Abstract
Corynebacterium diphtheriae, the main aetiological agent of diphtheria, is a re-emerging bacterial pathogen of public health concern, yet remains largely understudied globally. In this study, we analysed the population structure and antimicrobial resistance (AMR) of 210 C. diphtheriae isolates from Victoria, Australia, including 103 historical (1950-1970) and 107 contemporary clinical isolates (2004-2023), using whole-genome sequencing and phenotypic susceptibility testing. The diphtheria toxin gene (tox) was detected in 89 isolates, the majority of which (n=83; 93.3%) were historical. Population structure comprised two primary phylogenetic lineages, Mitis and Gravis, each containing multiple sublineages. Multi-locus sequence type (MLST) analysis revealed a highly diverse population structure with multiple novel MLST profiles and alleles. When placed within a global phylogenetic framework, Australian isolates were broadly distributed, reflecting substantial genetic diversity. Phenotypic susceptibility testing against 11 antimicrobials revealed that several contemporary isolates were resistant to multiple agents, including penicillin and erythromycin, first-line treatments of Corynebacterium infections. Eight contemporary isolates were multidrug-resistant (resistant to ≥3 antimicrobial classes), including five with resistance to both penicillin and erythromycin. Genomic analysis identified multiple genes and mutations conferring resistance among contemporary isolates. In contrast, no AMR phenotypes or genotypes were observed in historical isolates. Analysis of historical genomes provides valuable insights into a period of heightened diphtheria activity in Victoria prior to widespread immunization. Overall, these findings establish a baseline for ongoing genomic surveillance in the face of increasing global outbreaks, support informed empiric treatment strategies and contribute to the knowledge of the global population structure of C. diphtheriae.