Characterization of antibiotic resistance development of E. coli in synthetic and real wastewater

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 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 standard LB broth to a synthetic wastewater recipe and 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. Additionally, bacteria grown in synthetic wastewater and real wastewater samples had differing susceptibility to antibiotic pressure from Doxycycline, Ciprofloxacin, and Streptomycin. However, AMR development over time during continuous passaging under subinhibitory antibiotic pressure was similar in fold change across all media types. Thus, we find that while LB can act as a proxy for wastewater for AMR studies in E. coli, synthetic wastewater is a more accurate predictor of both E.coli growth and antibiotic resistance development. Moreover, we also show that antibiotic resistance can develop in real wastewater samples and components within wastewater likely have synergistic and antagonistic interactions with antibiotics. ImportanceAntimicrobial resistance (AMR) ranks among the leading global threats to public health and development. In 2019, bacterial AMR was estimated to have directly caused 1.27 million deaths worldwide and contributed to 4.95 million deaths overall (Murray, C. J., et al., (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet, 399(10325), 629-655.). With estimations of AMR only getting worse, it is imperative that we understand the complex dimensionalities that drive the genesis of antimicrobial resistance to where it begins-the environment. The paper investigates bacterial growth and AMR in real wastewater samples and highlights the importance of using a media that closely mimics real wastewater in AMR studies, compared to standard lab media like LB broth. This is crucial for understanding how E. coli and other bacteria develop AMR in environments similar to actual wastewater, which can inform more effective strategies to combat AMR in natural and engineered settings.