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 and may also be present in biofilms associated with these environmental sources. This study investigates the presence of S. Typhi and blaCTX-M genes in drinking water, wastewater, and their associated biofilms. 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 reports 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. The 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.

The authors have declared no competing interest.

This work was supported by the Bill & Melinda Gates Foundation Grant Number OPP1217099.

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