Identification of oral Actinomyces species using DNA probes

Oral Actinomyces comprise a major segment of both the supra- and subgingiva l microbiota; however, little is known about the distribution of individual species in different sites or clinical conditions. The purpose of the pres ent investigation was to develop DNA probes for suggested species and genot ypes of oral Actinomyces. Whole genomic DNA probes to 12 human oral species and/or serotypes were labeled with digoxigenin and used to seek cross-reac tions among the taxa using the checkerboard DNA-DNA hybridization assay. Th e Actinomyces formed three distinct groups: 1) Actinomyces georgiae, Actino myces meyeri and Actinomyces odontolyticus serotypes I and II; 2) Actinomyc es viscosus and Actinomyces naeslundii serotypes I, II, III and WVA 963; an d 3) Actinomyces gerencseriae and Actinomyces israelii. Cross-reactions amo ng taxa were detected and minimized by increasing the temperature of the po st-hybridization high-stringency wash to 80 degrees C. Despite the elevatio n in high stringency wash temperature, cross-reactions among strains of the A. naeslundii/A. viscosus group persisted. Probes for two of the three cur rently recognized genospecies in this group were prepared by removing the D NA in common between cross-reacting species using subtraction hybridization and polymerase chain reaction. Nine species and genospecies could be clear ly separated by a combination of whole genomic and subtraction hybridizatio n probes and by increasing the high-stringency wash temperature. A total of 195 fresh isolates of Actinomyces were grouped in a blind study using DNA probes and separately by SDS-PAGE protein profiles. Concordance between the two methods was 97.3%. The probes and hybridization conditions were tested for their ability to detect the Actinomyces species and genospecies in sam ples of supragingival and subgingival plaque from periodontitis subjects us ing checkerboard DNA-DNA hybridization. The probes detected the species in samples of supragingival and subgingival plaque, We concluded that whole ge nomic and subtraction hybridization DNA probes facilitate the detection and enumeration of species and genospecies of Actinomyces in plaque samples.