(Ballows 1991, Cowan and Steele 1993). Statistical analysis ... SUBGINGIVAL. SYSTEMIC. BASELINE 2V\^EKS 1 MO. 3 MO. 9 MO. 12 MO. Fig, 1, The median ...
Copyright
J ctin Periodontol 1995: 22: 380-384 Printed in Denmark . Alt rights reserved
© Munksgaard
1995
dinical podintoiojijr ISSN 0303-6979
Bacterial resistance following subgingival and systemic administration of minocycline
Hans R. Preus, Jergen Lassen\ Anne M.Aass and Sebastian G. Ciancio^ Department of Periodontology, School of Dentistry, University of Oslo, Norway, ^Department of Bacteriology, National Institute of Public Health, Oslo, Norway, ^Department of Periodontology, School of Dental Medicine, SUNY at Buffalo, USA
Preus HR, Lassen J, Aass AM, Cianeio SG: Bacterial resistance following subgingival and systemic administration of minoeydine. J Clin Periodontol 1995; 22: 380-384. © Munksgaard, 1995. Abstract. The aim of the present study was to compare total numbers of cultivable bacteria and prevalence of resistance to minocycline among periodontal bacteria following subgingival or systemic application of minocycline in patients suffering from periodontal disease. 10 adult patients were administered 2% minocycline ointment subgingivally into their periodontal pockets at baseline, week 2 and months 1, 3, 6 and 9. Patients had scaling/root planing at baseline and month 6. In addition, 10 patients undergoing scaling/root planing followed by a 10-day course of systemic minocychne therapy, were studied and compared with the subgingival application group. Bacterial samples were taken from the 4 deepest pockets before each subgingival application of the drug. The systemic administration group was sampled at baseline and at week 2, as well as months 1 and 3 after completing the antibiotic treatment. For each patient at each sampling, bacterial samples were pooled, diluted, seeded on parallel blood agar plates and incubated aerobically and anaerobically. After incubation, 30 colonies were picked at random and transferred to blood agar plates supplemented with 10 /^g/ml minocycline, to estimate prevalence of minocycline-resistant bacteria. The results of this study indicate that subgingival application of minocycline ointment resulted in an initial reduction in total numbers of cultivable bacteria, which then remained depressed during the full year of the study. No such observation was made in the systemic administration. Both in the subgingival and the systemic administration group, the % of cultivable aerobic and anaerobic minocyclineresistant bacterial strains increased transiently following administration of the drug, but returned to baseline levels within 3 months post-treatment. The median of cultivable aerobic minocycline-resistant bacterial strains decreased or remained unchanged in both groups during the study periods. Thus, subgingival application of minocycline seems no more likely than systemic administration of the drug to select for an increased proportion of resistant organisms in the periodontal pockets.
Tetracyclines possess broad spectrum bacteriostatic activity as a result of inhibition of protein synthesis. They have been used extensively for treatment of bacterial infections in humans and other animals, an give rise to little toxicity at therapeutic concentrations (Levy 1988). Moreover, drugs ofthe tetracycline class have been widely recommended for treatment of periodontal diseases, but their potential efficacy has been limited in recent years by the in-
creasing prevalence of bacterial antibiotic resistance (Levi 1988). Minocycline is a semisynthetic derivate of tetracycline which is commonly prescribed for treatment of various human oral and extraoral infections (O'Connor et al. 1990, Eady et al. 1990, Cullen et al. 1976). In vitro studies have shown that minocycline is capable of inhibiting many of the bacteria associated with periodontal diseases and may kill some of these bacteria after a compara-
Key words: local antibiotics; oral bacteria; antibiotic resistance Accepted for publication 21 May 1994
tively short exposure time (O'Connor et al. 1990). However, few studies have examined the potential for selection of resistant oral microorganisms during minocycline therapy (O'Connor et al. 1990, Baker et al. 1985a,b, Ciancio et al. 1982). Recently, minocycline has been formulated as an ointment (10 mg/0.5 g ointment) for subgingival application in patients with periodontal diseases. When introduced into the periodontal
Antibiotic resistance in periodontal bacteria pocket by a specially designed applicator, extremely high local concentration ofthe drug results. Satomi et al. (1987) measured gingival crevicular fluid (GCF) concentrations in excess of 1000 //g/ml 1 h after subgingivai application of 0.05 ml minocydine gel. This concentration is over 100 X the minimum inhibitory concentrationgo (MIC90) required for common periodontal pathogens. GCF concentrations in excess of 100 //g/ml were maintained through the first 6 h. Even at 48 h, the mean GCF minocydine concentration was sufficient to inhibit most, if not all, of the periodontopathogenic bacteria. The release of the drug into the oral cavity after deposition in the periodontal pocket, creates a steady decrease of the antibiotic concentration at the sites of application, so that low concentration of the drug persists subgingivally for several days. Thus, this subgingivai periodontal formulation could theoretically induce or select for microorganisms resistant to minocydine and other tetracychnes among the periodontal and oral flora. The aim of this study was to evaluate the potential of this new subgingivai formulation to select for minocyclineresistant subgingivai bacteria in patients suffering from periodontal disease. Material and Methods
10 adult patients (35-63 years of age) with moderate to severe periodontitis, who were enrolled in a chnicai trial designed to evaluate the safety and efficacy of 2% minocydine ointment, voluntered for the study. 10 patients (ageand sex-matched), undergoing scaling/ root planing with adjunct systemic minocychne therapy, were also studied as a comparison. None of the patients had received any antibiotic treatment during the last 6 months prior to enrollment. In each patient, 1 tooth in each quadrant with a periodontal pocket with loss of attachment (LA) ^5 mm was selected for the experiment. In the subgingivai treatment group, the patients had scahng/root planing at baseline and 6 months. 0.5-1 mg of minocydine was administered subgingivally into each of the 4 deepest pockets at baseline (after scaling/root planing), at 2 weeks and months 1, 3, 6 and 9. Bacterial samples were obtained immediately prior to application of the drug, by inserting two paper points into each of the 4 pockets.
At baseline and 6 months, the samples were taken after scaling/root planing. In the systemic administration group, the patients received scaling/root planing at baseline followed by a 10-day course of oral minocydine (50 mg in the morning and 100 mg in the evening). Bacterial samples were obtained at baseline, 2 weeks, 1 and 3 months post-treatment by inserting 2 paper points into each of the 4 selected pockets in each patient. Separate paper points from each of the four pockets, in each patient, were pooled in either PRAS Ringer or Ringers solution. After serial 10-fold dilutions, seeding was performed on to parallel plates of freshly made blood agar with or without 10 //g/ml mitiocycline, which were incubated aerobically (in air) for 3-5 days and anaerobically (10% H2, 10%, 80% N,) at 35°C for 5-7 days. 30 colonies were randomly picked from each non-selective plate, transferred to blood agar containing 10 //g/ ml minocydine, and incubated aerobically (3-5 days) and anaerobically for (5-7 days) days. After incubation, the CFU, indicating the number of minocycline-resistant bacteria in the samples were calculated, and the different bacterial species subcultured and identified at least to their genus level (Ballows 1991, Cowan and Steele 1993). Statistical analysis were performed using the ANOVA-test; split plot design (Fdwards 1979). The levd of statistical significance was set at 0.05. Results In the subgingivai application group, total numbers of bacteria showed an initial decrease from 10- per ml at baseline to 10"* per ml after 3 months, and remained at this magnitude for the rest of the study. The percentage of aerobic minocycline-resistant colony forming units (CFU) increased from 0.5% at baseline to 3.3% after 2 weeks, but then fell to 0.1% at 1, 9 and 12 months (Fig. 1). Corresponding figures for resistant anaerobes were 0.12%, 0.45%, 0.40%, 0.125%) and 0.14%. respectively (Fig. 2). Numbers of different aerobic bacterial species decreased from a median of 5.5 (range: 1-11) at baseline, to 2 or less (range 0-5) throughout the observation period (Table la). A similar pattern of reduction and stabilization of numbers of minocycline-resistant anaerobic species was recorded (Table 2a). In the systemic administration group.
381
Table 7. (a) The median and mean of different aerobic bacterial strains in the subgingivai application group, at baseline, 2 weeks, 1-, 9and 12 months Time baseline 2 weeks 1 month 9 months 12 months
Median
Mean
Range
5.5 2.0 1.0 1.5 1.5
5.9 1.8 1.1 1.9 1.8
l-ll 1-4 0^
1-5 1-5
ANOVA-paired comparison baseline - end; p
