ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1976, P. 128-131 Copyright 0 1976 American Society for Microbiology
Vol. 10, No. 1 Printed in U.S.A.
Antimicrobial Susceptibility of Group B Streptococci Isolated from a Variety of Clinical Sources CAROL J. BAKER,* BETTE J. WEBB, AND FRED F. BARRETT Departments of Pediatrics* and Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030 Received for publication 1 March 1976
The minimal inhibitory concentration of 10 antibiotics for 244 isolates of group B streptococci was determined. Susceptibility to penicillin G, ampicillin, cephalothin, chloramphenicol, and carbenicillin was uniform. Tetracycline and bacitracin resistance among these isolates was frequent (87.5 and 97.9%, respectively). Three strains (1.2%) failed to be inhibited by 100 pg of lincomycin or cindamycin per ml. Susceptibility of these 244 strains to the agents tested was unrelated to source of the isolate, year of isolation, or strain serotype. No apparent change in the suceptibility of group B streptococci to penicillin G has occurred during the past 2 decades. Strains of group B Streptococcus have become increasingly fiequent isolates from neonates and infants with serious bacterial infection during the past decade (1, 2, 4, 8). Because this microbial species has previously been noted to be uniformly susceptible to the penicillins (7, 9), penicillin G has been the recommended drug of choice for the treatment of group B streptococcal infections (3, 7). However, two recent reports suggest that certain strains are relatively resistant to penicillin G (M. J. Severin and J. L. Wiley, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 14th, San Francisco, Calif., Abstr. 251, 1974) or ampicillin (15), an observation that is believed to be related to time and that has important therapeutic implications if confirmed. In addition, serious puerperal infections and skin infection among diabetics have been associated with group B streptococci (2, 7), and certain patients with these infections require antimicrobial therapy with drugs alternative to penicillin G because of a history of allergy to the penicillins. Lincomycin and cindamycin may be useful for these patients on the basis of their uniformly good in vitro activity against Lancefield group B strains of streptococci (11). However, relatively few strains have been tested in vitro to clindamycin, and no data are available to date regarding its efficacy in the treatment of group B streptococcal infections. In the present study we determined the antimicrobial susceptibility of 244 strains of group B streptococci isolated from a variety of clinical sources between 1970 and 1975 as a means of defining resistance to penicillin G, useful alternative antibiotics for the treatment of penicil-
lin-allergic patients, and differences in the susceptibility of isolates on the basis of strain serotype, or source and year of isolation. MATERIALS AND METHODS Two hundred and forty-four strains of group B streptococci were studied. These strains were isolated from the following patient,groups: (i) blood or cerebrospinal fluid isolates from 45 infants and 5 adults (50 strains); (ii) vaginal isolates from 80 nonpregnant college women and 32 parturients at delivery (112 strains); (iii) throat, umbilical, or external auditory canal isolates from 34 asymptomatically, colonized neonates (34 strains); and (iv) vaginal or urethral isolates from 28 women and 20 men enrolled in a venereal disease clinic (48 strains). These strains were isolated in several geographical areas. Most of the "significant" isolates were from Houston, as were all the strains from asymptomatic pregnant women and neonates. All isolates from venereal disease clinic patients were from Boston, and all isolates from college women were from Rhode Island. Grouping and serotyping of these isolates were performed by the capillary precipitin method (12) with hyperimmune rabbit antisera prepared against formalinized vaccines ofstandard strains. Agar plate dilution tests of quantitative antibiotic susceptibility were performed on MuellerHinton agar (Difco Laboratories, Detroit, Mich.) containing 5% defibrinated sheep blood. Agar plates containing serial twofold dilutions of antibiotic at final concentrations from 0.00625 to 100 pgIml were inoculated by using the replicator technique of Steers et al. (14). The inoculum consisted of an undiluted overnight culture in Todd-Hewitt broth (Difco Laboratories) delivered in an aliquot of 0.002 ml; the inoculum was estimated to contain 107 viable organisms. The minimal inhibitory concentration (MWC) was defined as the lowest concentration of antibiotic in which no growth was visible after incu-
128
VOL. 10, 1976
,
GROUP B STREPTOCOCCI ANTIMICROBIAL SUSCEPTIBILITY
bation of the agar plates for 18 h at 37 C. Control plates without antibiotic were inoculated in a similar fashion in each series of tests to assess strain viability, and a reference strain of group B Streptococcus was included to assure reproducibility. Thirty-one of the isolates for which the MIC of penicillin G was 5 0.1 ,ug/ml were simultaneously tested, using identical methods and an inoculum of approximately 104 as well as 107 viable organisms on the same test day. The antibiotics tested were penicillin G, ampicillin, carbenicillin, cephalothin, chloramphenicol, gentamicin, bacitracin, lincomycin, clindamycin, and tetracycline. Standard epidemiological data, including the source and serotype of the isolate, the year of isolation, and the diagnosis of the patient, were recorded for each strain tested. These data were then correlated with in vitro susceptibility by appropriate statistical methods.
129
tococcus. All but three isolates (1.2% of strains) were inhibited by a concentration of 0.2 ,g or less of lincomycin and 0.1 ug or less of clindamycin per ml. The three resistant strains failed to have their growth inhibited by 100 ,ug of either agent per ml, and cross-resistance to erythromycin (disk diffusion method [5], zone of 6 mm) was uniform. These clindamycin-resistant strains were isolated from a neonate with septicemia (1972), a woman with puerperal sepsis (1973), and a vaginally colonized, asymptomatic parturient (1974). Of the antimicrobial agents tested, tetracycline was the only one for which there were significant numbers of both resistant and susceptible strains (87.5 and 12.5%, respectively). No relationship between susceptibility to tetracycline and the source, or the serotype of an RESULTS isolate or its year of isolation was apparent. To detect increasing resistance to penicillin G The results for the susceptibility of these 244 group B streptococci to 10 antibiotics are de- among the isolates tested on the basis of strain, picted graphically in Fig. 1 and are summa- serotype, or year of isolation, strains with an rized in Table 1. Penicillin G was the most MIC of 50.1 ug/ml were analyzed (Tables 2 and active antimicrobial agent tested; the median 3). It was apparent that serotype of infecting MIC was 0.05 ,ug/ml (0.083 units/ml). When strain was unrelated to penicillin susceptibilinoculum size was decreased to 104 viable orga- ity, since the distribution of serotypes for nisms, the median MIC of strains to penicillin strains inhibited at a concentration of 50.1 ug/ decreased to 0.025 ug/ml. All the strains tested ml was not significantly different from that were susceptible to penicillin G, ampicillin, among strains inhibited at a concentration of cephalothin, chloramphenicol, and carbenicil-
Vol. 10, No. 1 Printed in U.S.A.
Antimicrobial Susceptibility of Group B Streptococci Isolated from a Variety of Clinical Sources CAROL J. BAKER,* BETTE J. WEBB, AND FRED F. BARRETT Departments of Pediatrics* and Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030 Received for publication 1 March 1976
The minimal inhibitory concentration of 10 antibiotics for 244 isolates of group B streptococci was determined. Susceptibility to penicillin G, ampicillin, cephalothin, chloramphenicol, and carbenicillin was uniform. Tetracycline and bacitracin resistance among these isolates was frequent (87.5 and 97.9%, respectively). Three strains (1.2%) failed to be inhibited by 100 pg of lincomycin or cindamycin per ml. Susceptibility of these 244 strains to the agents tested was unrelated to source of the isolate, year of isolation, or strain serotype. No apparent change in the suceptibility of group B streptococci to penicillin G has occurred during the past 2 decades. Strains of group B Streptococcus have become increasingly fiequent isolates from neonates and infants with serious bacterial infection during the past decade (1, 2, 4, 8). Because this microbial species has previously been noted to be uniformly susceptible to the penicillins (7, 9), penicillin G has been the recommended drug of choice for the treatment of group B streptococcal infections (3, 7). However, two recent reports suggest that certain strains are relatively resistant to penicillin G (M. J. Severin and J. L. Wiley, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 14th, San Francisco, Calif., Abstr. 251, 1974) or ampicillin (15), an observation that is believed to be related to time and that has important therapeutic implications if confirmed. In addition, serious puerperal infections and skin infection among diabetics have been associated with group B streptococci (2, 7), and certain patients with these infections require antimicrobial therapy with drugs alternative to penicillin G because of a history of allergy to the penicillins. Lincomycin and cindamycin may be useful for these patients on the basis of their uniformly good in vitro activity against Lancefield group B strains of streptococci (11). However, relatively few strains have been tested in vitro to clindamycin, and no data are available to date regarding its efficacy in the treatment of group B streptococcal infections. In the present study we determined the antimicrobial susceptibility of 244 strains of group B streptococci isolated from a variety of clinical sources between 1970 and 1975 as a means of defining resistance to penicillin G, useful alternative antibiotics for the treatment of penicil-
lin-allergic patients, and differences in the susceptibility of isolates on the basis of strain serotype, or source and year of isolation. MATERIALS AND METHODS Two hundred and forty-four strains of group B streptococci were studied. These strains were isolated from the following patient,groups: (i) blood or cerebrospinal fluid isolates from 45 infants and 5 adults (50 strains); (ii) vaginal isolates from 80 nonpregnant college women and 32 parturients at delivery (112 strains); (iii) throat, umbilical, or external auditory canal isolates from 34 asymptomatically, colonized neonates (34 strains); and (iv) vaginal or urethral isolates from 28 women and 20 men enrolled in a venereal disease clinic (48 strains). These strains were isolated in several geographical areas. Most of the "significant" isolates were from Houston, as were all the strains from asymptomatic pregnant women and neonates. All isolates from venereal disease clinic patients were from Boston, and all isolates from college women were from Rhode Island. Grouping and serotyping of these isolates were performed by the capillary precipitin method (12) with hyperimmune rabbit antisera prepared against formalinized vaccines ofstandard strains. Agar plate dilution tests of quantitative antibiotic susceptibility were performed on MuellerHinton agar (Difco Laboratories, Detroit, Mich.) containing 5% defibrinated sheep blood. Agar plates containing serial twofold dilutions of antibiotic at final concentrations from 0.00625 to 100 pgIml were inoculated by using the replicator technique of Steers et al. (14). The inoculum consisted of an undiluted overnight culture in Todd-Hewitt broth (Difco Laboratories) delivered in an aliquot of 0.002 ml; the inoculum was estimated to contain 107 viable organisms. The minimal inhibitory concentration (MWC) was defined as the lowest concentration of antibiotic in which no growth was visible after incu-
128
VOL. 10, 1976
,
GROUP B STREPTOCOCCI ANTIMICROBIAL SUSCEPTIBILITY
bation of the agar plates for 18 h at 37 C. Control plates without antibiotic were inoculated in a similar fashion in each series of tests to assess strain viability, and a reference strain of group B Streptococcus was included to assure reproducibility. Thirty-one of the isolates for which the MIC of penicillin G was 5 0.1 ,ug/ml were simultaneously tested, using identical methods and an inoculum of approximately 104 as well as 107 viable organisms on the same test day. The antibiotics tested were penicillin G, ampicillin, carbenicillin, cephalothin, chloramphenicol, gentamicin, bacitracin, lincomycin, clindamycin, and tetracycline. Standard epidemiological data, including the source and serotype of the isolate, the year of isolation, and the diagnosis of the patient, were recorded for each strain tested. These data were then correlated with in vitro susceptibility by appropriate statistical methods.
129
tococcus. All but three isolates (1.2% of strains) were inhibited by a concentration of 0.2 ,g or less of lincomycin and 0.1 ug or less of clindamycin per ml. The three resistant strains failed to have their growth inhibited by 100 ,ug of either agent per ml, and cross-resistance to erythromycin (disk diffusion method [5], zone of 6 mm) was uniform. These clindamycin-resistant strains were isolated from a neonate with septicemia (1972), a woman with puerperal sepsis (1973), and a vaginally colonized, asymptomatic parturient (1974). Of the antimicrobial agents tested, tetracycline was the only one for which there were significant numbers of both resistant and susceptible strains (87.5 and 12.5%, respectively). No relationship between susceptibility to tetracycline and the source, or the serotype of an RESULTS isolate or its year of isolation was apparent. To detect increasing resistance to penicillin G The results for the susceptibility of these 244 group B streptococci to 10 antibiotics are de- among the isolates tested on the basis of strain, picted graphically in Fig. 1 and are summa- serotype, or year of isolation, strains with an rized in Table 1. Penicillin G was the most MIC of 50.1 ug/ml were analyzed (Tables 2 and active antimicrobial agent tested; the median 3). It was apparent that serotype of infecting MIC was 0.05 ,ug/ml (0.083 units/ml). When strain was unrelated to penicillin susceptibilinoculum size was decreased to 104 viable orga- ity, since the distribution of serotypes for nisms, the median MIC of strains to penicillin strains inhibited at a concentration of 50.1 ug/ decreased to 0.025 ug/ml. All the strains tested ml was not significantly different from that were susceptible to penicillin G, ampicillin, among strains inhibited at a concentration of cephalothin, chloramphenicol, and carbenicil-
