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Prevalence of Cytolethal Distending Toxin Production in Periodontopathogenic Bacteria

Departments of Bacteriology,¹ Orthodontics and Craniofacial Developmental Biology,² Periodontal Medicine, Hiroshima University Graduate School of Biomedical Sciences, Minami-ku, Hiroshima 734-8553,³ Department of Microbiology, Tokyo Dental College, Chiba 261-8502, Japan,⁴ Unite associee INRA de Microbiologie Moleculaire, Ecole Nationale Veterinaire de Toulouse, 31076 Toulouse cedex, France⁵

Received 19 July 2002/ Returned for modification 9 November 2002/ Accepted 6 January 2003

Cytolethal distending toxin (CDT) is a newly identified virulence factor produced by several pathogenic bacteria implicated in chronic infection. Seventy three strains of periodontopathogenic bacteria were examined for the production of CDT by a HeLa cell bioassay and for the presence of the cdt gene by PCR with degenerative oligonucleotide primers, which were designed based on various regions of the Escherichia coli and Campylobacter cdtB genes, which have been successfully used for the identification and cloning of cdtABC genes from Actinobacillus actinomycetemcomitans Y4 (M. Sugai et al., Infect. Immun. 66:5008-5019, 1998). CDT activity was found in culture supernatants of 40 of 45 tested A. actinomycetemcomintans strains, but the titer of the toxin varied considerably among these strains. PCR experiments indicated the presence of Y4-type cdt sequences in these strains, but the rest of A. actinomycetemcomitans were negative by PCR amplification and also by Southern blot analysis for the cdtABC gene. In the 40 CDT-positive strains, Southern hybridization with HindIII-digested genomic DNA revealed that there are at least 6 restriction fragment length polymorphism types. This suggests that the cdtABC flanking region is highly polymorphic, which may partly explain the variability of the CDT activity in the culture supernatants. The rest of tested strains of periodontopathogenic bacteria did not have detectable CDT production by the HeLa cell assay and for cdtB sequences by PCR analysis under our experimental conditions. These results strongly suggested that CDT is a unique toxin predominantly produced by A. actinomycetemcomitans among periodontopathogenic bacteria.

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