Environmental monitoring of antimicrobial resistant bacteria in North Carolina water and wastewater using the WHO Tricycle protocol in combination with membrane filtration and compartment bag test methods for detecting and quantifying ESBL E. coli

Antimicrobial resistance (AMR) threatens human and animal health; effective response requires monitoring AMR presence in humans, animals, and the environment. The World Health Organization Tricycle Protocol (WHO TP) standardizes and streamlines global AMR monitoring around a single indicator organism, extended-spectrum-β-lactamase-producing Escherichia coli (ESBL-Ec). The WHO TP culture-based method detects and quantifies ESBL-Ec by spread-plating or membrane filtration on either MacConkey or TBX agar (supplemented with cefotaxime). These methods require laboratories and trained personnel, limiting feasibility in low-resource and field settings. We adapted the WHO TP using a simplified method, the compartment bag test (CBT), to quantify most probable numbers (MPN) of ESBL-Ec in samples. CBT methods can be used correctly in the field by typical adults after a few hours’ training. We collected and analyzed municipal wastewater, surface water, and chicken waste samples from sites in Raleigh and Chapel Hill, NC over an 8-month period. Presumptive ESBL-Ec were quantified using MF on TBX agar supplemented with cefotaxime (MF+TBX), as well as using the CBT with chromogenic E . coli medium containing cefotaxime. Presumptive ESBL-Ec bacteria were isolated from completed tests for confirmation and characterization by Kirby Bauer disk diffusion tests (antibiotic sensitivity) and EnteroPluri biochemical tests (speciation). Both methods were easy to use, but MF+TBX required additional time and effort. The proportion of E . coli that were presumptively ESBL in surface water samples was significantly greater downstream vs upstream of wastewater treatment plant (WWTP) outfalls, suggesting that treated wastewater is a source of ESBL-Ec in some surface waters. The CBT and MF+TBX tests provided similar (but not identical) quantitative results, making the former method suitable as an alternative to the more complex MF+TBX procedure in some applications. Further AMR surveillance using MF+TBX and/or CBT methods may be useful to characterize and refine their performance for AMR monitoring in NC and elsewhere.