Validation of a Synthetic Wastewater Matrix as a Proxy for Municipal Wastewater for In Vitro Studies of E. coli.

Antimicrobial resistance (AMR) is a major threat to global health and resistant bacterial populations have been observed to develop and spread in and around wastewater. However, in vitro studies on AMR development are typically conducted in ideal rich media conditions, which can differ in composition and nutrient density from wastewater. In this study, we compare the growth and AMR development of E. coli in a synthetic wastewater recipe to autoclaved wastewater samples from the Massachusetts Water Resources Authority (MWRA). We found that synthetic wastewater and real wastewater samples both supported less bacterial growth compared to LB broth and had similar susceptibility profiles to antibiotic pressure from Doxycycline, Ciprofloxacin and Streptomycin. More importantly, unlike in LB broth, AMR development over time during continuous passaging under subinhibitory antibiotic pressure in pH-adjusted synthetic wastewater was similar in fold change to real wastewater samples. Thus, we suggest that synthetic wastewater is a more accurate predictor of both E. coli growth and antibiotic resistance development in wastewater systems than standard nutrient broths such as LB. The validation of synthetic wastewater as a better proxy for real wastewater will allow for further recapitulation of the wastewater environment in future in vitro studies of AMR in these conditions.