Genome-wide analysis inEscherichia coliunravels an unprecedented level of genetic homoplasy associated with cefotaxime resistance
Authors: Coolen Jordy P.M. , den Drijver Evert P.M. , Verweij Jaco J. , Schildkraut Jodie A. , Neveling Kornelia , Melchers Willem J.G. , Kolwijck Eva , Wertheim Heiman F.L. , Kluytmans Jan A.J.W. , Huynen Martijn A. 
Published: June 2020

ABSTRACTCefotaxime (CTX) is a commonly used third-generation cephalosporin (3GC) to treat infections caused byEscherichia coli. Two genetic mechanisms have been associated with 3GC resistance inE. coli. 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 theampCβ-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 resistantE. coliisolates 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 theampCpromoter. The 24 occurrences of a “T” at the −42 position rather than the wild type “C”, resulted from 18 independent C>T mutations in 5 phylogroups. The −42 C>T mutation was only observed inE. colilacking a plasmid-encodedampCgene. The association of the −42 C>T mutation with CTX resistance was confirmed to be significant (FDR < 0.05). To conclude, genome-wide analysis of homoplasy in combination with CTX resistance identifies the −42 C>T mutation of theampCpromotor as significantly associated with CTX resistance and underline the role of recurrent mutations in the spread of antibiotics resistance.Impact StatementIn 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 theampCgene. 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.Data summaryAll data is available under BioProject: PRJNA592140. Raw Illumina sequencing data and metadata of all 171E. coliisolates 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.CP046396.1and NCBI Reference Sequence: NZ_CP046396.1.