Clark Appling, Mark D Sobsey , Lisa M. Durso, Michael B. Fisher 

Antimicrobial resistance (AMR) is a growing threat to human and animal health, and efforts to combat it require widespread, robust and practical monitoring of AMR presence in humans, animals, and the environment. Because early AMR monitoring efforts were cumbersome, costly, and lacked standardization, the WHO Tricycle Protocol (WHO TP) was developed and released in 2021 to standardize and streamline global AMR monitoring by culturing a single indicator organism, extended-spectrum--lactamase-producing Escherichia coli (ESBL-Ec), The WHO TP culture-based method detects and quantifies ESBL-Ec by either spread plating or membrane filtration on either MacConkey or TBX agar (supplemented with cefotaxime), which are difficult methods to use in low-resource and field settings, and must be done mostly in lab settings by trained personnel. Therefore, we simultaneously detected and quantified ESBL-Ec in field samples using the WHO TP with membrane filtration (WHO TP MF) and also a simplified method, the compartment bag test (CBT), which quantifies different sample volumes as positive or negative for target bacteria and is easy for anyone to use in the field. 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 WHO TP MF on TBX agar supplemented with cefotaxime, 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 (for antibiotic sensitivity) and EnteroPluri tests (to speciate isolates). The WHO TP MF and the CBT methods were both easy to use, but the MF test 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 in both locations, suggesting that treated wastewater is a source of ESBL-Ec in some surface waters. The results of CBT and WHO TP MF tests were comparable, making the former method suitable as an alternative to the more complex WHO TP MF procedure. Further AMR surveillance using both the WHO TP MF and simpler CBT methods may be useful to further characterize and refine their performance for quantifying AMR occurrence in NC and elsewhere.