Wild raccoons (Procyon lotor) as a potential reservoir of cytolethal distending toxin-producing Providencia strains in Japan.

In view of increasing reports of infections due to virulent Providencia species including cytolethal distending toxin (cdt) gene-positive strains, it is important to identify the reservoirs and transmission routes of such pathogenic strains. Raccoons considered to be a source of zoonotic pathogens were monitored for the presence of Providencia species in Japan and analyzed for cdt genes. Of 384 wild raccoon rectal swabs analyzed, 60% were positive for Providencia species, of which 20% carried cdt-genes. Among seven Providencia species isolated (P. alcalifaciens, P. rustigianii, P. rettgeri, P. stuartii, P. heimbachae, P. vermicola, and P. huaxiensis), cdt genes were distributed in P. alcalifcaiens (63%), P. rustigianii (16%), and novel in P. rettgeri (21%). Complete cdt gene clusters were identified in P. alcalifaciens and P. rustigianii strains, whereas P. rettgeri had intact cdtB but truncated cdtA and cdtC genes. Phylogenetic analyses showed divergent pulsotypes among the cdt gene-positive Providencia strains. Cytotoxicity assay revealed that P. alcalifaciens and P. rustigianii produced CDT more toxic to eukaryotic cells compared to human clinical strains, which were neutralized by anti-PaCdtB serum. As expected, the P. rettgeri strains with truncated cdt genes had no biological activity. Molecular analysis revealed that all the cdt genes were located on plasmids as determined by S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern hybridization assay. Intriguingly, the cdtB gene in P. rustigianii strains was detected on dual plasmids. Notably, all the cdt gene-positive Providencia strains were found to carry plasmid-mediated T3SS-related genes. These results suggest that wild raccoons are possible reservoir of virulent Providencia strains in Japan.IMPORTANCEProvidencia species considered normal flora are occasionally associated with gastroenteritis in healthy humans. Cytolethal distending toxin (CDT), a bacterial virulence factor found in various Gram-negative bacteria and associated with gastroenteritis and extra-intestinal infection has also been reported in at least two Providencia species (P. alcalifaciens and P. rustigianii). Determination of the transmission routes of such virulent Providencia is crucial for the implementation of evidence-based control programs. In this study, we identified raccoons as the probable reservoir of the cdt gene-positive Providencia strains in Japan. Interestingly, CDTs produced by raccoon-derived Providencia strains exerted more toxic effects on the eukaryotic cells compared to the clinical Providencia strains. In addition, the identification of a novel cdt gene cluster in another species P. rettgeri isolated from raccoons suggests that Providencia may be categorized as an emerging zoonotic pathogen.