Genome-wide analysis in Escherichia coli unravels an unprecedented level of genetic homoplasy associated with cefotaxime resistance

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Cefotaxime (CTX) is a commonly used third-generation cephalosporin (3GC) to treat infections caused by <jats:italic>Escherichia coli</jats:italic>. Two genetic mechanisms have been associated with 3GC resistance in <jats:italic>E. coli</jats:italic>. The first is the conjugative transfer of a plasmid harboring antibiotic resistance genes. The second is the introduction of mutations in the promoter region of the <jats:italic>ampC</jats:italic> β-lactamase gene that cause chromosomal-encoded β-lactamase hyperproduction. A wide variety of promoter mutations related to AmpC hyperproduction have been described. However, their link to a specific 3GC such as CTX resistance has not been reported. Here, we measured CTX MICs in 172 cefoxitin resistant <jats:italic>E. coli</jats:italic> isolates 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 tailored-made 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 <jats:italic>ampC</jats:italic> promoter. The 24 occurrences of a “T” at the −42 position rather than the wild type “C”, resulted from 18 independent C&gt;T mutations in 5 phylogroups. The −42 C&gt;T mutation was only observed in <jats:italic>E. coli</jats:italic> lacking a plasmid-encoded <jats:italic>ampC</jats:italic> gene. The association of the −42 C&gt;T mutation with CTX resistance was confirmed to be significant (FDR &lt; 0.05). To conclude, genome-wide analysis of homoplasy in combination with CTX resistance identifies the −42 C&gt;T mutation of the <jats:italic>ampC</jats:italic> promotor as significantly associated with CTX resistance and underline the role of recurrent mutations in the spread of antibiotics resistance.</jats:p><jats:sec><jats:title>Impact Statement</jats:title><jats:p>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 E. coli chromosome-encoded β-lactam resistance can be caused by alterations in the promoter region of the <jats:italic>ampC</jats:italic> gene. To improve our understanding of how frequently these alterations occur, a comprehensive interpretation of the evolution of these mutations is essential. This study is the first to apply genome-wide homoplasy analysis to better perceive adaptation of the E. coli genome to antibiotics. Thereby, this study grants insights into how chromosomal-encoded antibiotic resistance evolves and, by combining genome-wide association studies with homoplasy analyses, provides potential strategies for future association studies into the causes of antibiotics resistance.</jats:p></jats:sec><jats:sec><jats:title>Data summary</jats:title><jats:p>All data is available under BioProject: PRJNA592140. Raw Illumina sequencing data and metadata of all 171 <jats:italic>E. coli</jats:italic> isolates used in this study is available from the Sequence Read Archive database under accession no. SAMN15052485 to SAMN15052655. Full reference chromosome of ampC_0069 is available via GenBank accession no. <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="gen" xlink:href="CP046396.1">CP046396.1</jats:ext-link> and NCBI Reference Sequence: NZ_CP046396.1.</jats:p></jats:sec>