Antibiotic Susceptibility and Mechanisms of Erythromycin Resistance in Clinical Isolates of Streptococcus agalactiae: French Multicenter Study

Abstract

Among 126 Streptococcus agalactiae isolates collected in 10 French laboratories in 1999, 27 (21.4%) had macrolide resistance related to the presence of erm(B) (11 strains), erm(A) subclass erm(TR) (10 strains), and mef(A) genes (2 strains) and the presence of combinations of erm(B) and erm(A) genes or mef(A) genes (3 strains).

Streptococcus agalactiae or group B streptococcus (GBS) is one of the pathogens most frequently responsible for peripartum maternal and neonatal infections. Aminopenicillin is recommended as a first-line intrapartum chemoprophylaxis for prevention of GBS infection in neonates. Erythromycin and clindamycin are alternatives to the penicillins in cases of intolerance (6). While aminopenicillins are still active against the vast majority of GBS strains, resistance to erythromycin has been reported as far back as 1962 in the United States and has emerged during the last decade in several countries (11, 15). In GBS, resistance to macrolides is conferred either by methylases encoded by erm genes, that modify the ribosomal target of macrolides or by pumps that efflux these antibiotics. Ribosomal modification by methylase was the first erythromycin resistance mechanism reported in GBS and results in cross-resistance to macrolide-lincosamide-streptogramin B (MLS_B). In streptococci, MLS_B resistance can be mediated by two classes of methylase genes, i.e., the conventional erm(B) (ermAM) determinant and the recently described erm(TR) gene, which is considered to be a subset of the erm(A) class (18). Macrolide efflux is mediated by a membrane-bound protein encoded by the mef(A) gene and gives rise to the so-called M phenotype characterized by resistance to 14- and 15-membered ring macrolides, while lincosamides, streptogramins, and 16-membered ring macrolides remain active even after induction with erythromycin (8). There have not been many studies investigating the macrolide resistance mechanisms in S. agalactiae (5, 17). The aim of this multicenter study was to determine the susceptibility to antibiotics of GBS isolated recently in the community and to characterize the mechanisms of macrolide resistance in erythromycin-resistant strains.

In February 1999, all consecutive clinical strains from outpatients identified as GBS and S. agalactiae by latex agglutination assay and API 20 STREPT gallery, respectively, were collected in 10 French private laboratories located in nine different cities in the Paris area and southeastern and southwestern France (one, two, and seven laboratories, respectively) and sent to the hospital Begin laboratory for investigation. The MICs of penicillin G, amoxicillin, cefotaxime, erythromycin, clindamycin, pristinamycin (a streptogramin antibiotic), tetracycline, and rifampin were determined by the agar dilution method with Mueller-Hinton medium supplemented with 5% defibrinated sheep blood (Bio-Rad, Marnes la Coquette, France) as recommended by the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM) (9). Results were interpreted according to the recommendations of the CA-SFM (breakpoints are shown in Table 1) (10).

TABLE 1.

In vitro activities of eight antibiotics for 126 S. agalactiae isolates

Antimicrobial agent	MIC (μg/ml)a				CA-FSM breakpointb (μg/ml)	Susceptibility (%)
	Range	Mean	50%	90%
Penicillin G	0.03–0.06	0.03	0.03	0.03	≤0.25	100
Amoxicillin	0.03–0.06	0.05	0.06	0.06	≤4	100
Erythromycin	0.03–>64	0.32	0.12	8	≤1	78.6
Clindamycin	0.03–>64	0.31	0.06	128	≤2	82.5
Pristinamycin	0.03–2	0.06	0.06	0.125	≤1	100
Tetracycline	0.03–>64	25.1	32	64	≤4	11.9
Co-trimoxazole	1–64	2.1	2	4	≤2/38	89.4
Rifampin	0.125–2	0.14	0.125	0.125	≤4	100

^a50 and 90%, MIC₅₀ and MIC₉₀, respectively. 

^bSusceptibility breakpoint recommended by the CA-SFM (8). 

The resistance phenotypes of erythromycin-resistant GBS were determined by the disk diffusion method on Mueller-Hinton agar supplemented with 5% defibrinated horse blood (Bio-Rad) on erythromycin and clindamycin disks. Blunting of the clindamycin inhibition zone proximal to the erythromycin disk indicated an inducible type of MLS_B resistance (12, 13, 19). Resistance to both erythromycin and clindamycin indicated an MLS_B cross-resistance. Susceptibility to clindamycin with no blunting defined the M phenotype (efflux mechanism) (8, 12).

The mef(A), erm(TR), erm(B), and msr(A) genes were detected after PCR amplification and hybridization as previously described (1, 20). Streptococcus pneumoniae HM28 containing the erm(B) gene, Staphylococcus aureus HM1054/R [erm(C)], S. aureus HM1051 [erm(A)], Streptococcus pyogenes UCN1 [erm(TR)], and Staphylococcus saprophyticus HM1053 [msr(A)] from our collection and S. pneumoniae O2J1175 [mef(A)] were used as controls in PCR experiments.

One hundred and twenty-six GBS strains were obtained, of which 63.4% were isolated from cases of genital tract infection or colonization, 16.1% were isolated from the urinary tract, and 6.5% were isolated from superficial pus. A summary of MIC data (geometric mean range, MIC at which 50% of strains tested are inhibited [MIC₅₀], and MIC₉₀) on 11 antibiotics for these strains are listed in Table 1. Twenty-seven GBS (21.4%) showed decreased susceptibility to erythromycin. The rates of resistance varied from 11 to 50% among the participant centers. Nineteen strains (70.4% of erythromycin-resistant strains) expressed the MLS_B phenotype, six strains (22.2%) expressed the inducible MLS_B phenotype, and two strains (7.4%) expressed the M phenotype. Distribution of erythromycin resistance genes according to erythromycin resistance phenotypes is reported in Table 2. The erm(B) and erm(TR) genes were prevalent, and three strains harbored combinations of resistance genes.

TABLE 2.

Distribution of erythromycin resistance phenotypes according to macrolide resistance genotype

Erythromycin resistance gene(s)	No. of strains with erythromycin resistance phenotype:			Total no. of strains
	MLSB	Inducible MLSB	M
erm(B)	10	1	0	11
erm(TR)	6	4	0	10
erm(B) + erm(TR)	1	0	0	1
mef(A)	0	0	2	2
erm(B) + mef(A)	2	0	0	2
Absencea	1	0	0	1
Total	19	6	2	27

^aAbsence, lack of amplification by PCR using primers specific for erm(B), erm(TR), mef(A), and msr(A) genes. 

One isolate resistant to erythromycin and clindamycin (MICs, 4 and 8 μg/ml, respectively) did not harbor any of the target genes. No hybridization with any probe was observed for five randomly selected erythromycin-susceptible strains used as controls. Our multicenter study confirms the high level of activity of penicillin G and amoxicillin against GBS (3, 14–16). In this species, beta-lactam resistance has rarely been described (4, 23). If beta-lactam susceptibility patterns have remained unchanged, then the rate of erythromycin resistance in GBS is increasing: 1.2% for the period 1980 to 1993 versus 18% for the period 1997 to 1998 in a North American study (15). Compared to this report and other recent studies, the rates of erythromycin and clindamycin resistance were high in our experience: respectively, 21.4% versus 4.9 to 20.2% for erythromycin and 18% versus 0.7 to 15% for clindamycin (3, 14–16).

Among the erythromycin-resistant strains, erm(B) genes and to a lesser extent erm(TR) genes were widely distributed. Resistance genes were combined in three strains. Combinations of erm(B) and mef(E) genes have been reported previously in pneumococci isolated in France (1). We did not study the relatedness of the strains by molecular techniques, but the erythromycin-resistant isolates were distributed in all the participant centers. The active efflux pump mediated by the mef(A) gene has already been reported in a previous French study (2) but is far less frequent in French GBS isolates than in isolates belonging to other beta-hemolytic streptococcal species, including S. pyogenes (12, 23). The presence of the mreA gene, described as a novel macrolide efflux gene (7), has not been investigated because it is an intrinsic gene that encodes riboflavin kinase and is found in all S. agalactiae strains (G. Clarebout and R. Leclercq, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 840, 1999). In our study, one erythromycin-resistant strain with the MLS_B phenotype had a negative PCR result. This strain might possess other macrolide resistance mechanisms such as mutation in 23S rRNA or one of the ribosomal proteins already described in S. pneumoniae (21).

The level of erythromycin and clindamycin resistance in French GBS isolates is of concern and leads to the recommendation that alternative prophylactic therapy for pregnant women who are penicillin intolerant should be guided by susceptibility testing.