Genome-wide analysis in Escherichia coli unravels a high level of genetic homoplasy associated with cefotaxime resistance.

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 ß-lactamase gene that cause chromosome-encoded ß-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.

Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC ß-lactamase production in Escherichia coli by elaborate phenotypic and genotypic characterization.

OBJECTIVES: AmpC-ß-lactamase production is an under-recognized antibiotic resistance mechanism that renders Gram-negative bacteria resistant to common ß-lactam antibiotics, similar to the well-known ESBLs. For infection control purposes, it is important to be able to discriminate between plasmid-mediated AmpC (pAmpC) production and chromosomal-mediated AmpC (cAmpC) hyperproduction in Gram-negative bacteria as pAmpC requires isolation precautions to minimize the risk of horizontal gene transmission. Detecting pAmpC in Escherichia coli is challenging, as both pAmpC production and cAmpC hyperproduction may lead to third-generation cephalosporin resistance. METHODS: We tested a collection of E. coli strains suspected to produce AmpC. Elaborate susceptibility testing for third-generation cephalosporins, WGS and machine learning were used to develop an algorithm to determine ampC genotypes in E. coli. WGS was applied to detect pampC genes, cAmpC hyperproducers and STs. RESULTS: In total, 172 E. coli strains (n=75 ST) were divided into a training set and two validation sets. Ninety strains were pampC positive, the predominant gene being blaCMY-2 (86.7%), followed by blaDHA-1 (7.8%), and 59 strains were cAmpC hyperproducers. The algorithm used a cefotaxime MIC value above 6 mg/L to identify pampC-positive E. coli and an MIC value of 0.5 mg/L to discriminate between cAmpC-hyperproducing and non-cAmpC-hyperproducing E. coli strains. Accuracy was 0.88 (95% CI=0.79-0.94) on the training set, 0.79 (95% CI=0.64-0.89) on validation set 1 and 0.85 (95% CI=0.71-0.94) on validation set 2. CONCLUSIONS: This approach resulted in a pragmatic algorithm for differentiating ampC genotypes in E. coli based on phenotypic susceptibility testing.