Culture-based 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

Authors: APPLING, KEITH - University Of North Carolina; SOBSEY, MARK - University Of North Carolina; Durso, Lisa; FISHER, MICHAEL - University Of North Carolina

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/4/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary: Antibiotic resistance infections are a serious health threat, where bacteria no longer respond to medicines that used to work - resulting in longer treatment times or even death where once a patient would have survived. In response to this crisis, there is a global effort to investigate antibiotic resistant bacteria not only in hospitals and animal clinics, but also in the environment. Because early antibiotic resistance monitoring efforts were cumbersome, costly, and lacked standardization, the WHO Tricycle Protocol was developed and released in 2021 to standardize and streamline global monitoring by culturing a single indicator organism, one of the top priority antibiotic resistant pathogens for human medicine. While the Tricycle Protocol has been adopted successfully in some countries worldwide, it has not been piloted in the United States previously. This project had two aims: (1) to count and describe ESBL-EC in North Carolina through the implementation of WHO’s newly published Tricycle Protocol and to compare it to the Compartment Bag Test, a simpler method that can be used in field settings without the need for specialized equipment, refrigeration, or incubators. The results of The Tricycle Protocol and the Compartment Bag Test were comparable, and the simpler Compartment Bag Test appears suitable as an alternative to the more complex Tricycle Protocol. Further antibiotic resistance surveillance using both methods may be useful to further characterize and refine their performance to track antibiotic resistance occurrence in NC and elsewhere.

Technical Abstract: 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 hazards 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-beta-lactamase-producing Escherichia coli (ESBL-Ec), The WHO TP culture-based method detects and quantifies ESBL-Ec on either MacConkey or TBX agar, which are difficult to use in low-resource and field settings and used 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 simpler method, the compartment bag test (CBT) which is easy for anyone to use in the field. We collected and analyzed municipal wastewater, surface water, and poultry waste samples from sites in Raleigh and Chapel Hill, NC, over an 8-month period. Presumptive ESBL-Ec concentrations were quantified using WHO TP MF on TBX agar supplemented with cefotaxime, as well as the CBT with cefotaxime. Presumptive ESBL-Ec bacteria were isolated from completed tests and further confirmed and characterized by Kirby Bauer disk diffusion tests for antibiotic sensitivity and EnteroPluri tests to identify and speciate. The WHO TP MF and the CBT methods were both easy to use, but the former test required additional time and effort du3 to MF prior to plating. 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, and the former method appears 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 to analyze AMR occurrence in NC and elsewhere.