Ultra-deep long-read sequencing detects IS-mediated gene duplications as a potential trigger to generate arrays of resistance genes and a mechanism to induce novel gene variants such as blaCTX-M-243.

Abstract

Extended-spectrum β-lactamases (ESBLs) are enzymes that can render their hosts resistant to various β-lactam antibiotics. CTX-M-type enzymes are the most prevalent ESBLs and the main cause of resistance to third-generation cephalosporins in Enterobacteriaceae. The number of described CTX-M types is continuously rising, currently comprising over 240 variants. During routine screening we identified a novel blaCTX-M gene. To characterize a novel blaCTX-M variant harboured by a multidrug-resistant Escherichia coli isolate of sequence type ST354. Antibiotic susceptibilities were determined using broth microdilution. Genome and plasmid sequences were reconstructed using short- and long-read sequencing. The novel blaCTX-M locus was analysed using long-read and Sanger sequencing. Plasmid polymorphisms were determined in silico on a single plasmid molecule level. The novel blaCTX-M-243 allele was discovered alongside a nearly identical blaCTX-M-104-containing gene array on a 219 kbp IncHI2A plasmid. CTX-M-243 differed from CTX-M-104 by only one amino acid substitution (N109S). Ultra-deep (2300-fold coverage) long-read sequencing revealed dynamic scaling of the blaCTX-M genetic contexts from one to five copies. Further antibiotic resistance genes such as blaTEM-1 also exhibited sequence heterogeneity but were stable in copy number. We identified the novel ESBL gene blaCTX-M-243 and illustrate a dynamic system of varying blaCTX-M copy numbers. Our results highlight the constant emergence of new CTX-M family enzymes and demonstrate a potential evolutionary platform to generate novel ESBL variants and possibly other antibiotic resistance genes.