Sulfadiazine on Streptococcus mutans and Lactobacilli in

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1976, P Copyright American Society for Microbiology Vol. 10, No. 2 Printed in U.S.A. Effect of Long-Term Therapies with Penicillin and Sulfadiazine on Streptococcus mutans and Lactobacilli in Dental Plaque H. G. WELD' AND H. J. SANDHAM* Department ofpreventive Dentistry, Faculty ofdentistry, University of Toronto, Toronto, Ontario M5G 1G6, Canada Received for publication 28 August 1975 Plaque samples were obtained from 13 children receiving long-term therapy with benzathine penicillin for the prevention of rheumatic fever recurrences, 31 children receiving oral sulfadiazine for the same purpose, and 29 untreated siblings. The therapies were found to have no effect upon the proportions of Streptococcus mutans or lactobacilli in dental plaque, upon the percentage of children harboring the organisms, nor upon the susceptibility of the organisms to penicillin and sulfadiazine. Of the S. mutans strains tested, 97% had a minimnal inhibitory concentration of penicillin G of less than 48 ng/ml and, of the lactobacillus strains tested, 96.8% had a minimal inhibitory concentration of less than 1,600 ng/ml. All strains of both organisms were profoundly resistant to sulfadiazine. Children receiving 200,000 U of oral penicillin daily for rheumatic fever prophylaxis have been reported to experience reductions in the incidence of dental caries of up to 56% as compared with their untreated siblings (13, 18). Handelman and Hawes have reported that the oral penicillin therapy had no effect on the number of salivary lactobacilli (12) and that the penicillin resistances of the total flora, the streptococci, and the staphylococci in the salivas of the antibiotic users were higher than those in the untreated siblings (11). They did not study the effect of oral penicillin on Streptococcus mutans; consequently, it is not known whether the caries-inhibiting effect of that agent (13) resulted from its action on that organism. Furthermore, they did not examine the dental plaque flora, which is more intimately associated with the development of the carious lesion than is the salivary flora. Treatment currently used at the Toronto Hospital for Sick Children for the prevention of rheumatic fever recurrences consists of either a monthly injection of 1.2 x 106 U of benzathine penicillin or a daily oral administration of 1 g of an etiological factor in dental caries (8). Lactobacilli have been implicated in dental caries etiology for many years (15). MATERIALS AND METHODS Study groups. The subjects examined were outpatients of the Division of Cardiology, Hospital for Sick Children, Toronto, and their siblings. All children attending the clinic in the 8-month period from June 1974 to February 1975 and whose parents consented were included in the study. The experimental groups consisted of a group of 13 children receiving one injection of benzathine penicillin per month and a second group of 31 receiving daily oral sulfadiazine. A group of 29 siblings served as controls. All subjects were between 7 and 21 years of age. An attempt was made to equalize the ages of children in the treatment and control groups by asking the parent to bring in the sibling nearest in age to the treated child. Plaque sampling. Because many children in the penicillin group received their injections from private practitioners, it was not possible to standardize the time interval between the penicillin injection and plaque sampling. In the majority of cases the interval was between 2 weeks and 1 month. The sulfadiazine. This report describes a study of plaques of sulfadiazine children were sampled approximately 6 h after they had received the the effects of these therapies on the proportions drug. Three plaque samples were obtained from each of and antimicrobial susceptibility of S. mutans the subjects: one from occlusal pits and fissures, one and lactobacilli isolated from dental plaque. S. from vestibular smooth surfaces, and one from approximal surfaces. Each sample consisted of plaque mutans was selected because it is a plaque organism that has been strongly implicated as pooled from all of the posterior teeth in the mandibular left quadrant. If two or more teeth were missing, the mandibular right quadrant was used. To ' Present address: Faculty of Dentistry, McGill University, P. 0. box 6070, Montreal, Quebec H3C 3G1, Canada. minimize salivary contamination, all surfaces were 200

2 VOL. 10, 1976 briefly air dried with a rubber bulb syringe before sampling. Samples, collected with sterile sharp explorers (Stardent no. 5, Star Dental Manufacturing Co., Philadelphia, Pa.), were immediately placed into reduced transport fluid (25). Microbiological methods. Plaque samples, returned to the laboratory within 2 h of collection, were dispersed under a 95% nitrogen-5% carbon dioxide mixture with a sonic probe (Biosonik IV, Bronwill Scientific Inc., Rochester, N.Y.) for 10 s at maximum power. Sample tubes were cooled in an ice water bath during the dispersion. Tenfold serial dilutions in reduced transport fluid were plated in duplicate on brain heart infusion agar (Difco) supplemented with 5% defibrinated sheep blood (BHI-blood agar), on mitis salivarius (MS) agar (Difco), and on Rogosa SL agar (Difco). These media were used for the enumeration of total cultivable flora, of total streptococci and S. mutans proportions, and of lactobacilli, respectively. A plate of mitis salivarius-bacitracin (MSB) agar (9) was also inoculated to obtain adequate numbers of S. mutans for susceptibility testing. All bacteria in the study were grown in airtight containers in an atmosphere of 95% nitrogen and 5% carbon dioxide at 37 C. MS and MSB agar plates were incubated for 18 h; BHI-blood agar and Rogosa SL plates were incubated for 72 h. MS and MSB agar plates were subsequently incubated aerobically at room temperature for 24 h before counting (16). Enumeration. Counting was carried out on plates containing between 75 and 150 colonies, whenever possible. On MS agar, both rough and mucoid colonies of S. mutans were counted (4, 14). On Rogosa SL agar, Lactobacillus colonies were counted as described previously (15). Susceptibility testing. To assess susceptibility of the organisms to penicillin and sulfadiazine, colonies were chosen at random from the MSB and Rogosa SL plates. Nine colonies ofs. mutans and six to nine colonies of lactobacilli were picked per subject, placed into 1 ml of Mueller-Hinton broth (Difco) supplemented with 0.1% glucose and 0.25% K2HPO4, and incubated for 18 h as described above. A suspension containing approximately 107 colony-forming units/ml was distributed with a replicator (model R- 2, Bio-Tech Co., Lynden, Ontario) onto plates of Mueller-Hinton medium (Difco), supplemented with 5% defibrinated sheep blood and dilutions of potassium penicillin G (Connaught Laboratories Ltd., Toronto), or sulfadiazine (Solu-Diazine, Poulenc, Montreal). Control plates free of the antimicrobial agents were also inoculated. The plates were incubated for 18 h and qualitatively assessed for growth (+) or no growth (-) (5). The lowest concentration of antimicrobial agent preventing growth was considered to be the minimal inhibitory concentration (MIC). Analysis of data. Quantitative data, such as the age and sex of the subjects and duration of therapy and the frequency of isolation and prevalence of streptococci and S. mutans, were compared by means of t tests. Qualitative data, such as the proportions of subjects and surfaces harboring either ANTIMICROBIAL THERAPY AND PLAQUE BACTERIA 201 lactobacilli or S. mutans, were compared by means of chi-square tests of confidence (22). Effects of age, sex, and duration of therapy on S. mutans proportions in plaque were analyzed by regression analysis (22). RESULTS Study groups. Despite our efforts to equalize the ages in the treatment and control groups, the mean ages of children in the treatment groups were approximately 2 years greater than that of the control group. The mean age in years, standard errors, and ranges of ages in the various groups were as follows: penicillin group, 14.3 ± 0.73 (7.4 to 18.5); sulfadiazine group, 14.5 ± 0.50 (9.8 to 20.4); and control group, 12.5 ± 0.52 (7.5 to 20.0). The mean duration of therapy was greater in the sulfadiazine group (6.1 ± 0.47 years; range, 1 to 13 years) than in the penicilllin group (4.1 ± 0.80 years; range, 1 to 8 years). In the sulfadiazine group, the numbers of males and females were nearly equal (51.8% males), but in the penicillin group males predominated (69.2%) and in the control group females predominated (58.6%). Effect of therapy on the frequency of isolation of streptococci. Comparing the mean percentages of streptococci in the plaques of subjects from the treatment and control groups (Table 1), the only statistically significant difference observed was that plaques from the penicillin-treated children contained a higher proportion of streptococci than did plaques from the control group (t = 2.49; P < 0.05). The differences on specific surfaces between either treatment group and the control group were not statistically significant. Effect on the incidence and prevalence ofs. mutans. S. mutans numbers, expressed as a percentage of the total streptococci (Table 2), in the groups receiving therapy did not differ significantly from those in the control group when either all plaques or plaques from specific tooth surfaces were compared. A lack of effect of therapy was also observed when S. mutans numbers were expressed as the percentage of the total cultivable flora. The proportions of S. mutans present on the occlusal surfaces in all groups (approximately 30% of the total streptococci) were greater than those for the vestibular and approximal surfaces (between 2.1 and 7.4%). Statistical analyses showed no significant correlation between (i) proportions of S. mutans in plaques of control children and age of the children, (ii) proportions of S. mutans in the plaques and sex of the subjects, (iii) propor-

3 202 WELD AND SANDHAM tions ofs. mutans in plaques of subjects in each of the treatment groups and length of therapy, and (iv) the percentage of subjects harboring S. mutans in one or more of their plaque samples and drug therapy. The percentage of children with S. mutans-infected plaque did not differ significantly among the control group (93.1%), the penicillin group (84.6%), and the sulfadiazine group (87.1%). Effect on the incidence and prevalence of lactobacilli. The percentage of the children harboring lactobacilli in one or more of their plaque samples (approximately 30%) did not differ significantly between treatment and control groups (P > 0.05). The percentage of specific tooth surfaces harboring lactobacilli also did not differ between groups. Neither age of subjects nor duration of therapy had any demonstrable effect on the proportions of subjects harboring lactpbacilli. Drug administration also had no effect on the proportions of lactobacilli, either in all plaques or in plaques from specific surfaces. The proportions of lactobacilli in plaques were low; in 61.3% of the samples from which lactobacilli were identified, they comprised less than 0.01% of the total cultivable flora. In no instance did they comprise more than 4.0% of the flora. Effect on the susceptibility of S. mutans and lactobacilli. A total of 80 strains of S. mutans from subjects receiving penicillin and 168 strains from control subjects were compared for their susceptibility to penicillin; no significant differences in susceptibility were observed. An MIC of 24 or 48 ng of penicillin per ml was observed for 100 and 96.4% of isolates from the respective groups. The growth of the ANTIMICROB. AGENTS CHEMOTHR. remaining strains was prevented by 100 ng/ml. All 18 strains of lactobacilli isolated from the penicillin group had an MIC of 1,600 ng of penicillin per ml, more than 30-fold higher than that ofs. mutans. Most of the 34 Lactobacilhl strains isolated from the control group also had an MIC of 1,600 ng/ml, but 8.8% had an MIC of 100 ng/ml and 29.4% had an MIC of 400 nglml. None of the lactobacilli had an MIC greater than 1,600 ng/ml. A total of 76 isolates of S. mutans and 59 isolates of lactobacilli were obtained from all study groups and tested for their susceptibility to sulfadiazine; all demonstrated a high level of resistance by growing in the presence ofconcentrations greater than 3.0 mg/ml. DISCUSSION The results of this study do not support the possibility that long-term therapy with benzathine penicillin or with sulfadiazine might decrease the cariogenicity of the plaque microflora. Neither drug caused a significant alteration in the proportions ofs. mutans or lactobacilli, the organisms most commonly implicated as etiological agents in dental caries (8, 15). The lack of effect of the berzathine penicillin therapy on the proportions of these bacteria in plaque, despite the high susceptibility to penicillin demonstrated by the organisms in vitro (see also 1, 6, 7), suggests that the therapy results in negligible levels of penicillin in saliva. The lack of development of resistance by the organismns to penicillin supports this contention. A similar conclusion was reached by previous workers (23) on the basis of findings that benzathine penicillin has an effect on the TABLE 1. Percentages of streptococci in the total cultivable flora ofplaque samples from children receiving various therapies Streptococci (%) on tooth surfaces: Group All ocelusal Vestibular Approximal Penicillin 34.7 ± 4.3" 47.1 ± ± ± 6.3 Sulfadiazine 26.7 ± ± ± ± 3.7 Control 22.7 ± ± ± ± 3.8 a Mean ± standard error. TABLz 2. Percentages ofs. mutans in the total streptococci ofplaque samples from children receiving various therapies S. mutans" (%) on various toothasurfices: Group All Occlusal Vestibular Approximal Penicillin 14.6 ± 4.0b 30.8 ± ± ± 3.7 Sulfadiazine 12.1 ± ± ± 1.9 Control 15.4 ± ± ± ± 2.7 a Expresed as a percentage of the total cultivable streptococci. b Mean ± standard error.

4 VOL. 10, 1976 penicillin resistance of the flora in the throat, saliva, and gingival margin (2, 19, 23). In contrast, Handelman and Hawes (11) have observed that, in children receiving daily oral penicillin, the penicillin resistance of the salivary flora, including the streptococci, is higher than that in untreated siblings. This suggests that the caries inhibitory effect of the therapy (13) was associated with appreciable salivary levels of the agent. They did not determine the penicillin resistance of lactobacilli in their subjects, but their observation that the numbers of lactobacilli were unaltered by the therapy suggests that organism was resistant to the concentration of penicillin present in the saliva. Since they did not examine the antibiotic resistance or numbers of S. mutans, the possibility that the caries inhibitory effect of the therapy (13) was due to its effect on that organism is unknown. Nevertheless, our observations that S. mutans is approximately 30-fold more susceptible to penicillin than are lactobacilli and that S. mutans is more intimately involved with the initiation of the carious lesion (15) lend weight to that premise. The mean length of time that the patients receiving benzathine penicillin had been under therapy in our study, 4.1 years, was considerably longer than that of the subjects receiving oral penicillin in the study of Handelman and Hawes (11) and should have been adequate to demonstrate any long-term effect. On the other hand, the small number of patients available in our study would have prevented the detection of small differences between the experimental groups. Imbalances were also present in ages and sex between the control and experimental groups but did not appear to significantly affect the results. An unexpected finding in the present study, that the penicillin group demonstrated a higher proportion of streptococci than did the controls, suggests that, if penicillin had an effect on the plaque flora, it was on components of the flora other than the streptococci. The reason for this observation is not apparent at the present time. Long-term sulfadiazine therapy did not have a significant effect on the proportions of S. mutans or lactobacilli in plaque. The result with S. mutans was particularly surprising since an earlier study (26), using an in vitro quantitative assessment of S. mutans growth, had shown that low levels of sulfadiazine (1 to 10 jig/ml) inhibited growth, even though the organisms were resistant to the agent as judged by usual qualitative susceptibility testing procedures (3, 20, 21). It would seem that, if such an inhibition occurs in vivo, it was not of sufflcient magnitude to significantly alter the proportions ANTIMICROBIAL THERAPY AND PLAQUE BACTERIA 203 of that organism in plaque. Whether the lack of effect of sulfadiazine therapy in our study may have been due in part to a lack of patient compliance was not determined. Lack of patient compliance to long-term therapeutic regimes is a well-known problem (24, 27, 28). The apparent lack of effect of prolonged benzathine penicillin and sulfadiazine therapies on the proportions of S. mutans and lactobacilli in plaque suggests, but does not prove, that the therapies are ineffective in reducing dental caries activity. More substantial proof must be provided by clinical caries studies. The present study also confirmed previous reports that occlusal pits and fissures are a major reservoir of S. mutans in permanent teeth (15), that the majority ofyoung people are infected with that organism (15), and that lactobacilli are a numerically minor component of dental plaque over noncarious sites, although they are present in the plaques of a substantial number of children (10, 15, 17). ACKNOWLEDGMENTS We thank R. D. Rowe, Langford Kidd, R. B. Ross, and the staff of the Division of Cardiology, Toronto Hospital for Sick Children, for their cooperation in the study. The assistance of J. Bennett in administering the clinical aspects of the study, R. P. Ellen and R. C. Burgess in reviewing the manuscript, G. W. Thompson in the statistical analyses, and M. Villari in typing is gratefully acknowledged. This study was assisted under the Province of Ontario Health Research grant no. P.R Interim support at the beginning of the study was provided by the R. Samuel McLaughlin Foundation. H.G.W. was the recipient of a fellowship from the Canadian Fund for Dental Education. LITERATURE CITED 1. Baker, C. N., and C. Thornsberry Antimicrobial susceptibility of Streptococcus mutans isolated from patients with endocarditis. Antimicrob. Agents Chemother. 5: Bentley, D. W., T. Frank, and J. J. Hahn Penicillin-resistant oral streptococci, p Antimicrob. Agents Chemother Carlsson, J A numerical taxonomic study of human oral streptococci. Odontol. Revy 19: Edward6son, S The caries-inducing property of variants of Streptococcus mutans. Odontol. Revy 21: Ericsson, H. M., and J. C. Sherris Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathol. Microbiol. Scand. Sect. B; Suppl Fitzgerald, R. J Inhibition of experimental dental caries by antibiotics. Antimicrob. Agents Chemother. 1: Fitzgerald, R. J., H. A. Zander, and H. V. Jordan The effects of a penicillin dentifrice on oral lactobacilli. J. Am. Dent. Assoc. 41: Gibbons, R. J., and J. van Houte On the formation of dental plaques. J. Periodontol. 44: Gold, 0. G., H. V. Jordan, and J. van Houte A selective medium for Streptococcus mutans. Arch. Oral Biol. 18:

5 204 WELD AND SANDHAM 10. Green, G. E., M. C. Dodd, and H. S. Inverso Comparative microflora of developing dental plaques in caries-immune and susceptible individuals. J. Dent. Res. 36: Handelman, S. L., and R. R. Hawes The effect of long-term antibiotic therapy on the antibiotic resistance of the salivary flora. J. Oral Ther. Pharmacol. 1: Handelman, S. L., and R. R. Hawes The effect of long-term systemic antibiotic administration on the numbers of salivary organisms. Arch. Oral Biol. 10: Handelman, S. L., J. R. Mills, and R. R. Hawes Caries incidence in subjects receiving long-term antibiotic therapy. J. Oral Ther. Pharmacol. 2: Ikeda, T., T. Mukasa, S. Yagi, K. Ochiai, Y. Doi, T. Shiota, and H. J. Sandham The characteristics of Streptococcus mutans PS-14. Nihon Univ. J. Oral Sci. 1: Ikeda, T., H. J. Sandham, and E. L. Bradley, Jr Changes in Streptococcus mutans and lactobacilli in plaque in relation to the initiation of dental caries in Negro children. Arch. Oral Biol. 18: Jordan, H. V., B. Krasse, and A. Moller A method of sampling human dental plaque for certain "caries-inducing" streptococci. Arch. Oral Biol. 13: Krasse, B The relationship between lactobacilli, Candida and streptococci and dental caries. Odontol. Revy 5: Littleton, N. W., and C. L. White Dental findings from a preliminary study of children receiving extended antibiotic therapy. J. Am. Dent. Assoc. 68: Patterson, P. Y., and G. M. Madden Occurrence and erythromycin susceptibility of penicillin-resistant viridans streptococci in rheumatic fever patients on oral penicillin prophylaxis, p Antimicrob. Agents Chemother ANTIMICROB. AGENTS CHEMOTHER. 20. Perch, B., E. Kjems, and T. Raun Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta Pathol. Microbiol. Scand. 82: Rogers, A. H The proportional distribution and characteristics of streptococci in human dental plaque. Caries Res. 3: Snedecor, G. W., and W. G. Cochran Statistical methods, 6th ed. Iowa State University Press, Ames. 23. Spencer, W. H., III, C. Thornsberry, M. D. Moody, and N. K. Wenger Rheumatic fever chemoprophylaxis and penicillin-resistant gingival organisms. Ann. Intern. Med. 73: Stollerman, G. H., J. H. Rusoff, and I. Hirschfeld Prophylaxis against group A streptococci in rheumatic fever patients by the use of single monthly injections of N,N' dibenzylethylenediamine dipenicillin G (bicillin). Antibiot. Annu. 1: Syed, S. A., and W. J. Loesche Survival of human dental plaque flora in various transport media. Appl. Microbiol. 24: Weld, H. G., and H. J. Sandham In vitro effects of low concentrations of penicillin and sulfadiazine on Streptococcus mutans. Antimicrob. Agents Chemother. 10: Wood, H. F., A. R. Feinstein, A. Taranta, J. A. Ep. stein, and K. Simpson Rheumatic fever in children and adolescents. II. Comparative effectiveness of three prophylaxis regimes in preventing streptococcal infections and rheumatic recurrences. Ann. Intern. Med. 60 (Suppl. 5): Wood, H. F., G. H. Stollerman, A. R. Feinstein, I. Hirschfeld, J. H. Rusoff, A. Taranta, R. C. Hoas, and J. A. Epstein A controlled study of three methods of prophylaxis against streptococcal infection in a population of rheumatic children. I. Streptococcal infections and recurrences of acute rheumatic fever in the first two years of the study. N. Engl. J. Med. 257: