Resolved genomes of wastewater ESBL-producing and metagenomic analysis of source wastewater samples.

Extended-spectrum beta-lactamase (ESBL)-producing pose a serious threat to human health because of their resistance to the most commonly prescribed antibiotics: penicillins and cephalosporins. In this study, we provide a genomic and metagenomic context for the determinant beta-lactam resistance genes of ESBL-positive isolated from various wastewater treatment utilities in Oregon, USA. Class A beta-lactamase genes on chromosomes (CTX-M, TEM) were clustered with antibiotic resistance genes associated with other classes of antibiotics (sulfonamides and aminoglycosides) along with insertional elements. ESBL genes such as CTX-M, TEM, and SHV were also detected on conjugable plasmids of IncF and IncI incompatibility types. One novel IncF plasmid (pSHV2A_ESBLF) was identified, which carried a multidrug resistance genotype (SHV-2A, , 3, 6, A, and 1) in addition to a (mercury resistance) operon, colicin, and aerobactin genes. Shotgun metagenomic analysis of the ESBL-producing -originating wastewater samples showed the presence of class A beta-lactamases; however, the ESBL genes identified in the genomes were below the detection limits. Other ESBL-associated genes (i.e., OXA.11, FOX.7, and GES.17) were identified in the wastewater samples, and their occurrences were correlated with the core microbial genera (e.g., ). In the genomes and wastewater samples, tetracycline, aminoglycoside, and beta-lactam resistance determinants frequently co-occurred. The combination of whole-genome and metagenomic analysis provides a holistic description of ESBL-producing organisms and genes in wastewater systems.IMPORTANCEUsing a hybrid sequencing and assembly strategy (short- and long-read sequencing), we identified the distribution of ARGs and virulence factors harbored on plasmids and chromosomes. We further characterized plasmids' incompatibility types and the co-occurrences of ARGs and virulence factors on plasmids and chromosomes. We investigated the transferability of plasmid-mediated beta-lactams via conjugation. Finally, using shotgun metagenomic analysis of the ESBL-producing -originated wastewater samples, we described the microbial community, the resistome composition, and the potential associations with plasmid-mediated beta-lactam genes and other ARGs.