First isolation of nontoxigenic tox gene-bearing Corynebacterium ulcerans from a diphtheria case in Minas Gerais, Brazil

Diphtheria is a severe, vaccine-preventable infection caused mainly by Corynebacterium diphtheriae, but other species within the C. diphtheriae complex, including C. ulcerans, can also carry the diphtheria toxin (DT) coding gene (tox). Strains classified as “nontoxigenic toxin-gene bearing” (NTTB) harbor the tox gene but do not produce functional DT, although reactivation remains possible. This study reports the first isolation of a NTTB C. ulcerans strain from a diphtheria case in Brazil. A bacterial strain was isolated in Minas Gerais (2019) and subjected to phenotypic, molecular, and phylogenetic analyses. Identification was performed using MALDI-TOF MS, multiplex PCR (mPCR), and sequencing of the 16S rRNA and rpoB genes, followed by phylogenetic inference. Toxigenicity was assessed by mPCR and the modified Elek test. Antimicrobial susceptibility was determined by the disk diffusion method according BrCAST guidelines. MALDI-TOF identified the isolate as C. ulcerans. The identification was confirmed by phylogenetic analysis of the rpoB gene. The strain carried the tox gene, but the Elek test did not show the production of DT, classifying the isolate as NTTB. Antimicrobial testing showed susceptibility to most drugs, with the susceptible, increased exposure profile to benzylpenicillin and ciprofloxacin. No resistance was detected. Although non-toxigenic, the isolation of a tox-positive C. ulcerans strain highlights the potential risk for re-emergence of diphtheria in Brazil. Continuous genomic surveillance, investigation of zoonotic reservoirs, and maintenance of high vaccination coverage remain crucial to prevent future outbreaks.