Macrolide- and Telithromycin-resistant Streptococcus pyogenes, Belgium, 1999–2003

We found a 13% macrolide resistance in 3,866 Streptococcus pyogenes isolated from tonsillopharyngitis patients; 59% macrolide-resistant isolates were distributed in 5 clones, suggesting the importance of both resistance gene transfer and clonal dissemination in the spread of these organisms. We also report one of the largest collections of telithromycin-resistant isolates.

We found a 13% macrolide resistance in 3,866 Streptococcus pyogenes isolated from tonsillopharyngitis patients; 59% macrolide-resistant isolates were distributed in 5 clones, suggesting the importance of both resistance gene transfer and clonal dissemination in the spread of these organisms. We also report one of the largest collections of telithromycin-resistant isolates.
S treptococcus pyogenes causes several million cases of upper respiratory tract infection each year. The problem of these infections is growing as resistance increases among S. pyogenes to the macrolide group of antimicrobial drugs commonly used to treat such infections (1)(2)(3)(4). S. pyogenes acquires resistance by 2 main mechanisms. The first is active drug efflux mediated by an ATP-binding cassette transporter wherein mef(A) encodes the transmembrane domains and msr(D) encodes the ATP-binding domains (5). This pattern of resistance is demonstrated by an M phenotype. In the second mechanism, gene products of erm(B) or erm(A) methylate the macrolide-binding site on 23S rRNA and stall bacterial protein synthesis. This pattern of resistance is demonstrated by either a constitutive (cMLS) or an inducible (iMLS) phenotype. A third, rare, mechanism is modification of the drug binding site on rRNA by mutation that is expressed as an M or a cMLS phenotype. The newest generation of macrolides, the ketolides, are also active against macrolide-resistant strains; however, few S. pyogenes strains of the cMLS phenotype have been found to be ketolide resistant (6).
In Belgium, the first ketolide to be used clinically, telithromycin, was approved in October 2002 to treat community-acquired respiratory infections in patients >12 years of age. We investigated the temporal trends in resistance and clonality among macrolide (including telithromycin)-resistant S. pyogenes recovered from patients with tonsillopharyngitis during surveillance studies conducted in Belgium.

The Study
During 1999-2003, a total of 4,031 nonduplicate, putative S. pyogenes isolates were collected from 10 Belgian provinces at the reference center with the date of isolation, specimen source, and patient's age and residential address. By using a battery of tests, for example, β-hemolysis on blood agar, Gram stain, catalase production, pyrrolidonyl arylamidase, presence of Group A antigen, and bacitracin susceptibility, 3,866 isolates were confirmed to be S. pyogenes. The age of the patient was known in 3,654 cases. Population statistics are detailed in the first half of Table 1.
By using erythromycin (78 µg) and clindamycin (25 µg) double-disk diffusion (Neo-Sensitab discs; Rosco, Taastrup, Denmark), all 3,866 S. pyogenes isolates were further screened for a phenotypic expression of macrolide resistance, which was identified in 506 (13%) isolates. The proportion of macrolide-resistant isolates among the total S. pyogenes isolated from each of the 10 Belgian provinces fluctuated from 0% to 40% over the 5 years studied. The 3 known phenotypes, cMLS, iMLS, and M, were identified in 209 (41%), 18 (4%), and 279 (55%) isolates, respectively. Changes in prevalence of the 3 phenotypes among macrolide-resistant S. pyogenes over 5 years are presented in the second half of Table 1.
We next studied the genotype for the 3 macrolide-resistant phenotypes. Polymerase chain reaction was performed for erm(A), erm(B), and mef(A) (1,9,10). Isolates negative for all 3 genes were screened for ribosomal mutations in L4, L22, and portions of 23S RNA genes by using published primers (11). Amplimers were analyzed by direct double-strand sequencing (3730 DNA Analyzer, Applied Biosystems, Foster City, CA, USA) with the BigDye Terminator Version 3.1 Kit (Applied Biosystems). Nucleotide sequence alignment was done with SeqMan (DNASTAR Inc., Madison, WI, USA). Phenotypic and genotypic profiles of the macrolide-resistant S. pyogenes were generally consistent; however, 3% of the resistant iso-lates carried 2 resistance genes. Of the 209 cMLS isolates, 199 carried erm(B), 9

carried erm(B)+mef(A), and 1 carried erm(B)+erm(A). Of the 279 M phenotype isolates, 273 carried mef(A), 1 carried erm(B)+mef(A), and 4 carried mef(A)+erm(A)
. The 1 isolate that was negative for all 3 genes carried a single A2059G (Escherichia coli numbering system) mutation in the 23S rRNA gene. The A2059G mutation, although quite frequent in S. pneumoniae, has been rarely observed in S. pyogenes. Finally, of the 18 iMLS isolates, 5 carried erm(B) and 13 carried erm(A).
Ten percent of the macrolide-resistant strains harboring erm(B) alone or with mef(A) were also telithromycinresistant, and telithromycin has additional binding sites on 23S rRNA. Therefore, we hypothesized that either mutations in the erm gene promoter region have upregulated methylase expression or that mutations in the coding region have changed the methylase specificity to include the additional binding sites of telithromycin. Alternatively, mutations at the additional binding sites on the 23S rRNA genes might also disable the binding arm; however, a recent study described only a low level of telithromycin resistance in the presence of these mutations (12). Utilizing 3 pairs of overlapping primers (primer sequences available on request), DNASTAR software, and sequence data of Tn1545 (National Center for Biotechnology Information, Rockville, MD, USA, accession no. X52632), the entire erm(B) gene, including the promoter and control peptides, were sequenced from 10 telithromycin-resistant isolates. In addition, L4, L22, and portions of 23S rRNA genes were also amplified as above. Analysis of the sequencing data showed a single H118R (A677G) substitution in the erm(B) coding region of all 10 telithromycin-resistant isolates. While our study was ongoing, the H118R substitution in erm(B) was also confirmed independently for 2 telithromycin-resistant isolates (6).

Conclusions
We demonstrated in this study that overall macrolide resistance in Belgium is driven by an epidemic spread of a few major clones as well as by resistance gene transfer among genetically diverse S. pyogenes. On average, we demonstrated a 2-fold (13%) increase in macrolide resistance in Belgium from 1999 to 2003, compared to that observed from 1995 to 1997 (6.5%) (1). Although, a general increase in macrolide-resistance in Europe has been observed during the last few years, resistance levels tend to differ considerably between countries. For instance, while resistance rates in Germany (6) and Poland (4) were similar to those observed in Belgium, considerably higher resistance levels were observed in Spain and Portugal (2), as well as in Italy (3). Provincial variations in macrolideresistance observed in Belgium have also been reported in other countries (3); however, the precise causes underlying such variations within or between countries are not fully understood. Macrolide consumption might be one factor that explains the regional variations in macrolide-resistant S. pyogenes in Spain and Finland (13,14), especially when consumption surpasses a critical threshold (15). However, in France, one of the highest macrolide consumption within Europe is not paralleled by an equally high resistance in S. pyogenes (16,17). The identification of telithromycinresistant S. pyogenes in our study, many of which were already present in the community before the introduction of telithromycin in Belgium, also suggest that antimicrobial drug use and development of resistance might be dissociated to some extent. Clearly, other factors like natural fluctuations in prevalence of clones (18), patient compliance with antimicrobial drug regimens, fitness costs of drug resistance, or even tetracycline consumption (tetracycline and macrolide-resistance genes cosegregate) (19) might be important determinants for the development and spread of macrolide-resistant S. pyogenes. Thus, any direct link between macrolide use and resistance in S. pyogenes should be interpreted cautiously.