In Vitro Activities of the Novel Ketolide Telithromycin (HMR 3647) against Erythromycin-Resistant StreptococcusSpecies

RESULTS AND DISCUSSION

In this study, the in vitro activities of telithromycin and six other antibiotics against 184 erythromycin-resistant and 51 erythromycin-susceptibleStreptococcus strains were studied (Table 1 and Fig.1). In general, telithromycin was more active than 14- and 15-member-ring macrolides (erythromycin, azithromycin, and clarithromycin) against erythromycin-resistant Streptococcus strains. MICs of telithromycin were ≤0.5 μg/ml for all but eight S. pyogenes strains (see below). Quinupristin-dalfopristin, a mixture of streptogramin A and B antibiotics, showed activity similar to that of telithromycin, although MICs were somewhat higher (MIC at which 90% of the isolates are inhibited [MIC₉₀], 0.5 to 4 μg/ml). Clindamycin (a lincosamide) was active against strains with an inducible erm methylase gene (MIC₉₀, 0.063 to 0,5 μg/ml) and was even more active than telithromycin against strains with amef gene (MIC₉₀, 0.063 to 0.125 μg/ml). The clindamycin MICs for strains with a constitutive ermmethylase gene were high (MIC₉₀, >64 μg/ml). Also,S. pneumoniae strains with the novel resistance phenotype were intermediate or resistant to clindamycin (MIC₉₀, 4 μg/ml). Spiramycin (a 16-member-ring macrolide) showed activity similar to that of telithromycin against strains with a mef resistance gene. However, the spiramycin MICs for all strains with an erm methylase gene (inducible or constitutive) and for S. pneumoniae strains with the novel resistance phenotype were elevated (MIC₉₀, 4 to >64 μg/ml).

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Fig. 1.

Distribution of macrolide resistance genes of S. pyogenes (A), GCS (B), GGS (C), and S. pneumoniae (D) strains according to telithromycin MICs Abbreviations: ERY, erythromycin; c, constitutive; i, inducible; erm, erythromycin ribosome methylation genes; mef, macrolide efflux genes. See text for details of the novel resistance phenotype.

The eight S. pyogenes strains for which the telithromycin MICs were elevated included one strain with an inducible erm(B) gene (MIC, 1 μg/ml), one strain with two resistance genes [an inducible erm(B) gene and amef(A) gene; MIC, 4 μg/ml], and, most importantly, six strains with a constitutive erm(B) gene (MIC₉₀, 64 μg/ml). These six S. pyogenes strains belonged to six different serotypes (T12M22 opacity factor positive, T12M12 opacity factor negative, T1M1 opacity factor negative, T12M66 opacity factor positive, T6M6 opacity factor negative, and T28R28 opacity factor positive), six different Vir types, and six different random amplified polymorphic DNA types, indicating that the strains were not of the same clonal origin. In contrast to the case for S. pyogenesstrains, telithromycin MICs for gene S. pneumoniae strains with the constitutive erm(B) were low (MIC₉₀, 0.25 μg/ml). This finding is concordant with a previous study where telithromycin MICs for S. pneumoniae strains with the constitutive MLS_Bphenotype of erythromycin resistance were low (MIC₉₀, 0.25 μg/ml) (16). Similar to the case for S. pyogenes, telithromycin MICs for constitutively erythromycin-resistant S. aureus strains have been shown to be elevated (6). Telithromycin MICs for S. pyogenes and GGS strains which had an inducible erm(TR) gene were low (MIC₉₀, 0.063 μg/ml). In contrast to the case for the constitutive erm(B) gene, constitutive expression of the erm(TR) gene in one S. pyogenes strain did not increase the telithromycin MIC.

An interesting question is why the Erm(B) methylase does not protectS. pneumoniae as well as it protects S. pyogenesagainst telithromycin. The Erm(B) methylases of S. pneumoniae and S. pyogenes are nearly identical (99% similarity at the amino acid level [21]), so it is unlikely that the differences in the erm(B) genes can explain differences in susceptibility to telithromycin. Structural differences in the 23S rRNA molecules or other ribosomal components are more likely explanations. Ketolides as well as other macrolides interact with the peptidyl transferase loop in the V domain of the 23S rRNA. For example, both telithromycin and erythromycin protect positions A2058, A2059, and G2505 of the 23S rRNA against chemical modification (7, 28). It has also been shown that hairpin 35 in domain II of the 23S rRNA constitutes a part of the binding site of macrolides and ketolides (7,28). Telithromycin protects against chemical modification, and erythromycin enhances chemical modification, of residue A752 in hairpin 35 (7). Actually, using different ketolide derivatives it has been shown that interaction with hairpin 35 is essential for the antimicrobial activity of ketolides (4). Methylation of A2058 of the peptidyl transferase loop in the V domain confers resistance to MLS_B antibiotics by inhibiting the binding of the antibiotics to the ribosomes. However, as we have shown here withS. pneumoniae and as has been shown with several other bacteria (6, 18), this methylation does not protect against telithromycin. It has been proposed that although methylation of A2058 weakens the binding of macrolides and ketolides to the ribosomes by interfering with the interaction between the antibiotic and the residues on the peptidyl transferase loop, it does not prevent the strong interaction of ketolides with hairpin 35. This interaction therefore is probably enough to bind the antibiotic to the ribosome and to prevent protein synthesis (28). The same is true with mutations in the peptidyl transferase loop, which can inhibit binding of macrolides but not ketolides to the ribosomes if ketolides have alkyl-aryl 11/12 lactone ring extensions, which can make contact with hairpin 35 and the drug (4). It might be that in S. pyogenes the interaction of telithromycin with hairpin 35 is weaker than that in S. pneumoniaeand because of that, telithromycin can not bind to the ribosomes of S. pyogenes if A2058 is methylated. Structural differences in the ribosomes of the two bacteria may thus explain the differences in the interactions between telithromycin and hairpin 35.

Quite recently, Tait-Kamradt et al. (23) demonstrated that in S. pneumoniae mutations in the 23S rRNA or ribosomal protein L4 can cause resistance to macrolides. Mutations in the pepdityl transferase loop of the 23S rRNA caused phenotypes that were similar but not identical to those described in this work for S. pneumoniae strains without any known macrolide resistance genes. The type, number, and positions of mutations in the peptidyl transferase loop affect the phenotype, so it is possible that the strains we describe here also carry resistance-causing mutations in 23S rRNA molecules.

Telithromycin is a novel ketolide that belongs to the macrolide family of antibiotics. Our work in addition to several other studies, indicates that the new ketolides are active in vitro against various gram-positive bacteria, including strains that are resistant to other macrolides (6, 16). Telithromycin MICs were high only forS. pyogenes strains with the constitutive erm(B) resistance gene. In recent years these strains have comprised about 10% of all erythromycin-resistant S. pyogenes strains in Finland (11) and several other countries (10). Although the presence of a constitutiveerm(B) gene in S. pyogenes varies in different countries (10), it seems that telithromycin has good in vitro activity against most S. pyogenes strains.