Virulence and mutations analysis based on the whole genome of a Brazilian Corynebacterium diphtheriae strain isolated from a cutaneous infection

Max Roberto Batista Araújo^1,2*Louisy Sanches dos Santos³Fernanda Diniz Prates^1,2Hugo Perini⁴Jailan Sousa Silva¹Juliana Nunes Ramos³Sérgio Bokermann⁵Cláudio Tavares Sacchi⁶Ana Luíza Mattos Guaraldi³Karoline Campos⁶Tayná do Carmo Sant’Anna Cardoso³Diogo Luiz de Carvalho Castro¹Marcos Andrade Silva³Mireille Ângela Bernardes Sousa²Verônica Viana Vieira⁷Marlon Benedito Nascimento Santos⁶Carlos Henrique Camargo⁵Bruno Silva Andrade⁸Marcos Vinicius Da Silva⁴Lincoln de Oliveira Sant'Anna³Marcus Vinicius Canário Viana¹Vasco Azevedo^1*

• ¹Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
• ²Operational Technical Nucleus, Microbiology, Hermes Pardini Institute, Vespasiano, Brazil
• ³Rio de Janeiro State University, Rio de Janeiro, Brazil
• ⁴Federal University of Triângulo Mineiro, Uberaba, Brazil
• ⁵Center for Bacteriology, Adolfo Lutz Institute, Sao Paulo, Sao Paulo, Brazil
• ⁶Strategic Laboratory, Adolfo Lutz Institute, São Paulo, Brazil
• ⁷Oswaldo Cruz Institute, Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro, Paraná, Brazil
• ⁸State University of Southwest Bahia, Jequié, Brazil

Corynebacterium diphtheriae is the main etiological agent of diphtheria, a potentially fatal disease whose most severe signs and symptoms result from the action of an exotoxin, the diphtheria toxin (DT). Although non-toxigenic C. diphtheriae strains have been associated with several diseases, including cutaneous infections and endocarditis, they are not monitored in many countries, and their mechanisms of virulence and antimicrobial resistance remain underexplored. Therefore, this study aimed to provide a comprehensive characterizationthrough genomic, in vitro, and in vivo analyses -of a non-toxigenic C. diphtheriae strain (46855) isolated from a leg lesion, highlighting its pathogenic potential and resistance profile.The isolate was assigned to a novel sequence type (ST-925) and was found to be resistant to tetracycline and rifampin. Multiple antimicrobial resistance genes were predicted in the genome, such as tet(33), rbpA, and rpoB2, in addition to mutations in the rpoB gene. A diverse set of virulence-associated genes related to adhesion, iron uptake systems, gene regulation, and post-translational modification was also identified. The isolate was able to form biofilm in vitro and exhibited strong virulence in Galleria mellonella larvae and A549 human pneumocyte cells. Finally, the structural analysis of the rpoB gene, carried out for the first time in this study, linked the observed mutations to rifampin resistance in C. diphtheriae. In summary, the data revealed that C. diphtheriae 46855, although non-toxigenic, harbors multiple genes associated with antimicrobial resistance and virulence, emphasizing the need for greater surveillance and functional studies on non-toxigenic strains.