COMPARISON OF 6 TYPING METHODS FOR ACTINOBACILLUS-ACTINOMYCETEMCOMITANS

Actinobacillus actinomycetemcomitans is an important pathogen in the e tiology of severe periodontitis. For epidemiological studies on the pr evalence of certain pathogenic clones and transmission of this bacteri um, adequate typing methods are necessary. The purpose of this study w as to compare six different typing methods for A. actinomycetemcomitan s. Five reference strains and 27 fresh clinical isolates from periodon titis patients were used. Serotyping showed 12 serotype a strains, 13 type b strains, 6 type c strains, and 1 nontypeable strain. Biotyping on the basis of the fermentation of mannose, mannitol, and xylose resu lted in six biotypes. Antibiogram typing was evaluated by measuring th e inhibition zones of seven antibiotics in agar diffusion tests. With this method eight main types which could be further differentiated int o 15 subtypes were found. Sodium dodecyl sulfate-polyacrylamide gel el ectrophoresis patterns of outer membrane proteins were similar among a ll isolates tested. Restriction endonuclease analysis (REA) of whole c hromosomal DNA resulted in five main types. These five main types were further differentiated into 24 subtypes on the basis of DNA fragment differences in the high-molecular-weight region. Hybridization of DNA fragments, with ribosomal DNA (ribotyping) resulted in 22 to 24 differ ent types, depending on the restriction endonuclease used. Ribotype pa tterns were easy to interpret and provided an univocal distinction bet ween different strains compared with REA results. When applied to epid emiologically related isolates, all methods were able to discriminate two clonal types among five isolates from five children from one famil y. We conclude that serotyping, biotyping, and outer membrane patterns were reproducible but had a low discriminatory potential. REA and rib otyping were reproducible and gave the highest number of distinct type s. When the DNA typing methods were compared, all strains tested could be distinguished. These findings confirm the heterogeneity found with in the species A. actinomycetemcomitans.