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Antimicrob Agents Chemother. May 2001; 45(5): 1595–1598.
PMCID: PMC90516
Note

Serotype 19F Multiresistant Pneumococcal Clone Harboring Two Erythromycin Resistance Determinants [erm(B) and mef(A)] in South Africa

Lesley McGee,1,* Keith P. Klugman,1 Avril Wasas,1 Thora Capper,1 Adrian Brink,2 and The Antibiotics Surveillance Forum Of South Africa

Abstract

One hundred eighteen erythromycin-resistant Streptococcus pneumoniae (ERSP) strains (MICs of ≥0.5 μg/ml) from five laboratories serving the private sector in South Africa were analyzed for the genes encoding resistance to macrolides. Sixty-seven ERSP strains (56.8%) contained the erm(B) gene, and 15 isolates (12.7%) contained the mef(A) gene. Thirty-six isolates (30.5%) harbored both the erm(B) and mef(A) genes and were highly resistant to erythromycin and clindamycin. DNA fingerprinting by BOX-PCR and pulsed-field gel electrophoresis identified 83% of these strains as belonging to a single multiresistant serotype 19F clone.

Resistance to erythromycin in Streptococcus pneumoniae has been observed since 1967 (3) and was first reported for South African multiresistant pneumococcal strains in 1978 (7). Macrolide resistance in the pneumococcus has increased considerably over the last 5 years in several geographic areas (1, 4, 5, 9, 15).

Until recently, the unique mechanism of macrolide resistance in streptococci was target modification by 23S rRNA methylases encoded by erm genes, which convey cross-resistance to macrolides, lincosamides, and the streptogramin B compounds (MLSB phenotype) (12). With the increase of erythromycin resistance, a new phenotype designated M, consisting of resistance to 14- and 15-member-ring macrolides but susceptibility to 16-member-ring macrolides, lincosamides, and type B streptogramins, emerged. S. pneumoniae strains with the M phenotype carry the mef(A) gene, which codes for an efflux mechanism (22). [The mef(A) and mef(E) genes for Streptococcus pyogenes and S. pneumoniae have been classified into one group, mef(A) (19).] In Canada (8) and the United States (L. K. McDougal and F. C. Tenover, Program Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-77b, p. 59, 1997) the M phenotype represents the prevailing mechanism of macrolide resistance, while in Spain (M. Lantero, A. Portillo, M. J. Gastanares, F. Ruiz-Larrea, M. Zarazaga, I. Olarte, et al., Program Abstr. 4th Int. Conf. Macrolides Azalides Streptogramins Ketolides, abstr. 3.10, p. 34, 1998) an MLSB phenotype is observed almost exclusively. Tait-Kamradt et al. (23) have recently described a third mechanism of resistance to macrolides in the pneumococcus, where wild-type strains and laboratory mutants lacking the erm and mef genes show mutations in 23S rRNA genes or in the ribosomal protein L4.

We have recently documented the emergence of the M phenotype in erythromycin-resistant S. pneumoniae (ERSP) strains isolated from the public sector in South Africa (24). In the work described here we determined the prevalence of mef(A) and erm(B) genes in 118 ERSP isolates from the private sector in South Africa, where macrolides are more frequently prescribed than in the public sector (24).

S. pneumoniae clinical strains isolated between July and September 1999 in five laboratories serving the private sector in four cities in South Africa were initially screened for erythromycin resistance by disk diffusion according to NCCLS criteria (16). Clinical isolates collected from the following sites were included in the study: cerebrospinal fluid (CSF), middle ear, blood, sinus tract, and sputum. Based on the NCCLS criteria, 118 ERSP strains were identified and included in the study (Table (Table1).1). MICs of erythromycin (Sigma, St. Louis, Mo.), penicillin (Sigma), and clindamycin (Sigma) were determined for all strains by the broth dilution method according to NCCLS guidelines (17). Additional MICs of chloramphenicol (Sigma), tetracycline (Sigma), trimethoprim (Glaxo Wellcome, Green- ford, United Kingdom)-sulfamethoxazole (Sigma), rifampin (Sigma), and levofloxacin (Hoechst Marion Roussel, Romainville, France) were determined for strains carrying both erm(B) and mef(A) genes. All 118 isolates were resistant (MICs, ≥ 1 μg/ml) to erythromycin, with 92 (78%) of these isolates exhibiting high-level resistance (>64 μg/ml). One hundred three isolates (87.3%) were resistant to clindamycin (MICs, ≥1 μg/ml), and 102 (86.4%) were resistant to penicillin (MICs, ≥0.12 μg/ml). Serotyping showed that the majority (78.8%) of isolates belonged to serotypes 19F (46.6%), 6B (18.6%), and 14 (13.6%). The majority of strains were isolated from the upper respiratory tract, with 34.7% from sputum and 33.8% from the ear. Two strains were isolated from the CSF and blood. Overall, 22% of the strains were from adults and 75.4% of the strains were from children under 12 years of age. For three isolates the patient's age was unknown.

TABLE 1
Distribution and resistance mechanisms of 118 ERSP isolates from five private laboratories in South Africa

All 118 ERSP strains were screened for the presence of the erm(B) and mef(A) resistance determinants using PCR (21, 24). The erm(B) gene alone was amplified in 67 isolates (56.8%), and the mef(A) gene was amplified in 15 isolates (12.7%). We identified 36 isolates (30.5%) in which both the erm(B) and mef(A) genes were amplified, which was confirmed by sequencing (20). Isolates carrying the erm(B) gene alone were all highly resistant to both erythromycin (MICs, ≥8 μg/ml) and clindamycin (MICs, ≥4 μg/ml), while those carrying the mef(A) gene alone showed low-level resistance to erythromycin (MICs, 1 to 4 μg/ml) and no resistance to clindamycin, which is typical of the M phenotype. The isolates harboring both resistance genes showed resistance patterns identical to those of strains carrying the erm(B) gene alone, i.e., high-level resistance to both erythromycin (MICs, ≥8 μg/ml) and clindamycin (MICs, ≥4 μg/ml). These erm(B) mef(A) strains were multiply resistant, showing in addition high-level penicillin resistance as well as resistance to chloramphenicol, tetracycline and trimethoprim-sulfamethoxazole.

DNA fingerprinting by BOX-PCR (11) and pulsed-field gel electrophoresis (PFGE) (13) identified 43 types among the 118 strains. Thirty of the 36 isolates identified as having both the erm(B) and mef(A) genes, which were isolated in all four cities in South Africa (Fig. (Fig.1),1), were shown to belong to a single multiply resistant 19F clone (Fig. (Fig.2).2).

FIG. 1
Geographic locations of laboratories and distribution of isolates belonging to a serotype 19F clone in South Africa.
FIG. 2
DNA fingerprint patterns for representative isolates of the 19F multiresistant clone with erm(B) and mef(A) genes by BOX-PCR (a) and PFGE (b). Lanes: 1, R6 (unencapsulated laboratory strain); 2 to 4, isolates PA7062, D1, and J5, respectively; 5 to 7, ...

Resistance to erythromycin in the pneumococcus was first reported in South Africa in the late 1970s (7), and although the rates of resistance of strains isolated from the public sector have increased from 2.5% in 1987 to 1991 (A. Wasas, R. E. Huebner, and K. P. Klugman, 7th Joint Biennial Congr. STD ID Soc. South. Afr., abstr. IDP18, 1999) to 4.9% in 1995 to 1998 (24), these rates are relatively low. In South Africa, macrolide use in the public sector is estimated at 56% of that in the private sector (24). Erythromycin resistance rates in the private sector have increased from 13.3% in 1986 (10) to 38.8% in 1999 (6), a rate far higher than that observed in the public sector. An essential factor in the increase of resistance is the availability and use of pediatric services and antibiotics for children in the private sector.

Virtually all clinical isolates of macrolide-resistant pneumococcal strains that have been examined for macrolide resistance mechanisms have contained either mef(A) or erm(B), with the mef(A) gene predominant in some countries (McDougal and Tenover, 37th ICAAC) and the erm(B) gene the major resistance determinant in others (Lantero et al., Int. Conf. Macrolides Azalides Streptogramins Ketolides). Pneumococcal strains that contain both genes have occasionally been reported (2, 14) and a recent study from Tokyo, Japan (18), reports that 16.1% of macrolide-resistant strains isolated from a single hospital harbored both genes simultaneously. In the present study, a surprisingly high percentage (30.5%) of isolates harbored both genes. In a recent study conducted on strains isolated in the public sector in South Africa, the M phenotype was reported as having increased significantly as a percentage of macrolide-resistant strains from 1987 to 1991 (0.8%) compared with 1992 to 1996 (19.7%) in blood and CSF isolates (24). No strains harboring both genes were detected in that study, however.

Susceptibilities to erythromycin and clindamycin for the erm(B) mef(A) strains were identical to those for strains carrying erm(B) alone, which suggests that erm(B) is sufficient on its own to express resistance and that the presence of the mef(A) gene cannot be inferred from the phenotypic expression of MIC. The clinical impact of erm(B) plus mef(A) is likely to be similar to that of erm(B) alone, as the high-level resistance phenotype is the same. DNA fingerprinting revealed that 25.4% of all ERSP strains in this study belonged to a single multiresistant serotype 19F clone. The clone appears to have disseminated in both the adult and child populations, suggesting that this clone is circulating in the community and contributing to macrolide resistance in the private sector, where macrolide consumption is high. Corso et al. (2) describe a serogroup 19 macrolide-resistant clone (3.3% of macrolide-resistant strains), harboring erm(B) and mef(A) genes, in the United States which appears to be related to this clone based on PFGE patterns.

The results from this study suggest that although the MLSB phenotype is still predominate in macrolide resistance in South Africa, the M phenotype, which is relatively new, appears to be emerging as an important factor in erythromycin-resistant pneumococci. The majority of erm(B) mef(A) strains belong to a multiresistant serotype 19F clone which is circulating throughout South Africa, contributing to high levels of resistance to erythromycin and clindamycin, especially in children under 5 years of age, and may be present in the United States. The molecular relatedness of the resistant erm(B) and mef(A) strains should be determined, and if these strains are confirmed to be an identical clone, this would represent the global emergence of this clone.

Acknowledgments

Collection of S. pneumoniae isolates from the five laboratories was made possible by a grant from Abbott Laboratories.

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