No direct measurement was performed in this study. It should be framed more cautiously. Statistical reporting is basic; effect sizes, replicates, and confidence intervals should be provided. Sole reliance on p-values is not sufficient. The terminology ("all-or-nothing" for NaClO) is imprecise and should be replaced with more scientific language. The introduction under-represents the broad range of alternative disinfectants (peracetic acid, electrolysed water, hydrogen peroxide) that are already used in food production. This weakens the rationale for focusing exclusively on CAW. Figures are verbose and would benefit from simplification. Raw data in supplementary files should be integrated into the results more clearly. No mention is made of CAW's cost, stability, safety, or compatibility with food and equipment surfaces, all of which are critical for adoption.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Line commentary: - Methods: - Overall, it should include the companies where the materials were taken from and their main branch location. - Line 101-102: More information about the bacterial strains used would be best. - Line 114-121: Have different conditions in a table, not as individual lines. Then finish the paragraph by discussing all four conditions. - Line 128-140: Needs to be in paragraph format and have a flow through rather than bullet points listed as a protocol. - Line 125: When it means FAC is above guaranteed value, is this good or bad for consumers then? Is it good to have a chlorine concentration value above the recommended threshold? - Line 141: Rationale for 25C? Is this the temperature of slaughterhouses or packaging areas? - Line 147: I am unsure what drug treatment was …
Comments to Author
Line commentary: - Methods: - Overall, it should include the companies where the materials were taken from and their main branch location. - Line 101-102: More information about the bacterial strains used would be best. - Line 114-121: Have different conditions in a table, not as individual lines. Then finish the paragraph by discussing all four conditions. - Line 128-140: Needs to be in paragraph format and have a flow through rather than bullet points listed as a protocol. - Line 125: When it means FAC is above guaranteed value, is this good or bad for consumers then? Is it good to have a chlorine concentration value above the recommended threshold? - Line 141: Rationale for 25C? Is this the temperature of slaughterhouses or packaging areas? - Line 147: I am unsure what drug treatment was used, is it not a chemical treatment? - Line 155: why were the bean sprouts autoclaved? Rationale would be needed, as a simple UV exposure could kill/reduce most of the bacterial load to test. As autoclaving doesn't get all spores either unless doing low pressure. - Line 166: Consider input from the table indices about how means were calculated to be added to the methods. Also, including how many trials were conducted is important for replication events. - Where is the negative control? Such as bacterial strains just growing and being tested at these concentrations? - Results: - Line 177: Supplementary table 1: where are the other time points for Organic Matter free? Such as 5, 10, 30, and 60 minutes? - Line 174-177: Would suggest reporting all findings and numbers. - Line 224: mention of a 10 minute sampling result, but not exactly what that result was. Overall, need to report on all findings then potentially wrap up each paragraph with the key finding for that experiment. - Intro/discussion: - line 76-78: Include additional rationale for why it is important/different from other sanitizers. - From a food safety and public health point-of-view, is this safe for retailers to sell to consumers post treatment? - Discussion: do not need to cite the figures, as you have done that in the results. - Line 237-251: This group of lines seem more like results rather than discussion with current literature about finding similar results. - Line 263: I would like to know the exact levels of FAC that are acceptable, especially in the Introduction to understand that the research conducted is safe to be used in the industry. Summary: The study, Bactericidal effect of chlorous acid water against enterohemorrhagic Escherichia coli, focused on three major foodborne serogroups of E. coli against chlorous acid water treatment. Several conditions were tested against CAW and another chemical agent, NAClO, where results showed promise with CAW being used in the food and packaging industry. However, the study needs to focus on rationalizing their methods and making clear the testing they conducted with number of trial events and controls used. Results had a major message about reduction but felt lacking with all that was reported compared to the supplementary tables and general figures. Discussion needs to include more literature analysis to wrap up their findings with previous studies to make consistent conclusions or relevant connections. Overall, I found this study promising to be used in the industry but there needs to additional work to be considered complete.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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