AbstractBackgroundCorynebacterium 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.MethodsHere, we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production, and genomic features inC. 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.ResultsPhylogenetic analysis revealed multiple deep-branching sublineages, grouped into a Mitis lineage strongly associated with diphtheria toxin production and a largely toxin gene-negative Gravis lineage with few toxin-producing isolates 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. Seventeen (10.4%) prospective isolates were multidrug-resistant (≥ 3 antimicrobial categories), including four isolates resistant 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. Genepbp2mis widespread in otherCorynebacteriumspecies, and its expression inC. glutamicumdemonstrated its effect against several beta-lactams. A novel 73-kbC. diphtheriaemultiresistance plasmid was discovered.ConclusionsThis work uncovers the dynamics of antimicrobial resistance inC. diphtheriaein the context of phylogenetic structure, biovar, and diphtheria toxin production and provides a blueprint to analyze re-emerging diphtheria.