Members of the genus Actinomyces are Gram-positive, generally facultatively anaerobic rods commonly isolated from the human mouth .As for the classification of these oral Actinomyces spp., phenotypical tests including the API ZYM tests , serological tests and monoclonal antibodies (, the protein profiles (SDS–PAGE) comparison, DNA–DNA hybridization and 16 S rDNA sequence comparison have been generally employed. However, most of these methods are relatively time-consuming, and there are no reliable phenotypic tests that rapidly and completely differentiate among the oral Actinomyces spp. especially between A. naeslundii genospecies 1 and 2. In addition, specific antisera are not available commercially.

16S rDNA PCR-RFLP has proved a useful typing technique for a number of groups of organisms, and can be used to identify species within some genera .

Here, reference strains, including the ATCC type strains, of oral Actinomyces spp., i.e., A. israelii, A. gerencseriae, A. naeslundii genospecies 1 and 2, A. odontolyticus, A. meyeri and A. georgiae, and 23 Actinomyces strains isolated from human dental plaque, were studied by 16S rDNA PCR-RFLP to assess the usefulness of the method for the differentiation of oral Actinomyces spp..

Strains, , were cultured on brain-heart infusion–yeast extract–blood (sheep) agar plates , and incubated at 37°C for 3 days in an anaerobic glove box (Model AZ-Hard, Hirasawa, Tokyo) containing 80% N₂, 10% H₂ and 10% CO₂. Twenty-three clinical strains were taken from our departmental collection for this study. They had been identified to the species/genospecies level by the biochemical and serological tests . In brief, they were Gram-positive facultatively anaerobic rods; acetate, lactate, succinate and/or pyruvate were produced as acid end-products in peptone–yeast extract broth with 1% glucose (pH 7.0) . Isolates assigned to A. gerencseriae were catalase-negative, urease-negative, utilizing raffinose, and not utilizing arabinose . Isolates assigned to A. odontolyticus were catalase-negative, urease-negative, not utilizing raffinose, and their colonies red or pink . Isolates assigned to A. naeslundii genospecies 1 and 2 were identified serologically by the method described.

From 3-day-old cultures, DNA was extracted by the InstaGene Matrix (BioRad), according to the manufacturer’s instructions. The 16S rRNA gene sequences were amplified by PCR with primers: 63f and 1387r (J. R. Marchesi and A. J. Weightman, 1995, personal communication) and Taq DNA polymerase (TaKaRa Premix Taq, TaKaRa Biomedicals, Shiga, Japan), according to the manufacturer’s instructions. The primer sequences were: 63f, 5?-CAG GCC TAA CAC ATG CAA GTC-3?; and 1387r, 5?-GGG CGG WGT GTA CAA GGC-3?. There were 35 cycles, each consisting of template denaturation for 1 min at 94°C, primer annealing for 1 min at 52°C, and extension for 2 min at 72°C.

PCR products were purified by SpinBind PCR Purification System (FMC BioProducts, ME, USA). Purified 16S rRNA genes were digested singly with restriction endonucleases, i.e., HaeIII, HpaII, MnlI (New England Biolabs, MA, USA) or CfoI (NBL Gene Sciences Limited, UK), according to the manufacturer’s instructions. Digestion products were separated on 2% agarose gels of NuSieve 3:1 agarose (FMC BioProducts, ME, U.S.A.), stained with ethidium bromide and photographed under ultraviolet light.

RFLP patterns for the 16S rDNA PCR products from the seven reference strains were obtained with the four enzymes, i.e., MnlI, HaeIII, CfoI and HpaII. Among the four restriction endonucleases, MnlI produced seven RFLP patterns for seven reference strains . In contrast, HaeIII produced five patterns, since the profiles for A. odontolyticus, A. meyeri and A. georgiae were the same . CfoI produced five patterns, since the profiles for A. israelii and A. odontolyticus were the same, and those for A. gerencseriae and A. georgiae were the same . HpaII produced five patterns for seven species, as the profiles for A. gerencseriae and A. naeslundii were the same, as were those for A. odontolyticus and A. meyeri . Thus, MnlI discriminated the respective reference strains. Data from MnlI were used in a further RFLP analysis for clinical samples, and the results are summarized The clinical isolates were assigned to one of the species, i.e., A. israelii (= PCR-RFLP profile I), A. gerencseriae (= profile II), A. naeslundii genospecies 1 (= profile III) and 2 (= profile IV) and A. odontolyticus (= profile V), on the basis of their restriction profiles by single digestion with MnlI.