A novel Enrichment-free, Low-volume filtration and Rapid lysis method (ELR) in combination with real-time PCR for detection of Shiga toxin-producing Escherichia coli (STEC) in water
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Consequences of Shiga toxin-producing Escherichia coli (STEC) infection can range in severity from asymptomatic infection to haemolytic uraemic syndrome (HUS), renal failure, and death. Groundwater-derived drinking water is an important route for STEC transmission. Detection of STEC in water is crucial for timely response and public health interventions, however currently used culture-based methods are time-consuming and laborious. Therefore, there is a need for rapid methods that maintain high sensitivity and specificity. We describe a novel sensitive, enrichment-free water filtration method using a convenient sample volume (100 mL) to detect DNA markers of STEC serogroups and virulence factors within 6 hours. Quantitative real-time Polymerase Chain Reaction (qPCR) was used to detect and quantify the most common STEC infection-associated serogroups globally, O157 and O26. Real-time PCR was used to detect genetic determinants of STEC virulence (stx1,stx2 and eae genes) and specific marker genes for the clinically relevant serogroups O111, O103, O145 and O104. Results showed that the novel method can detect as low as 5 CFU/mL of STEC in water. The limit of detection for O157 and O26 qPCR assays was 2 and 6 copies, respectively. Groundwater and surface water samples (n=28) were collected and processed using the novel method. STEC O157 and O26 serogroups were detected in 23/28 (82.1%) samples (mean 5.2x104 copies/reaction) and 19/28 (67.9%) samples (mean 7.83x104 copies/reaction), respectively. Shiga toxin genes stx1 or stx2 were detected in 15/28 (53.6%) and 9/28 (32.1%) samples, respectively. Virulence factor intimin gene eae was detected in 24/28 (85.7%) samples. STEC serogroups O111, O103, O145 and O104 were detected in 15/28 (53.6%), 10/28 (35.7%), 11/28 (39.3%) and 15/28 (53.6%) samples, respectively. This novel method reproducibly detects low copies of STEC in low volume fresh water and has the potential to be used for detection and quantification of waterborne bacterial pathogens.
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One reviewer has raised concerns that the validation of this novel method is insufficient, as it has only been compared with the capE method, and not any commercially available kits. Please respond to this in your revisions. Please address each reviewer comment point by point, and provide a tracked changes version of the manuscript to flag any revisions.
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Comments to Author
The manuscript generally meets the requirements of the target journal, including structure, formatting, and scope. It presents a novel method for detecting STEC in water. The research has clear implications for improving water quality monitoring and public health interventions. In this study, the authors have developed and validated a new method (ELR) for detecting STEC in water samples. The ELR method offers a rapid approach to STEC detection in water by combining enrichment-free, low-volume filtration with rapid lysis and real-time PCR. The comparison with the existing CapE method further strengthens the study's validity by providing a benchmark for performance evaluation. The following questions need to be answered before acceptance. 1. The author compared the ELR method with the CapE method, …
Comments to Author
The manuscript generally meets the requirements of the target journal, including structure, formatting, and scope. It presents a novel method for detecting STEC in water. The research has clear implications for improving water quality monitoring and public health interventions. In this study, the authors have developed and validated a new method (ELR) for detecting STEC in water samples. The ELR method offers a rapid approach to STEC detection in water by combining enrichment-free, low-volume filtration with rapid lysis and real-time PCR. The comparison with the existing CapE method further strengthens the study's validity by providing a benchmark for performance evaluation. The following questions need to be answered before acceptance. 1. The author compared the ELR method with the CapE method, which was developed in the lab previously. I would suggest you compare the ELR method with one method using a commercial kit or culture-based methods 2. Table 2 presents the detection and quantification results of the ELR and CapE methods for STEC O157 and O26 serogroups. While the data are informative, it would be beneficial to include basic water quality parameters (such as pH, temperature, and turbidity) for the samples analyzed. This additional information would provide a more comprehensive understanding of the environmental conditions that may influence STEC presence and detection. Moreover, would the water quality impact the results from the ELR method? The authors need to clarify this. 3. In the discussion section, the authors compare the ELR method with the CapE method. This comparison could be expanded to include a more detailed discussion of the implications of the observed differences in STEC O157 and O26 copy numbers between the two methods and how these differences might affect the interpretation of water quality results. 4. Please check out the format of the manuscript, including Figures and Tables. 5. Please clarify whether this approach is economically feasible.
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?
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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Comments to Author
The authors present a novel method for enrichment-free detection of STEC, specifically in Irish groundwater samples. The study describes a filtration concentration method followed by qPCR detection of virulence/serotype genes. The authors provide a limit of detection for O157 and O26 serotypes. This method appears promising, with applications for ground water detection and beyond. These potential applications are succinctly described in the discussion. I have some recommendations: 1) Line 29: insert comma after novel 2) Line 53: insert carriage after asymptomatic 3) Line 292-298: Here, the influence of culture bias in detection is discussed, in reference to Conrad et al., (2016). I feel this could be expanded- definining what the 'big six' non-o157 serotypes are and what the percentages mean in the …
Comments to Author
The authors present a novel method for enrichment-free detection of STEC, specifically in Irish groundwater samples. The study describes a filtration concentration method followed by qPCR detection of virulence/serotype genes. The authors provide a limit of detection for O157 and O26 serotypes. This method appears promising, with applications for ground water detection and beyond. These potential applications are succinctly described in the discussion. I have some recommendations: 1) Line 29: insert comma after novel 2) Line 53: insert carriage after asymptomatic 3) Line 292-298: Here, the influence of culture bias in detection is discussed, in reference to Conrad et al., (2016). I feel this could be expanded- definining what the 'big six' non-o157 serotypes are and what the percentages mean in the context, which I don't think is currently very clear. Are some serotypes more difficult to isolate than others in general, or only following enrichment? Finally, the figures and tables accuratley report the authors' findings, but I feel a colour scheme would make both Table 1 and 2 more easy to interperate.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
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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