Identification and whole genome analysis of cefotaxime resistant Escherichia coli recovered from a tertiary treatment wastewater treatment plant discharge point

Wastewater treatment plants (WWTPs) act as reservoirs for antibiotic resistant bacteria (ARB) and may facilitate transmission of antibiotic resistance genes (ARGs) between bacterial populations including pathogens that in turn may persist in wider aquatic environments. ESBL-producing Escherichia coli (E. coli) cause both hospital and community-acquired infections globally and may serve as a proxy of a wider problem. The aim of this research was to investigate the abundance and diversity of ESBL-producing E. coli released from a municipal WWTP to the river Almond in West Lothian, Scotland. Further, whole genome sequence (WGS) analysis of ESBL-producing E. coli isolates was undertaken to gain insight to the potential contribution of this WWTP to the resistome in the adjacent aquatic environment. A culture-based study found approximately 2.0 x102 CFU ml-1 cefotaxime resistant E. coli in the effluent outflow from the WWTP. Isolates were confirmed as E. coli by gadA and uidA targeted primers and further characterised to five E. coli phylogroups (B1, B2, C, D, and F) and 5 REP-PCR types. Representatives of each REP type were confirmed to carry Group 1 and 9 CTX-M enzymes. All but 1 isolate exhibited multi drug resistance (MDR) when challenged with representatives from different antibiotic classes. Whole genome sequencing (WGS) and analysis was performed on four resistant E. coli isolates. ARGs, mobile genetic elements, and genetic lineages were identified using web-based bioinformatic tools. All resistant strains were confirmed to carry CTX-M genes. ARGs conferring resistance to beta-lactams, aminoglycosides, macrolides, fluoroquinolones, tetracyclines, folate pathway agonists, phenicols, and disinfectants were also identified amongst the resistant strains. ESBL resistance was mobilised, either by IS6 family composite transposons maintained in IncFII or Inc1I plasmids. Other MDR regions were mobilised by Int1 integrons. Importantly, 3 of the 4 isolates belong to globally recognised lineages associated with bacteraemia in humans, including sequence type (ST)131 and ST69. In conclusion, the presence of ESBL-producing, and indeed globally important multi-drug resistant E.coli strains in the effluent indicate a potential role of the WWTP in the dissemination of ARB. These findings support the case for active surveillance of ARB and ARGs released from municipal WWTPs.