
Tetracyclines are broad-spectrum antibiotics that exhibit activity against a wide range of bacteria [1]. They are a family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor site. Tetracyclines also have a number of non-antibacterial effects, such as inhibition of collagenase, which was found to be therapeutically useful in periodontitis [2]. However, the extensive use of these agents has led to the emergence of antibiotic resistance.
Resistance to tetracycline occurs at high frequency among clinical isolates of both gram-positive and gram-negative bacteria [3]. The first tetracycline-resistant bacterium was isolated in 1953 [4]. Tetracycline-resistance genes mediate resistance mainly by two different mechanisms: active efflux or ribosomal protection. The tetracycline-resistance genes
The
Dental plaque is comprised of more than 700 species of bacteria, including bacteria related to periodontal diseases [12]. Because oral streptococci constitute the major bacterial species found in human dental plaque [13], tetracycline-resistance genes present in oral streptococci may be a source for transferring tetracycline resistance to other bacteria present in the oral cavity. Although many clinical oral streptococcal isolates are tetracycline resistant, the genetics of tetracycline-resistance genes have not been extensively studied in oral streptococci [14,15]. Here, we examined the genetic diversity of
Streptococci were isolated from the supragingival plaque samples of healthy persons. All volunteers willing to donate their plaques for this study were informed about the procedure and gave written consent for inclusion in the study. This study was approved by the Institutional Review Board of Gangneung-Wonju National University Dental Hospital (IRB 2011-2). Isolates were identified to the species level using the Rapid ID 32 Strep system and a mini API reader (bioMerieux, Marcy-l‘Etoile, France). Seven specific viridans-group streptococcal species (
To determine the minimal inhibitory concentration (MIC) of the antibiotic, stock antibiotic solution of tetracycline (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) was prepared. The MICs were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines using a microdilution method in cation-adjusted Mueller-Hinton broth supplemented with lysed horse blood. Using streptococcal colonies taken directly from sheep-blood agar plates (KOMED, Seongnam, Korea), which were incubated at 37°C for 18 hours in aerobic conditions, a suspension equivalent to that of the 0.5 McFarland standard (~1×108 CFU/mL) in cation-adjusted Mueller-Hinton broth was prepared. The bacteria were inoculated into serially diluted antibiotic solutions in 96-well microtitration plates at final concentrations of 5×105 CFU/mL. The microtitration plates were incubated in an ambient-air incubator at 37°C for 24 hours. The microtitration plates were read visually and the minimum concentration of the antibiotics that produced no turbidity was recorded as the MIC. Antibiotic resistance was determined by interpretive standard concentrations from CLSI guidelines [16] and tests were repeated at least twice. The range of concentrations tested for each antibiotic was from 0.001–1,024 μg/mL.
Genomic DNA was extracted from the tetracycline-resistant strains using an
Among the strains containing
Tetracycline-susceptibility tests were performed for 635 streptococcal isolates, with 148 isolates being determined to be tetracycline resistant. Among the 148 tetracycline-resistant oral streptococcal strains, 68 (46%) contained
Comparison of only polymorphic sites among the sequences is shown in Fig. 1. It was revealed that genetic variation in
To examine the sequence divergence of
Several studies reported the prevalence of tetracycline resistance in oral streptococci, with the severity of the prevalence varying among studies conducted in different countries. In London, Stapleton et al. [11] found
The
Previous studies demonstrated the heterogeneity of the
In our data, the
Streptococci make up a large contingent of oral bacteria. It is assumed that they are potential sources of tetracycline-resistance genes for other bacteria, including the bacteria that cause periodontal disease. When the transposon containing a tetracycline-resistance gene moves between the chromosomes of bacteria in the oral cavity, transfer of tetracycline resistance may occur. However, further studies are necessary to reveal the possibility of transferring genes associated with tetracycline resistance between oral bacterial species.
The data presented in our study provide basic information about the transposition process associated with
The authors declare that they have no competing interests.