Dynamics and virulence of Enterobacteriaceae reservoirs harboring bla CTX-M group 1 in community wastewater

ABSTRACT Extended-spectrum beta-lactamase (ESBL)-producing bacteria are ubiquitous and can cause serious infections. Here, we examined untreated community wastewater influent as a reservoir for bla CTX-M group 1 organisms and their virulence potential. Raw influent samples ( n = 268) were collected from four wastewater treatment plants (WWTPs) representing dense urban populations. We observed that bla CTX-M group 1 levels were high at all WWTPs and only ~1–2 log 10 lower and not correlated to common human-specific microbiome fecal markers, Lachno3 and HF183, indicating a lack of connection to human fecal inputs. Concentrations of bla CTX-M group 1 genes and markers for presumptive host organisms Escherichia coli and Klebsiella pneumoniae were influenced by travel time and season. Amplicon sequencing revealed high diversity of bla CTX-M group 1–9 genes, with 63% belonging to group 1. Selective culture and 16S rRNA gene sequencing showed bla CTX-M group 1 isolates were 26% E. coli , 26% K. pneumoniae , 40% other Enterobacteriaceae , and 8% Aeromonas . Overall, E. coli averaged 3.6E7 cells/L, with 3% of all E. coli found to contain bla CTX-M group 1. Whole-genome sequencing of bla CTX-M group 1 E. coli from wastewater revealed resistance and virulence gene profiles similar to clinical isolates and distinct from other wastewater ESBL-resistant and non-resistant E. coli . Interpretation of wastewater data needs to consider both the existence of environmental reservoirs that contain potentially pathogenic organisms and the strong influence the dynamics of the conveyance system can have on final concentrations measured at the WWTP. IMPORTANCE The CTX-M enzyme family is highly abundant in nosocomial, community, and environmental settings and is leading to treatment of infections with carbapenem antibiotics, a last-line therapeutic option. The progressive increase of the clinically relevant bla CTX-M group 1 resistance genes in the human population warrants investigation, particularly to understand the establishment and dynamics of environmental reservoirs. This study utilized molecular and culture methods to gain insight into the possible origin, abundance, and dynamics of bla CTX-M group 1 genes in untreated wastewater influent samples. We found extremely high levels of these genes, with Escherichia coli as a major host organism that closely resembled clinical strains, suggesting they are seeded and propagate in sewer pipe systems. The significance of our research is in developing approaches to monitor antimicrobial resistance reservoirs in community wastewater, which could shed light on global burdens and potential transmission cycles and indicate increasing inputs of clinically relevant strains originating from human populations.