Targeted Sequencing of CTX-M Alleles 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, which 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 36 samples from three Seattle area wastewater treatment plants 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 detected 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 the CARD database. This method provides information (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.