118 Chapter 7 Abstract Cefotaxime (CTX) is a third-generation cephalosporin (3GC) commonly used to treat infections caused by Escherichia coli. Two genetic mechanisms have been associated with 3GC resistance in E. coli. The first is the conjugative transfer of a plasmid harbouring antibiotic-resistance genes. The second is the introduction of mutations in the promoter region of the ampC beta-lactamase gene that cause chromosome-encoded beta-lactamase hyperproduction. A wide variety of promoter mutations related to AmpC hyperproduction have been described. However, their link to CTX resistance has not been reported. We recultured 172 cefoxitin-resistant E. coli isolates with known CTX minimum inhibitory concentrations and performed genome-wide analysis of homoplastic mutations associated with CTX resistance by comparing Illumina whole-genome sequencing data of all isolates to a PacBio sequenced reference chromosome. We mapped the mutations on the reference chromosome and determined their occurrence in the phylogeny, revealing extreme homoplasy at the −42 position of the ampC promoter. The 24 occurrences of a T at the −42 position rather than the wild-type C, resulted from 18 independent C>T mutations in five phylogroups. The −42 C>T mutation was only observed in E. coli lacking a plasmid-encoded ampC gene. The association of the −42 C>T mutation with CTX resistance was confirmed to be significant (false discovery rate <0.05). To conclude, genome-wide analysis of homoplasy in combination with CTX resistance identifies the −42 C>T mutation of the ampC promotor as significantly associated with CTX resistance and underlines the role of recurrent mutations in the spread of antibiotic resistance. Impact Statement In the past decades, the worldwide spread of extended-spectrum beta-lactamases (ESBLs) has led to a substantial increase in the prevalence of resistant common pathogens, thereby restricting available treatment options. Although acquired resistance genes, e.g. ESBLs, get most attention, chromosome-encoded resistance mechanisms may play an important role as well. In Escherichia coli, chromosome-encoded beta-lactam resistance can be caused by alterations in the promoter region of the ampC gene. To improve our understanding of how frequently these alterations occur, a comprehensive interpretation of the evolution of these mutations is essential. In the current study, we apply genome-wide homoplasy analysis to better perceive adaptation of the E.
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