AWrMICROBIAL AGozm AND CHzmOTHHRAP, Aug. 1976, p. 274-276 Copyright © 1976 American Society for Microbiology
Vol. 10, No. 2 Printed in U.S.A.
Susceptibility of Streptococcus mutans to Antimicrobial Agents JOSEPH J. FERRETTI* AND MARGIE WARD Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 Received for publication 23 February 1976
Fifty strains of Streptococcus mutans, including defined strains and clinical isolates, were tested for susceptibility to 20 different antimicrobial agents. Minimal inhibitory concentrations were determined by a liquid microtiter procedure. Antibiotics that were most effective in concentrations below 0.1 ,ug/ml included penicillin, ampicillin, erythromycin, cephalothin, and methicillin. Antibiotics effective in concentrations between 0.1 ug and 10 ,ug/ml included rifampin, lincomycin, thiostrepton, spiromycin, vancomycin, streptolydigan, novobiocin, tetracycline, chloramphenicol, spectinomycin, and gentamicin. Antibiotics effective at higher concentrations ranging from 10 ,g/ml to 400 ,ug/ml included the aminoglycosides kanamycin, neomycin, streptomycin, and kasugamycin. Although most antibiotics exhibited inhibitory effects in a narrow range of concentrations, antibiotics such as tetracycline, thiostrepton, and spiromycin had a 1,000-fold range from the lowest to highest concentrations required for growth inhibition.
Interest in the organism Streptococcus mutans is closely associated with its involvement in dental caries. This organism is also involved as a causative agent of endocarditis (1, 3). Knowledge of the antibiotic susceptibility patterns of S. mutans is of importance for proper therapy in the cases of endocarditis because S. mutans endocarditis has been *in some instances confused with group D enterococcal endocarditis (3). In one recent report, approximately 5% of streptococcal endocarditis isolates studied were S. mutans. (2). Baker and Thornsberry (2) have reported the patterns of susceptibility of S. mutans, isolated from patients with endocarditis, to 11 antibiotics. In this study we have determined the susceptibility of 50 strains of S. mutans, isolated from dental plaque, to 20 different antimicrobial agents. MATERIALS AND METHODS Bacterial strains. The bacterial strains used in this study were obtained from the dental clinics of the University of Oklahoma College of Dentistry and from C. Schaechtele, the University of Minnesota. Of the 50 strains of S. mutans, 6 were defined strains representing each known serotype and included the following: LM7, SL-1, 6715, Ingbritt, BHT, and OMZ-61. The remaining strains were isolated from samples of dental plaque and were identified according to (i) colony morphology on Mitis Salivarius agar, (ii) fermentation of mannitol and
sorbitol, and (iii) extracellular polysaccharide production in 5% sucrose broth. Antibiotics. The antibiotics used and the sources from which they were obtained were: erythromycin, kanamycin, neomycin, novobiocin, and tetracycline (Sigma Chemical Co., St. Louis, Mo.); ampicillin, kasugamycin, and methicillin (Bristol Laboratories, Syracuse, N.Y.); lincomycin, spectinomycin, and streptolydigan (Upjohn Co., Kalamazoo, Mich.); cephalothin and vancomycin (Eli Lilly & Co., Indianapolis, Ind.); penicillin and streptomycin (Nutritional Biochemical Corp., Cleveland, Ohio); rifampin (Becton-Dickinson & Co., Rutherford, N.J.); chloramphenicol (Parke, Davis & Co., Detroit, Mich.); spiromycin (Rhodia Inc., New York); gentamicin (Schering Laboratories, Bloomfield, N.J.); and thiostrepton (E. R. Squibb & Sons, New York). Media. The standard liquid medium used in this study was proteose peptone broth. It contained 6% proteose peptone no. 3 (Difco), 0.3% NaCl, and 0.38% Na2HPO4. After sterilization the broth was completed by the addition of three more components at the following final concentrations: 0.02% CaCl2, 0.05% glucose, and 5% normal horse serum (GIBCO). Trypticase soy broth (BBL) was also used in the initial portions of the study. Susceptibility tests. Each strain of S. mutans was streaked onto Mitis Salivarius agar plates, incubated at 37°C in an anaerobic jar for 24 h, and then allowed to incubate at 37°C aerobically for an additional 24 h. Several colonies with similar morphology were picked and inoculated into proteose peptone broth. After overnight incubation at 37°C, 0.1 ml of each culture was inoculated into a tube containing 10 ml of proteose peptone broth. To sterile 274
VOL. 10, 1976
ANTIMICROBIAL SUSCEPTIBILITY OF S. MUTANS
microdilution plates containing appropriate serial dilutions of antibiotic in proteose peptone broth, 0.05 ml of the overnight diluted culture was added. Each plate was covered with clear plastic and incubated at 37°C for 48 h. Unless otherwise specified, all incubations were performed aerobically. In instances when anaerobic conditions were maintained, anaerobic Gas-Pak (BBL) jars were utilized. The minimal inhibitory concentration (MIC) was the lowest concentration of antibiotic that caused complete inhibition of growth. RESULTS
275
concentration required for growth inhibition. With two of the antibiotics, rifampin and lincomycin, there was a 250-fold range from the lowest to highest concentration required for growth inhibition. In Fig. 2 the concentration of antibiotics ranged from 0.01 to 10 ,ug/ml and included streptolydigan, thiostrepton, spiromycin, kanamycin, and novobiocin. A 1,000-fold range in the concentration for inhibition of growth was observed with thiostrepton and spiromycin. In Fig. 3 the concentration of antibiotics ranged from 0.05 to 400 ,ug/ml and included tetracycline, chloramphenicol, spectinomycin, gentamicin, kanamycin, neomycin, streptomycin, and kasugamycin. A 1,000-fold range in the concentrations of tetracycline required for inhibition of growth was observed, whereas there was a more narrow range of inhibition for all other antibiotics.
Growth of the various strains of S. mutans supported best in proteose peptone broth containing 5% horse serum. In comparison with other media, including Trypticase soy broth, growth of all bacterial strains was sufficient in 24 h to determine the MIC of a particular antibiotic within one dilution. To insure that maximal growth occurred, however, final MICs were routinely determined after 48 h of incubation. DISCUSSION There was some question whether there were any differences in growth or inhibition by antiThe purpose of this investigation was to test biotics when the organism was incubated under the susceptibility of strains of S. mutans isoaerobic or anaerobic conditions. To answer this la question the MICs of several antibiotics (cephalothin, methicillin, and vancomycin) were determined with 50 different strains of S. mutans C/, incubated under both aerobic and anaerobic z conditions. The results from these experiments C/,Iare given in Table 1. The average MICs obtained for each antibiotic were essentially iden- -im tical with either aerobic or anaerobic incubation and indicated that the atmosphere had little effect on the growth of the organism or its cn be susceptibility to antibiotics. The MICs for inhibition of the 50 strains, plotted as a function of the percentage of susceptible strains, are shown in Fig. 1 through 3. .0005 .001 .002 .004 .008 .015 .031 .063 .125 .250 .5 1.0 2.0 In Fig. 1 the concentration of antibiotics ranged MINIMAL INHIBITORY CONCENTRATION from 0.0005 to 2 jig/ml and included penicillin, OF ANTIBIOTIC (ig/ml) ampicillin, erythromycin, cephalothin, methiFIG. 1. Susceptibility ofS. mutans strains to pencillin, rifampin, and lincomycin. The inhibitory icillin (), erythromycin (0), ampicillin (a), cephaconcentrations of most of the antibiotics cov- lothin (a), rifampin (A), methicillin (A), and lincoered a 10-fold range from the lowest to highest mycin (*). was
L&J
C.)
TABLE 1. Minimal inhibitory concentrations (MICs) ofthree antibiotics for 50 strains of S. mutans incubated under aerobic and anaerobic conditions Antibiotic Percentage of strains with MIC (yg/ml) of: Cephalothin (0.25) (0.125) (0.063) (0.031) (0.015) (0.008) (0.004) (0.002) (0.001) (0.0005) 0 0 Aerobic 4 40 0 13 19 24 0 0 0 0 0 0 Anaerobic 2 2 36 15 30 15 Methicillin (0.25) (0.125) (0.063) (0.031) (0.015) (0.008) (0.004) (0.002) (0.001) (0.0005) 0 0 Aerobic 34 0 0 0 0 13 43 10 0 0 0 0 42 0 Anaerobic 11 2 0 45 Vancomycin (10) (5) (2.5) (1.25) (0.625) (0.313) (0.157) (0.08) (0.04) (0.02) Aerobic Anaerobic
0 0
0 0
0 0
2 2
4 8
28 26
36 21
28 32
2 11
0 0
276
FERRETTI AND WARD
ANTIMICROB. AGENTS CHEMOTHER.
endocarditis strains due to prior chemotherapy. Further, it excludes antimicrobial susceptibility patterns as contributory to the ability of plaque-forming organisms to establish infection in the heart. The results of this study also increase the number of antimicrobial agents cn60 with known efficacy against these organisms. CLJ Antimicrobial agents that were particularly effective at low concentrations (below 0.1 ug/ ml) included penicillin, ampicillin, erythromy(n) cin, cephalothin, and methicillin. Each ofthese antibiotics inhibited all of the strains tested in a narrow concentration range. Two antibiotics that inhibited S. mutans at low concentrations, rifampin and lincomycin, had a 250-fold concentration range of effectiveness. Even more strik.01 .02 .04 .08 .16 .31 .62 1.25 2.5 5.0 10.0 ing were the wide ranges of effectiveness of MINIMAL INHIBITORY CONCENTRATION tetracycline, thiostrepton, and spiromycin. OF ANTIBIOTIC (jig/ml) Each of these antibiotics had a 1,000-fold range FIG. 2. Susceptibility of S. mutans strains to spi- from the lowest to highest concentrations reromycin (O), streptolydigan (0), novobiocin (O), quired for growth inhibition. vancomycin (a), and thiostrepton (A). Among group A streptococci there has been a world-wide increase in the number of tetracycline-resistant strains observed (4, 6). Since tetracycline is a frequently used antibiotic, the incidence of tetracycline resistance among S. mutans may also be increasing. However, the aR other two antibiotics with wide ranges of MICs, thiostrepton and spiromycin, are not frequently used and one would not expect much increase in the incidence ofresistant organisms. There was U-) no evidence of multiple drug resistance among C,, any of the strains of S. mutans used in this 80-
z
I
m
W
S
40
0
cfl
z
C,,
study.
MINIMAL INHIBITORY CONCENTRATION OF ANTIBIOTIC (jig/ml)
FIG. 3. Susceptibility of S. mutans strains to tetracycline (0), chloramphenicol (0), gentamicin (A), spectinomycin (A), kanamycin (0), neomycin (a), streptomycin (*), and kasugamycin (0).
lated from dental plaque to 20 different antimicrobial agents. In a previous study of 11 antibiotics, Baker and Thornsberry (2) determined the antimicrobial susceptibility patterns of strains of S. mutans known to cause endocarditis. The MICs observed in our study are in essential agreement with those observed by Baker and Thornsbery and indicate that there is no difference in the susceptibility patterns of S. mutans isolated from endocarditis patients and from dental plaque. This observation is significant as it excludes the possibility that antimicrobial resistance has been induced in the
ACKNOWLEDGMENT This work was supported by Public Health Service grant DE03697 from the National Institute of Dental Research.
LITERATURE CITED 1. Abercombie, G. F., and W. M. Scott, 1928. A case of infective endocarditis due to Streptococcus mutans. Lancet 2:697-699. 2. Baker, C. N., and C. Thornsberry. 1974. Antimicrobial susceptibility of Streptococcus mutans isolated from patients with endocarditis. Antimicrob. Agents Chemother. 5:268-271. 3. DeMoor, C. E., J. D. DeStoppelaar, and J. van Houte. 1972. The occurrence of S. mutans and S. sanguis in the blood of endocarditis patients. Caries Res. 6:73. 4. Fallon, R. J. 1974. Tetracycline-resistant group A streptococci. Br. Med. J. 3:741. 5. Neefe, L. I., J. H. Chretien, E. C. Delaha, and V. F. Garagusi. 1974. Streptococcus mutans endocarditis. J. Am. Med. Assoc. 230:1298-1299. 6. Ubakata, K., M. Konno, and R. Fujii. 1973. Group A beta-hemolytic streptococci resistant to tetracycline, chloramphenicol, macrolides, lincomycin, and clindamycin. Jpn. J. Pediatr. 26:1451-1458.
Vol. 10, No. 2 Printed in U.S.A.
Susceptibility of Streptococcus mutans to Antimicrobial Agents JOSEPH J. FERRETTI* AND MARGIE WARD Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 Received for publication 23 February 1976
Fifty strains of Streptococcus mutans, including defined strains and clinical isolates, were tested for susceptibility to 20 different antimicrobial agents. Minimal inhibitory concentrations were determined by a liquid microtiter procedure. Antibiotics that were most effective in concentrations below 0.1 ,ug/ml included penicillin, ampicillin, erythromycin, cephalothin, and methicillin. Antibiotics effective in concentrations between 0.1 ug and 10 ,ug/ml included rifampin, lincomycin, thiostrepton, spiromycin, vancomycin, streptolydigan, novobiocin, tetracycline, chloramphenicol, spectinomycin, and gentamicin. Antibiotics effective at higher concentrations ranging from 10 ,g/ml to 400 ,ug/ml included the aminoglycosides kanamycin, neomycin, streptomycin, and kasugamycin. Although most antibiotics exhibited inhibitory effects in a narrow range of concentrations, antibiotics such as tetracycline, thiostrepton, and spiromycin had a 1,000-fold range from the lowest to highest concentrations required for growth inhibition.
Interest in the organism Streptococcus mutans is closely associated with its involvement in dental caries. This organism is also involved as a causative agent of endocarditis (1, 3). Knowledge of the antibiotic susceptibility patterns of S. mutans is of importance for proper therapy in the cases of endocarditis because S. mutans endocarditis has been *in some instances confused with group D enterococcal endocarditis (3). In one recent report, approximately 5% of streptococcal endocarditis isolates studied were S. mutans. (2). Baker and Thornsberry (2) have reported the patterns of susceptibility of S. mutans, isolated from patients with endocarditis, to 11 antibiotics. In this study we have determined the susceptibility of 50 strains of S. mutans, isolated from dental plaque, to 20 different antimicrobial agents. MATERIALS AND METHODS Bacterial strains. The bacterial strains used in this study were obtained from the dental clinics of the University of Oklahoma College of Dentistry and from C. Schaechtele, the University of Minnesota. Of the 50 strains of S. mutans, 6 were defined strains representing each known serotype and included the following: LM7, SL-1, 6715, Ingbritt, BHT, and OMZ-61. The remaining strains were isolated from samples of dental plaque and were identified according to (i) colony morphology on Mitis Salivarius agar, (ii) fermentation of mannitol and
sorbitol, and (iii) extracellular polysaccharide production in 5% sucrose broth. Antibiotics. The antibiotics used and the sources from which they were obtained were: erythromycin, kanamycin, neomycin, novobiocin, and tetracycline (Sigma Chemical Co., St. Louis, Mo.); ampicillin, kasugamycin, and methicillin (Bristol Laboratories, Syracuse, N.Y.); lincomycin, spectinomycin, and streptolydigan (Upjohn Co., Kalamazoo, Mich.); cephalothin and vancomycin (Eli Lilly & Co., Indianapolis, Ind.); penicillin and streptomycin (Nutritional Biochemical Corp., Cleveland, Ohio); rifampin (Becton-Dickinson & Co., Rutherford, N.J.); chloramphenicol (Parke, Davis & Co., Detroit, Mich.); spiromycin (Rhodia Inc., New York); gentamicin (Schering Laboratories, Bloomfield, N.J.); and thiostrepton (E. R. Squibb & Sons, New York). Media. The standard liquid medium used in this study was proteose peptone broth. It contained 6% proteose peptone no. 3 (Difco), 0.3% NaCl, and 0.38% Na2HPO4. After sterilization the broth was completed by the addition of three more components at the following final concentrations: 0.02% CaCl2, 0.05% glucose, and 5% normal horse serum (GIBCO). Trypticase soy broth (BBL) was also used in the initial portions of the study. Susceptibility tests. Each strain of S. mutans was streaked onto Mitis Salivarius agar plates, incubated at 37°C in an anaerobic jar for 24 h, and then allowed to incubate at 37°C aerobically for an additional 24 h. Several colonies with similar morphology were picked and inoculated into proteose peptone broth. After overnight incubation at 37°C, 0.1 ml of each culture was inoculated into a tube containing 10 ml of proteose peptone broth. To sterile 274
VOL. 10, 1976
ANTIMICROBIAL SUSCEPTIBILITY OF S. MUTANS
microdilution plates containing appropriate serial dilutions of antibiotic in proteose peptone broth, 0.05 ml of the overnight diluted culture was added. Each plate was covered with clear plastic and incubated at 37°C for 48 h. Unless otherwise specified, all incubations were performed aerobically. In instances when anaerobic conditions were maintained, anaerobic Gas-Pak (BBL) jars were utilized. The minimal inhibitory concentration (MIC) was the lowest concentration of antibiotic that caused complete inhibition of growth. RESULTS
275
concentration required for growth inhibition. With two of the antibiotics, rifampin and lincomycin, there was a 250-fold range from the lowest to highest concentration required for growth inhibition. In Fig. 2 the concentration of antibiotics ranged from 0.01 to 10 ,ug/ml and included streptolydigan, thiostrepton, spiromycin, kanamycin, and novobiocin. A 1,000-fold range in the concentration for inhibition of growth was observed with thiostrepton and spiromycin. In Fig. 3 the concentration of antibiotics ranged from 0.05 to 400 ,ug/ml and included tetracycline, chloramphenicol, spectinomycin, gentamicin, kanamycin, neomycin, streptomycin, and kasugamycin. A 1,000-fold range in the concentrations of tetracycline required for inhibition of growth was observed, whereas there was a more narrow range of inhibition for all other antibiotics.
Growth of the various strains of S. mutans supported best in proteose peptone broth containing 5% horse serum. In comparison with other media, including Trypticase soy broth, growth of all bacterial strains was sufficient in 24 h to determine the MIC of a particular antibiotic within one dilution. To insure that maximal growth occurred, however, final MICs were routinely determined after 48 h of incubation. DISCUSSION There was some question whether there were any differences in growth or inhibition by antiThe purpose of this investigation was to test biotics when the organism was incubated under the susceptibility of strains of S. mutans isoaerobic or anaerobic conditions. To answer this la question the MICs of several antibiotics (cephalothin, methicillin, and vancomycin) were determined with 50 different strains of S. mutans C/, incubated under both aerobic and anaerobic z conditions. The results from these experiments C/,Iare given in Table 1. The average MICs obtained for each antibiotic were essentially iden- -im tical with either aerobic or anaerobic incubation and indicated that the atmosphere had little effect on the growth of the organism or its cn be susceptibility to antibiotics. The MICs for inhibition of the 50 strains, plotted as a function of the percentage of susceptible strains, are shown in Fig. 1 through 3. .0005 .001 .002 .004 .008 .015 .031 .063 .125 .250 .5 1.0 2.0 In Fig. 1 the concentration of antibiotics ranged MINIMAL INHIBITORY CONCENTRATION from 0.0005 to 2 jig/ml and included penicillin, OF ANTIBIOTIC (ig/ml) ampicillin, erythromycin, cephalothin, methiFIG. 1. Susceptibility ofS. mutans strains to pencillin, rifampin, and lincomycin. The inhibitory icillin (), erythromycin (0), ampicillin (a), cephaconcentrations of most of the antibiotics cov- lothin (a), rifampin (A), methicillin (A), and lincoered a 10-fold range from the lowest to highest mycin (*). was
L&J
C.)
TABLE 1. Minimal inhibitory concentrations (MICs) ofthree antibiotics for 50 strains of S. mutans incubated under aerobic and anaerobic conditions Antibiotic Percentage of strains with MIC (yg/ml) of: Cephalothin (0.25) (0.125) (0.063) (0.031) (0.015) (0.008) (0.004) (0.002) (0.001) (0.0005) 0 0 Aerobic 4 40 0 13 19 24 0 0 0 0 0 0 Anaerobic 2 2 36 15 30 15 Methicillin (0.25) (0.125) (0.063) (0.031) (0.015) (0.008) (0.004) (0.002) (0.001) (0.0005) 0 0 Aerobic 34 0 0 0 0 13 43 10 0 0 0 0 42 0 Anaerobic 11 2 0 45 Vancomycin (10) (5) (2.5) (1.25) (0.625) (0.313) (0.157) (0.08) (0.04) (0.02) Aerobic Anaerobic
0 0
0 0
0 0
2 2
4 8
28 26
36 21
28 32
2 11
0 0
276
FERRETTI AND WARD
ANTIMICROB. AGENTS CHEMOTHER.
endocarditis strains due to prior chemotherapy. Further, it excludes antimicrobial susceptibility patterns as contributory to the ability of plaque-forming organisms to establish infection in the heart. The results of this study also increase the number of antimicrobial agents cn60 with known efficacy against these organisms. CLJ Antimicrobial agents that were particularly effective at low concentrations (below 0.1 ug/ ml) included penicillin, ampicillin, erythromy(n) cin, cephalothin, and methicillin. Each ofthese antibiotics inhibited all of the strains tested in a narrow concentration range. Two antibiotics that inhibited S. mutans at low concentrations, rifampin and lincomycin, had a 250-fold concentration range of effectiveness. Even more strik.01 .02 .04 .08 .16 .31 .62 1.25 2.5 5.0 10.0 ing were the wide ranges of effectiveness of MINIMAL INHIBITORY CONCENTRATION tetracycline, thiostrepton, and spiromycin. OF ANTIBIOTIC (jig/ml) Each of these antibiotics had a 1,000-fold range FIG. 2. Susceptibility of S. mutans strains to spi- from the lowest to highest concentrations reromycin (O), streptolydigan (0), novobiocin (O), quired for growth inhibition. vancomycin (a), and thiostrepton (A). Among group A streptococci there has been a world-wide increase in the number of tetracycline-resistant strains observed (4, 6). Since tetracycline is a frequently used antibiotic, the incidence of tetracycline resistance among S. mutans may also be increasing. However, the aR other two antibiotics with wide ranges of MICs, thiostrepton and spiromycin, are not frequently used and one would not expect much increase in the incidence ofresistant organisms. There was U-) no evidence of multiple drug resistance among C,, any of the strains of S. mutans used in this 80-
z
I
m
W
S
40
0
cfl
z
C,,
study.
MINIMAL INHIBITORY CONCENTRATION OF ANTIBIOTIC (jig/ml)
FIG. 3. Susceptibility of S. mutans strains to tetracycline (0), chloramphenicol (0), gentamicin (A), spectinomycin (A), kanamycin (0), neomycin (a), streptomycin (*), and kasugamycin (0).
lated from dental plaque to 20 different antimicrobial agents. In a previous study of 11 antibiotics, Baker and Thornsberry (2) determined the antimicrobial susceptibility patterns of strains of S. mutans known to cause endocarditis. The MICs observed in our study are in essential agreement with those observed by Baker and Thornsbery and indicate that there is no difference in the susceptibility patterns of S. mutans isolated from endocarditis patients and from dental plaque. This observation is significant as it excludes the possibility that antimicrobial resistance has been induced in the
ACKNOWLEDGMENT This work was supported by Public Health Service grant DE03697 from the National Institute of Dental Research.
LITERATURE CITED 1. Abercombie, G. F., and W. M. Scott, 1928. A case of infective endocarditis due to Streptococcus mutans. Lancet 2:697-699. 2. Baker, C. N., and C. Thornsberry. 1974. Antimicrobial susceptibility of Streptococcus mutans isolated from patients with endocarditis. Antimicrob. Agents Chemother. 5:268-271. 3. DeMoor, C. E., J. D. DeStoppelaar, and J. van Houte. 1972. The occurrence of S. mutans and S. sanguis in the blood of endocarditis patients. Caries Res. 6:73. 4. Fallon, R. J. 1974. Tetracycline-resistant group A streptococci. Br. Med. J. 3:741. 5. Neefe, L. I., J. H. Chretien, E. C. Delaha, and V. F. Garagusi. 1974. Streptococcus mutans endocarditis. J. Am. Med. Assoc. 230:1298-1299. 6. Ubakata, K., M. Konno, and R. Fujii. 1973. Group A beta-hemolytic streptococci resistant to tetracycline, chloramphenicol, macrolides, lincomycin, and clindamycin. Jpn. J. Pediatr. 26:1451-1458.
