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. 2001 Feb;45(2):636–638. doi: 10.1128/AAC.45.2.636-638.2001

Mechanisms of Macrolide Resistance in Clinical Pneumococcal Isolates in France

Frederic Fitoussi 1, Catherine Doit 1, Pierre Geslin 2, Naima Brahimi 1, Edouard Bingen 1,*
PMCID: PMC90344  PMID: 11158772

Abstract

The genetic basis of macrolide resistance was investigated in a collection of 48 genotypically unrelated clinical isolates of Streptococcus pneumoniae obtained between 1987 and 1997 in France. All strains were resistant to erythromycin, clindamycin, and streptogramin B, exhibiting a macrolide-lincosamide-streptogramin B resistance phenotype, and harbored the erm(B) gene. None of the strains carried the mef(A) or erm(A) subclass erm(TR) gene.

Antibiotic resistance in Streptococcus pneumoniae, especially to β-lactam antibiotics, has been a matter of growing concern in the last two decades. Resistance in this species has also been noted with tetracyclines, chloramphenicol, cotrimoxazole, and macrolides. In the United States, 19 to 34% of pneumococcal isolates are currently resistant to macrolides (8; M. R. Jacobs, D. Felminghan, P. C. Appelbaum, and The Alexander Project Group, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1044, p. 151, 1999). In Western Europe, the low prevalence of macrolide resistance in Germany, Austria, Portugal, The Netherlands, and Switzerland (1.5 to 4.6%) contrasts with the high rates observed in Spain, Italy, and Belgium (33, 24, and 31%, respectively) (1, 17; D. Felmingham and R. N. Gruneberg, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1790, p. 108, 2000). In France, the prevalence of pneumococcal resistance to erythromycin was 53% in 1997 (10). Four mechanisms of macrolide resistance have been described in S. pneumoniae. The first is a target modification involving a ribosomal methylase, associated with the erm(B) gene (15, 25). A macrolide-specific efflux mechanism encoded by the mef(A) gene was described in 1996 (21, 23). The erm(B) gene is associated with high-level resistance to macrolides, lincosamides, and streptogramin B (MLSB phenotype), while the mef(A) gene is associated with low-level resistance to 14- and 15-membered-ring macrolides (M phenotype). More recently, erythromycin resistance in clinical isolates of S. pneumoniae harboring the erm(A) subclass erm(TR) gene has been described (3; G. A. Syrogiannopoulos, I. N. Grivea, A. Tait-Kamradt, G. D. Katopodis, N. G. Beratis, J. Sutcliffe, P. C. Appelbaum, and T. A. Davies, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 139, p. 65, 2000). Finally, macrolide-resistant pneumococcal strains with mutations in the 23S rRNA and ribosomal protein L4 or L22 have been selected by macrolide passage in vitro (22; A. Canu, B. Malbruny, M. Coquemont, T. A. Davies, P. C. Appelbaum, and R. Leclercq, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1927, p. 118, 2000; J. Sutcliffe, A. Tait-Kamradt, A. Walker, and J. Petitpas, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1925, p. 117, 2000) or have been found in clinical isolates (T. A. Davies, P. C. Appelbaum, W. Hryniewicz, L. Drukalska, H. Hupkova, J. Kolman, J. Mieivleviciene, M. Pana, L. Setchanova, A. Tambic, M. K. Thege, J. Trupl, and P. Urbaskova, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 138, p. 65, 2000; A. Tait-Kamradt, T. Davies, L. Brennan, F. Depardieu, P. Courvalin, J. Duignan, J. Petitpas, L. Wondrack, M. Jacobs, P. Appelbaum, and J. Sutcliffe, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. LB 8, p. 15, 2000).

The mechanisms of pneumococcal resistance to macrolides have been investigated in several countries, but very few data deal with the French situation (C. Arpin, M. H. Canron, P. Noury, and C. Quentin, Letter, J. Antimicrob. Chemother. 44:133–134, 1999; P. Angot, M. Vergnaud, and R. Leclercq, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1221, p. 158, 1999). The aim of this study was to determine the genetic mechanisms of S. pneumoniae macrolide resistance in a collection of genotypically unrelated clinical isolates obtained between 1987 and 1997 in French hospitals.

Forty-eight invasive strains of S. pneumoniae resistant to erythromycin by the disk diffusion method were collected throughout France between 1987 and 1997. The 48 isolates were previously shown to be of different clonal origins by means of restriction fragment length polymorphism analysis of the rRNA gene regions and of the pbp2b and 2x genes (9). The serotypes were 6B (n = 10), 6A (n = 4), 19A (n = 1), 14 (n = 9), 15A (n = 3), 24 (n = 2), 23F (n = 9), and 19F (n = 9); one strain was nontypeable. The MICs of erythromycin, azithromycin, josamycin, clindamycin, streptogramin B, and penicillin G were determined by the agar dilution method, using Mueller-Hinton medium supplemented with 5% sheep blood and incubation in room air (7).

The mef and erm genes were detected after PCR amplification, as previously described (4, 12, 18, 20). Streptococcus pyogenes 02C 1061, S. pyogenes 02C 1110, and S. pyogenes 02C 1064 were used as positive PCR controls for the erm(B), erm(A) subclass erm(TR), and mef(A) genes, respectively (4–6).

The 48 pneumococcal isolates displayed a high level of resistance to erythromycin and related agents (Table 1), which is typical of the MLSB resistance phenotype. Seventy-six percent of the strains were intermediately susceptible or resistant to penicillin. PCR amplification with primers specific for the erm and mef genes yielded results in keeping with the high level of resistance to MLSB. Indeed, all the strains harbored the erm(B) gene, and none bore the mef or erm(A) subclass erm(TR) gene.

TABLE 1.

MICs of macrolides and related agents and of penicillin G for 48 erythromycin-resistant S. pneumoniae isolates

Antimicrobial agent MIC (μg/ml)a
50% 90% Range
Erythromycin >128 >128 >128
Azithromycin >128 >128 >128
Josamycin >128 >128 >128
Clindamycin >128 >128 64–>128
Streptogramin B 128 >128 2–>128
Penicillin G 0.5 4 0.016–8
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a

50 and 90%, MICs at which the growth of 50 and 90% of isolates, respectively, was inhibited. 

The prevalence of erythromycin resistance in North American pneumococcal isolates ranges from 2.9% in Canada to 34% in the United States (8, 11, 24). This erythromycin resistance is associated with the M phenotype and the mef(A) gene in 56 to 63% of cases (11, 19, 24). In South Africa the prevalence of erythromycin-resistant pneumococci with the M phenotype has increased from 1 to 20% in the last 10 years (26). In Western Europe the prevalence of pneumococcal macrolide resistance varies geographically (1, 17; Felmingham et al., 40th ICAAC). Mechanisms of resistance have been investigated in countries with high prevalence rates. In Spain, 98% of erythromycin-resistant strains were found to have the MLSB phenotype (2). In Italy and Belgium, more than 90% of strains carried the erm(B) gene (13, 16). The spread of multiresistant clonal epidemic pneumococcal strains has been recently reported in France (9). To avoid biasing our results, we determined the genetic mechanisms of macrolide resistance only in genotypically unrelated isolates, which were representative of the strains usually recovered in France during that period (9). All strains possessed the erm(B) gene. The location of the erm(B) gene on a transposon may explain the interstrain spread of erythromycin resistance. None of our isolates harbored the mef(A) gene, which is rare in France (Arpin et al., letter; Angot et al., 39th ICAAC). Macrolide resistance due to mutations in ribosomal protein L4, as recently reported for clinical isolates (Davies et al., 40th ICAAC), was not investigated for our strains. Our results contrast with the epidemiology of the mechanisms of resistance described for North American isolates; this may result from the use of 16-membered-ring macrolides in France, which tends to select strains with the MLSB phenotype, conferring resistance to 14-, 15-, and 16-membered-ring macrolides (14; D. Guillemot, C. Carbon, N. Thibult, H. Lecoeur, P. Weber, and E. Eschewege, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1863, p. 463, 2000).

Acknowledgments

We thank Joyce Sutcliffe for providing S. pyogenes reference strains 02C 1061, O2C 1064, and 02C 1110 and R. Leclercq for helpful discussions.

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