Detection and Incidence of the Tetracycline Resistance Determinant tet(M) in the Microflora Associated with Adult Periodontitis

Abstract

Subgingival plaque samples were collected from 68 patients with adult periodontitis, enumerated on Trypticase-soy blood agar plates, with and without tetracycline at 4 micrograms/ml, and incubated anaerobically for 5 days. Each different colony morphotype was enumerated, and a representative colony was subcultured for identification and examined for the tetracycline resistance gene tet(M). Both PCR amplification and DNA hybridization, using a fragment of tet(M) from Tn1545, were used to detect tet(M). The PCR primers (5'-GACACGCCAGGACATATGG-3' and 5'-TGCTTTCCTCTTGTTCGAG-3') were chosen to amplify a 397 bp region of tet(M). Tetracycline-resistant bacteria represented approximately 12% of the total viable count. The percentage of tet(M)-positive bacteria in the tetracycline resistant microflora varied from < or = 0.05 to 83% (mean of 10%). tet(M) was detected in 60% of 204 tetracycline-resistant strains subcultured and identified. The tet(M) containing strains consisted of streptococci (55%, mainly S. intermedius, S. oralis, S. sanguis, and Streptococcus SM4), Actinomyces D01 (14%), Bifidobacterium D05 (11%), and Veillonella spp. (10%). Tetracycline-resistant strains in which tet(M) was not detected included the Prevotella and Bacteroides species (41%, mainly Bacteroides D28, P. intermedia, P. nigrescens, and P. oris). These results suggest that tet(M) is widely spread in the adult periodontal microflora, but it appears, with the exception of S. intermedius, to be mainly associated with microorganisms not considered to be periodontopathogens. Assessment of other tetracycline-resistant genes in oral organisms is needed to fully evaluate the nature of resistance to this antibiotic in the oral flora.