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Review 2: "Detection of Salmonella Typhi and blaCTX-M Genes in Drinking Water, Wastewater, and Environmental Biofilms in Sindh Province, Pakistan"

Overall, both the reviewers find that this study builds on previous work on the role of biofilm as a pathogen reservoir in urban water systems. However, they expressed concerns about the strength of evidence to support its claims.

Published onMay 23, 2024
Review 2: "Detection of Salmonella Typhi and blaCTX-M Genes in Drinking Water, Wastewater, and Environmental Biofilms in Sindh Province, Pakistan"
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key-enterThis Pub is a Review of
Detection of Salmonella Typhi and blaCTX-M Genes in Drinking Water, Wastewater, and Environmental Biofilms in Sindh Province, Pakistan
Detection of Salmonella Typhi and blaCTX-M Genes in Drinking Water, Wastewater, and Environmental Biofilms in Sindh Province, Pakistan
Description

Abstract Typhoid fever poses a significant public health risk, particularly in low- and middle-income countries where access to clean water and improved sanitation may be limited. In Pakistan, this risk is especially serious given the emergence of an extensively drug-resistant (XDR) Salmonella Typhi strain, a strain attributed to S. Typhi acquisition of the blaCTX-M-15 gene. The now-dominant XDR S. Typhi strain, non-XDR S. Typhi, and blaCTX-M genes are readily disseminated via drinking water and wastewater in Pakistan and may also be present in biofilms associated with these environmental sources. This study investigates the presence of S. Typhi and blaCTX-M genes within these environmental compartments. Drinking water (n=35) or wastewater samples (n=35) and samples of their associated biofilms were collected from Karachi and Hyderabad, Pakistan. Samples were tested by PCR for S. Typhi and blaCTX-M group 1 genes as a proxy for blaCTX-M-15. Heterotrophic plate counts (HPC) were conducted to assess microbial load. S. Typhi was detected by PCR in one bulk wastewater sample and one drinking water biofilm. BlaCTX-M group 1 genes were detected in all sample types and were detected more frequently in bulk wastewater (n=13/35) than in drinking water (n=2/35) and more frequently overall in biofilm samples (n=22/70) versus bulk water (n=15/70). Detection of blaCTX-M in biofilm was not significantly associated with detection in the associated bulk water sample. This study marks the first detection of S. Typhi in drinking water biofilms and the first report of blaCTX-M genes in environmental biofilms in Pakistan. Environmental biofilms, particularly in drinking water systems, may serve as reservoirs for human exposure to S. Typhi and drug resistance genes. This study underscores the importance of expanding surveillance strategies to include biofilm sampling, providing valuable insights into pathogen dissemination in water systems, and informing targeted public health interventions to prevent waterborne diseases.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: This study investigates the presence of S. Typhi and blaCTX-M genes in drinking water, wastewater and the associated biofilm samples in Pakistan via PCR. Heterotrophic plate counts (HPC) were also conducted to assess microbial load. BlaCTX-M group 1 genes were found to appear more frequently in biofilm samples than bulk water, more in wastewater than in drinking water. Moreover, detection of blaCTX-M in biofilm was not significantly associated with detection in the associated bulk water sample. Microbial pollutants could be detected in biofilms while not in the associated bulk water sample. This indicate that environmental biofilms, particularly in drinking water systems, may serve as reservoirs for human exposure to pathogens and drug resistance genes. Hence, we should expand water surveillance strategies to include biofilm sampling to provide more insights into pathogen dissemination in water systems and inform public health interventions. The conclusions made in this manuscript could be justified and supported by the experimental testing results, but the PCR detections may subject to false positive or false negative. It is better to conduct duplicates or even triplicates for the PCR detection. Also, quantitative PCR (qPCR) was mentioned to detect S. Typhi genes, but seems the results was not shown in copy numbers in the preprint. If qPCR resource is available, it is recommended to apply qPCR to both targets to provide quantitative information.

This manuscript confirms previous work on the role of biofilm as a pathogen reservoir in urban water system (Li et al, ‘Impact of sewer biofilms on fate of SARS-CoV-2 RNA and wastewater surveillance’, Nature Water, vol. 1, no. 3, pp. 272-80; Zhang et al, ‘The Reduction of SARS-CoV-2 RNA Concentration in the Presence of Sewer Biofilms’, Water, vol. 15, no. 11, p. 2132; Morales et al, ‘Accumulation of SARS-CoV-2 RNA in sewer biofilms’, ACS ES&T Water, vol. 2, no. 11, pp. 1844-51.). However, this manuscript did not cite sufficient current literature or discuss limitations thoroughly. The work was presented clearly in the well-written manuscript, but Figure 2 may need some improvement. It looks more like a table in the current form. A major revision would be helpful to enhance evidencing the main claim with more statistical analysis, in-depth discussion, and relevant literature. Overall, the results and claims in this research are likely to be similar to the hypothetical ideal study. This work could be useful to prevent waterborne diseases and has the potential to impact the implementation of policy or programs, particularly in low- and middle-income countries.

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