Population structure and antimicrobial resistance of Corynebacterium diphtheriae in Victoria, Australia

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 eleven 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 antimicrobial resistance 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 immunisation. 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 global population structure of C. diphtheriae. Impact statement Corynebacterium diphtheriae is a re-emerging pathogen of public health concern with limited genomic data available to support surveillance and public health interventions. This study provides a contemporary understanding of C. diphtheriae from Australia in the era of genomic surveillance and helps us better understand baseline genomic diversity and antimicrobial resistance. Additionally, these findings enhance preparedness for potential future incursions or outbreaks, including those involving drug-resistant strains, as recently observed in parts of Africa and Europe. Data summary Genome sequences are deposited in GenBank under BioProject PRJNA870170. Sample data and accession numbers are included in the Supplementary Table S1. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Competing Interest Statement The authors have declared no competing interest. Abbreviations - AMR - antimicrobial resistance - tox - diphtheria toxin gene - NTTB - non-toxigenic tox gene-bearing - MDU PHL - Microbiological Diagnostic Unit Public Health Laboratory - PCR - polymerase chain reaction - MLST - multi-locus sequence type - ST - sequence type - MIC - minimum inhibitory concentration - MDR - multidrug-resistant - CLSI - Clinical and Laboratory Standards Institute - EUCAST - European Committee on Antimicrobial Susceptibility Testing