Targeted Sequencing of CTX-M Alleles Encoding for Extended-Spectrum Beta-Lactamases in Seattle Area Wastewater

Extended-spectrum beta-lactamases (ESBLs) are a growing group of antimicrobial resistance (AMR) enzymes that can result in severe clinical outcomes. The CTX-M gene (≈ 876 base pairs) encodes for ESBLs in bacteria, confers resistance to third-generation cephalosporins, and is of high clinical concern. We developed a targeted, long-read sequencing method utilizing unique molecular identifiers to generate accurate, full length CTX-M gene sequences from wastewater. We characterized CTX-M in Seattle area wastewater influent from April 2020 to March 2021. We identified a core community of alleles that persisted across time and treatment plant. The CTX-M-15 containing protein variant (CTX-M-15/216/28) was detected in all but three samples and made up, at most, 30% of CTX-M alleles. We observed significant diversity across the CTX-M gene at the nucleic acid level, although most nucleotide mutations were synonymous─resulting in two to three amino acid variants across 19 loci. By average relative abundance, 23% of protein variants were novel, defined as those not represented in CARD. This method provides full length gene sequences that cannot be obtained through other culture-independent methods. This flexible approach can be expanded to additional targets and implemented in settings where AMR surveillance is a priority, such as hospital wastewater.