Comparative Genomics of Multidrug-Resistant Enterococcus spp. isolated from Wastewater Treatment Plants

Background Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. E. faecalis and E. faecium are recognized indicators of fecal contamination in water. Genome comparisons of enterococci isolated from a conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs were conducted. Results VRE isolates, including E. faecalis (n=24), E. faecium (n=11), E. casseliflavus (n=2) and E. gallinarum (n=2), were selected for sequencing based on source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to mobilome had a positive correlation with genome size in E. faecium (p < 0.001) and E. faecalis (p < 0.001) and with the number of AMR genes in E. faecium (p = 0.005). The AMR phenotype generally aligned with genotype. Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent groups of functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum genomes, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on source but not based on WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers. Conclusions There was no discernable difference between enterococcal genomes from the CAS and BAF WWTPs either before or after treatment. Understanding the impact of WWTPs on the dissemination of AMR in the environment will require knowledge of the mobility and upregulation of genes and the characterization of mobilomes within WWTPs and surrounding environments.