Impact of Minocycline Ointment for Periodontal Treatment of Oral Bacteria

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1 Jpn. J. Infect. Dis., 64, , 2011 Short Communication Impact of Minocycline Ointment for Periodontal Treatment of Oral Bacteria Ryoma Nakao*, Satoko Takigawa 1, Naoyuki Sugano 1, Ryosuke Koshi 1, Koichi Ito 1, Haruo Watanabe, and Hidenobu Senpuku Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo ; and 1 Department of Periodontology, Nihon University School of Dentistry, Tokyo , Japan (Received November 12, Accepted January 25, 2011) SUMMARY: Topical tetracyclines, such as minocycline ointment, are frequently used for the treatment of periodontal infection. We investigated the influence of minocycline ointment use on oral bacteria, using supragingival plaque samples from adults who had not taken any antibiotics for 6 months. Initially we investigated the effect of topical minocycline administration on the emergence of tetracycline-resistant oral bacteria in four healthy adults. The isolation frequency of tetracycline-resistant oral bacteria to total viable bacteria increased substantially on day 6 after treatment, although it returned to baseline on day 25. Subsequently we investigated the isolation frequency of tetracycline-resistant oral streptococci (TOS) as a representative oral bacterium, using samples from 41 subjects with periodontal diseases. The percentage of TOS (of the total oral streptococci) increased significantly (from 11.9 ± 15.6% to 34.2 ± 24.0%) after minocycline treatment. Various TOS species were identified; S. mitis, S. salivarius, S. sanguinis, and S. oralis were frequently isolated. PCR and Southern blotting allowed us to identify tetm on the Tn916-like elements as the gene responsible for tetracycline-resistance. These findings suggest that the potential risk of the spread of similar genetic elements through bacteria in the oral cavity should be considered. Periodontal diseases are infectious diseases that cause the destruction of supporting connective tissue and bone, and ultimately tooth loss. The initiation and progression of periodontal diseases is the result of complex interactions between the colonizing bacteria in the periodontal pockets and the host immune and inflammatory responses. Periodontal biofilms in the subgingival pockets are formed by a mixture of many pathogens rather than a single pathogenic species, and are generally resistant to antibacterial treatment. Antibiotic therapy is therefore regarded as a supplementary means of supporting the conventional mechanical therapy to remove biofilms and dental calculus, i.e., brushing, scaling, and root planing. Tetracyclines are commonly used in dental practice as a prophylactic agent and for the treatment of oral infections (1) as it has been shown that tetracyclines have an inhibitory effect on most periodontitis-associated bacteria in vitro (2) and in vivo (3). Furthermore, antimicrobial pastes that can be applied to the oral cavity have been developed to cure oral bacterial infection, especially infection by periodontopathic bacteria. The emergence of antimicrobial resistance is currently posing a major global challenge, with an increasing number of strains, including commensal and pathogenic oral bacteria, becoming resistant to commonly used antimicrobials. In the present study, we investigated the effect of using minocycline ointment on oral bacteria by examining supragingival plaque samples from healthy adults and periodontal disease patients. Ethical approval for this epidemiological study was obtained from the Research and Ethics Committee of the National Institute of Infectious Diseases, and each subject *Corresponding author: Mailing address: Department of Bacteriology I, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo , Japan. Tel: , Fax: , ryoma73@nih.go.jp signed an informed consent form. Supragingival plaque samples were obtained from 15 medically healthy adults who were not suffering from any oral disease and from 41 patients with periodontal disease attending the periodontology section of the Nihon University School of Dentistry Dental Hospital. These 41 patients (25 female and 16 male) were aged 26 to 87 years (56.4 ± 14.0 shown as average ± SD) and presented with 4 mm of attachment loss on at least two teeth. None of the individuals had received any antibiotics during the previous 6 months. Systemic disease and a history of medication therapy within the last 6 months that could influence the outcome of this study were considered to be criteria for exclusion from the study. The plaque samples were collected from the gingival margin of the teeth, using CultureSwab Plus (BD Diagnostics, Sparks, Md., USA) as a bacterial-collection and -transport system. Plaque samples taken after minocycline administration were collected from the gingival margin of the teeth treated with minocycline. Samples were taken by dentists from the periodontology section who were well acquainted with the plaque-sampling technique. These samples were placed into transport fluid in the ClutureSwab Plus for analysis. Samples were also taken from 4 healthy adults and 12 of 41 patients after treatment with minocycline. Minocycline was administered to patients after fundamental treatment for periodontal disease, including instruction on tooth brushing and mechanical scaling. For the four healthy adults in the intervention study, 0.2 g of Periofeel 2% (w/w) minocycline ointment (Showa Yakuhin Kako Co., Tokyo, Japan) was administered to the gingival pockets of the upper right posterior teeth on day 0, then plaque samples were collected on days 6 and 25. For the 12 patients with periodontitis, plaque samples were collected at different time points (7 60 days; 21.3 ± 13.8) after administration of the minocycline ointment. These plaque samples were re- 156

2 suspended and dispersed in 2 ml of phosphate buffered saline (PBS) by ultrasonication for 10 s before serial 10- fold dilution. The samples were then spread on a culture plate. Brain heart infusion (BHI) agar plates, with or without tetracycline, were used for tetracycline-resistant oral bacteria or total oral bacteria, whereas mitis-salivarius (MS) agar plates, with or without tetracycline, were used for tetracycline-resistant oral streptococci (TOS) or total oral streptococci. Tetracycline-HCl was used at a concentration of 8 g/ml (Sigma, St Louis, Mo., USA) for the discrimination of tetracycline-resistant strains in the population, according to guideline M07-A07 from the Clinical and Laboratory Standards Institute (CLSI). All plates were incubated anaerobically at 37 C for 3 days using the BBL GasPak system (BD Diagnostics). Colonies on plates were discriminated on the basis of morphological differences after growth on MS agar, and the number of each type of colony was counted. One representative colony of each type was identified by its biological properties and/or partial 16S rdna gene sequencing along with Gram-staining and colony morphology. Biological identification was performed using a Rapid ID 32 Strep API (BioMerieux, Marcy l Etoile, France) according to the manufacturer s instructions. For identification by 16S rdna sequencing, bacterial genomic DNA was first extracted from a 2-ml culture of each isolate using a ChargeSwitch gdna mini bacteria kit (Invitrogen, Carlsbad, Calif., USA). The entire 16S rdna sequence (approximately 1.5 kb) was PCR-amplified using universal primer pairs, 16S-f1 (forward, 5 -AGAGTTTGATCCTGGCTCAG-3 ) and 16S-r1 (reverse, 5 -AAGGAGGTGATCCAGCC-3 ) and then cloned into pmd20-t using the Mighty Mix DNA ligation kit (Takara Bio, Shiga, Japan). The first 0.5 kb at the 5 end of the gene was DNA-sequenced (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems, Foster City, Calif., USA) in both the sense and anti-sense directions using the universal primers, 16S-f1 and F15 (4). The insert DNA was analyzed using the 16S rdna database from the DNA Data Bank of Japan (DDBJ) periodical release. In order to define a phylotype, we used a cutoff of 99% identity among the first 0.5 kb at the 5 end of the 16S rdna, where the most variable region exists among bacterial species. A total of 49 isolates from 16 subjects were tested for genes encoding ribosomal protection proteins (tetm, teto, tetq, tets, tett, and tetw) and efflux pumps (teta, tetb, tetc, tete, tetk, and tetl) by PCR, as described previously (5,6). Likewise, several TOSs were tested for the presence of the int and xis genes of Tn916-like elements using Southern blot, as described previously (7). Statistical analysis was performed using the Mann- Whitney U test. P values of 0.05 or less were considered to be statistically significant. Initially we investigated the isolation frequency of tetracycline-resistant oral bacteria in supragingival samples from five healthy adults (Fig. 1). Four of the five healthy volunteers received treatment with minocycline ointment on day 0 (Fig. 1. A D: intervention group), whereas the remaining volunteer did not receive the treatment (Fig. 1. E: control). The percentage of tetracycline-resistant oral bacteria in the intervention group before treatment was 4.0 ± 4.4% on day 0 (shown as % of average ± SD). The percentage was found to have increased significantly (approximately 10-fold; 40.4 ± 12.9%; P = ) on day 6 after treatment, whereas by day 25, the percentage recovered to the level of the baseline CFU on BHIT/CFU on BHI 100 (%) A B C D E: control Days post treatment Fig. 1. Isolation frequency of tetracycline-resistant bacteria. Four of five healthy adults received administration of minocycline ointment on day 0 (A D: intervention group). One of five did not receive the treatment (E). The isolation frequency was formulated as a percentage of the number of colony forming units (CFUs) on BHI agar plates with tetracycline (BHIT) to CFUs on BHI agar plates without tetracycline (BHI). (3.3 ± 3.2%; P = ). The isolation frequency for the control without administration of minocycline on day 0 (Fig. 1. E) did not change at the two different time points (days 0 and 6). Although a minor population of tetracyclineresistant bacteria expanded on day 6, due to minocycline selection, this was followed by rapid restoration of the population to baseline on day 25. In order to assess the impact of minocycline treatment on oral bacteria in actual patients, the isolation frequency of TOS as a representative oral bacterium was examined in 41 subjects with periodontal disease. The percentage of TOS among patients after minocycline therapy increased significantly (three-fold) compared to baseline on day 0 (34.2 ± 24.0% [n = 36] versus 11.9 ± 15.6% [n = 12]; P = ) (Table 1). Samples were obtained from seven of 41 subjects (Pt-17, 18, 19, 20, 21, 22, and 23) both before and after treatment and all seven, except Pt-21, showed an increased frequency of TOS isolation after treatment. Some patients (Pt-18, 22, and 33) maintained high TOS isolation frequencies for more than 3 weeks after minocycline therapy. Although these frequencies varied widely among individuals, we did not find any correlation between post-treatment TOS isolation frequency and any factors that influenced changes in the spatial volume of subgingival pockets, such as the depth of the periodontal pockets or the number of teeth with deep pockets (Table 1). The variation among individuals may therefore be due to differences in the amount of ointment administered to each patient and/or individual differences in physiological properties, such as clearance efficacy by saliva or gingival crevicular fluid. In addition, the isolation frequency of TOS before minocycline therapy was not less than 30% in some patients (Pt-1, 8, 18, and 31) despite their lack of antibiotic use within the previous 6 months. This suggests that the fact that the isolation frequency of TOS may be high in some patients regardless of recent antibiotic use must be taken into account. To identify the species of TOS, we examined 44 isolates from 25 subjects, including 15 patients. A total of 35 of these isolates (79.5%) could be identified by biochemical methods and/or 16s rdna sequencing (Table 2). The most frequently identified species were S. salivarius, S. mitis, S. oralis, and S. sanguinis (Table 2). PCR using primer pairs to detect 19 different tet genes suggested that all TOS iso- 157

3 Table 1. Isolation frequency of TOS before and after administration of minocycline ointment Before treatment % of TOS After treatment Days after treatment Depth of No. of teeth periodontal with deep pockets 1) pockets 2) Pt-1 30 Pt Pt Pt Pt Pt Pt Pt-8 43 Pt Pt Pt Pt Pt Pt Pt Pt Pt A 3 Pt A 5 Pt B 23 Pt B 22 Pt A 3 Pt B 25 Pt B 12 Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt A 23 Pt Pt A 16 Pt Pt A 3 Pt B 22 Pt Pt A 13 Pt Average ± SD (%) 11.9 ± ± 24.0 Total number of patients ) : The depth of the deepest periodontal pocket: A, <6 mm; B, 6 mm. 2) : The number of teeth with deep pockets of 4 mm or more in depth. lates examined in this PCR analysis (49 TOS isolates from 16 individuals) possessed tetm (data not shown). Only tetm of the other tet genes was detected by PCR. Southern blot analysis using five TOS isolates revealed the presence of other Tn916-like elements responsible for the excision of Tn916, namely int and xis genes (data not shown). A previous study using saliva samples from healthy adults showed that tetracycline-resistant bacteria accounted for an average of 11% of the total viable oral microflora and that the most common tet gene identified was tetm (8). Our study supports this previous report regarding tetracycline-resistant oral bacteria and expands the current knowledge about TOS, namely that various TOS species can be isolated from both patients and healthy adults and that TOS frequently possesses the tetm gene along with other Tn916-like elements. It is noteworthy that three of the four dominant TOS strains, S. mitis, S. oralis, and S. sanguinis, are regarded as causative bacteria in heart diseases such as infectious endocarditis. Many different species of bacteria form complex communities on tooth surfaces, oral mucosa, and periodontal pockets, in other words oral biofilms. It has been suggested that such biofilms promote intimate contact between bacteria where genetic exchange frequently occurs. Various genetic elements, including Tn916, often carry both antibiotic- 158

4 Table 2. TOS isolated in this study ID tet-resistant streptococcus % Colony morphology Identification 1) Hv-1 ND 98.9 Small colonies with or without halos S. salivarius 1.1 Large, blue, dome-shaped mucoid gumdrop colonies b, s Hv-2 S. milleri group 0.4 Large, blue, dome-shaped mucoid gumdrop colonies b S. mitis 99.6 Small colonies s Hv-3 S. oralis Flat, transparent colonies b Hv-4 S. sanguinis 66.0 Small colonies with rough edges without halos b S. mitis 34.0 Flat and granulated colonies without halos b Hv-5 S. salivarius 1.7 Large and mucoid dome-shaped colonies s S. massiliensis 9.3 Small, flat and blue colonies s S. mutans 89.0 Small and rough colonies s Hv-6 S. mutans Small and rough colonies s Hv-7 ND no data Large and mucoid dome-shaped colonies S. sanguinis no data Small and domed colonies s Hv-8 S. salivarius 11.8 Large and mucoid dome-shaped colonies s S. sanguinis 88.2 Small and granulated colonies s Hv-9 S. mutans 86.0 Small and granulated colonies s ND 14.0 Small and flat colonies Hv-10 S. gordonii Small colonies; adhere tightly to MS agar s Pt-1 ND 21.8 Relatively large and granulated colonies ND 76.8 Small and domed colonies E. faecalis 1.4 Small and white colonies with halo b, s Pt-2 S. salivarius 46.5 Flat and large colonies s S. gordonii 2.5 Flat and small colonies s S. gordonii 51.0 Small and granulated colonies s Pt-3 S. salivarius Relatively large colonies with a hollow centers b, s Pt-4 S. oralis Small and granulated colonies b Pt-5 ND 16.9 Large, blue, dome-shaped mucoid gumdrop colonies S. oralis 83.1 Granulated colonies b Pt-10 ND 9.8 Large colonies S. salivarius 36.8 Large and flat colonies b S. oralis 53.4 Small colonies b Pt-11 S. oralis Small and flat colonies b Pt-12 S. mitis 90.6 Small and flat colonies b ND 9.4 Large colonies Pt-15 S. mitis Small colonies with rough edges b Pt-17 S. mitis Small colonies with rough edges b Pt-19 S. mitis Small colonies with rough edges s Pt-20 S. salivarius Flat, large, and mucoid dome-shaped colonies s Pt-21 S. salivarius 0.8 Large and flat colonies with halos s S. sanguinis 99.2 Small and flat colonies s Pt-22 ND 58.8 Small colonies with rough edges S. salivarius 35.3 Flat colonies; adhere tightly to MS agar b S. salivarius 5.9 Small and dome-shaped colonies b Pt-37 S. salivarius Large colonies s 1) : b, isolates identified biochemically. s, isolates identified by 16s rdna sequencing. resistance genes as well as other genes required for their excision from the host chromosome, circularization, conjugative transfer, and reinsertion into the chromosome of a new host. Oral bacteria that carry antibiotic-resistance genes on similar genetic elements therefore have the opportunity to propagate and transfer these genes to other pathogenic species in the oral environment. Although most patients with periodontal disease respond 159

5 well to conventional mechanical therapy, the empirical use of antibiotics against periodontal disease is still currently performed routinely. Our study strongly suggests that empirical antibiotic therapies for periodontal disease must be modified and that strict regulations governing the usage of antibiotics in dentistry are urgently needed. Acknowledgments We thank Dr. Souichi Furukawa for providing the information about bacterial identification. We also thank Dr. Taketo Kawarai and Dr. Saori Yoneda for technical assistance. This study was supported in part by Grants-in-Aid from the Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology of Japan ( and ), the Ministry of Health, Labour and Welfare (H19 Iryo-Ippan-007), and the Japan Health Science Foundation (No. KHC3308). Conflict of interest None to declare. REFERENCES 1. Chopra, I. and Roberts, M. (2001): Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev., 65, O Connor, B.C., Newman, H.N. and Wilson, M. (1990): Susceptibility and resistance of plaque bacteria to minocycline. J. Periodontol., 61, van Steenberghe, D., Rosling, B., Soder, B.O., et al. (1990): A 15- month evaluation of the effects of repeated subgingival minocycline in chronic adult periodontitis. J. Periodontol., 70, Paster, B.J., Boches, S.K., Galvin, J.L., et al. (2001): Bacterial diversity in human subgingival plaque. J. Bacteriol., 183, Aminov, R.I., Garrigues-Jeanjean, N. and Mackie, R.I. (2001): Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. Appl. Environ. Microbiol., 67, Ng, L.K., Martin, I., Alfa, M., et al. (2001): Multiplex PCR for the detection of tetracycline resistant genes. Mol. Cell Probes, 15, Roberts, A.P., Cheah, G., Ready, D., et al. (2001): Transfer of Tn916- like elements in microcosm dental plaques. Antimicrob. Agents Chemother., 45, Villedieu, A., Diaz-Torres, M.L. and Hunt, N. (2006): Prevalence of tetracycline resistance genes in oral bacteria. Antimicrob. Agents Chemother., 47,