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Antimicrobial Agents and Chemotherapy, August 2001, p. 2400-2402, Vol. 45, No. 8
Association de Formation Continue en
Pathologie Infectieuse des Biologistes, 75005 Paris,1 Hôpital d'Instruction des
Armées, 94163 Saint-Mandé,2 and
Centre Hospitalier Universitaire Côte de Nacre, 14033 Caen Cedex,3 France
Received 7 February 2001/Returned for modification 7 April
2001/Accepted 9 May 2001
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 (MLSB). In
streptococci, MLSB 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).
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2400-2402.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Antibiotic Susceptibility and Mechanisms of
Erythromycin Resistance in Clinical Isolates of Streptococcus
agalactiae: French Multicenter Study
ABSTRACT
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TABLE 1.
In vitro activities of eight antibiotics for 126 S. agalactiae isolates
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 MLSB resistance (12, 13, 19). Resistance to both erythromycin and clindamycin indicated an MLSB 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 MLSB
phenotype, six strains (22.2%) expressed the inducible
MLSB 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.
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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 MLSB 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.
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ACKNOWLEDGMENTS |
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We thank Bruno Moutinho for technical assistance, and we are grateful to Joyce Sutcliffe for providing S. pneumoniae reference strain O2J1175.
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FOOTNOTES |
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* Corresponding author. Mailing address: Département de Biologie Clinique, Hôpital d'Instructions des Armées Bégin, 69 avenue de Paris, 94163 Saint-Mandé Cedex, France. Phone: (33) (1) 43 98 42 37. Fax: (33) (1) 43 98 53 36. E-mail: hia-begin-biologie{at}worldonline.fr.
Members of the Association de Formation Continue en Pathologie
Infectieuse des Biologistes (AFICORPI-BIO) Network and their locations
(in France) are as follows: J. P. Arzouni, Martigues; J. L. Berges,
Perpignan; J. P. Bouilloux, Rodez; N. Charbit, Tonnerre; D. De Mouy,
Paris; S. Fleutiaux, Tarbes; J. P. Galinier, Toulouse; A. Gayon,
St-Gaudens; G. Larribet, St-Médard-en-Jalles; and J. P. Lepargneur, Toulouse.
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Antimicrobial Agents and Chemotherapy, August 2001, p. 2400-2402, Vol. 45, No. 8
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2400-2402.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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