Panunzi, L. G. | bioRxiv Science

bioRxiv2020

Population genomics and antimicrobial resistance in Corynebacterium diphtheriae

Corynebacterium diphtheriae, the agent of diphtheria, is a genetically diverse bacterial species. Although antimicrobial resistance has emerged against several drugs including first-line penicillin, the genomic determinants and population dynamics of resistance are largely unknown for this neglected human pathogen. Here we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production and genomic features in C. diphtheriae. We used 247 strains collected over several decades in multiple world regions, including the 163 clinical isolates collected prospectively from 2008 to 2017 in France mainland and overseas territories. Phylogenetic analysis revealed multiple deep-branching sublineages, grouped into a Mitis lineage strongly associated with diphtheria toxin production, and a tox-negative Gravis lineage with few tox+ exceptions including the 1990s ex-Soviet Union outbreak strain. The distribution of susceptibility phenotypes allowed proposing ecological cutoffs for most of the 19 agents tested, thereby defining acquired antimicrobial resistance. Penicillin resistance was found in 17.2% of prospective isolates. Four isolates were multidrug resistant (>8 agents), including to penicillin and macrolides. Homologous recombination was frequent (r/m = 5) and horizontal gene transfer contributed to the emergence of antimicrobial resistance in multiple sublineages. Genome-wide association mapping uncovered genetic factors of resistance, including an accessory penicillin-binding protein (PBP2m) located in diverse genomic contexts. Gene pbp2m is widespread in other Corynebacterium species and its expression in C. glutamicum demonstrated its effect against several beta-lactams. A novel 73-kb C. diphtheriae multi-resistance plasmid was discovered. This work uncovers the dynamics of antimicrobial resistance in C. diphtheriae in the context of phylogenetic structure, biovar and diphtheria toxin production, and provides a blueprint to analyze re-emerging diphtheria.

microbiology↗

bioRxiv2019

Optimization and validation of a quadruplex real-time PCR assay for the diagnosis of diphtheria

Diphtheria is caused by toxigenic strains of Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin (tox)-positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysa et al. J Med Microbiol. 2016 65(12):1521-1527). Here, we present an improvement of the quadruplex method, in which a 16S rRNA gene target was added as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays. This improved qPCR method was validated using 36 bacterial isolates and 16 clinical samples. The method allows detection of the tox gene and distinguishing C. diphtheriae (including the newly described species C. belfantii) from C. ulcerans and C. pseudotuberculosis. Complete diagnostic specificity, sensitivity and experimental robustness of the method to temperature and reagent concentration variations were demonstrated. The lower limit of detection for C. diphtheriae, C. ulcerans and tox targets was 1.86 genome copies per 5 L reaction volume. Finally, the method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England - National Infection Service, London, UK). This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.

microbiology↗