High-Level Telithromycin Resistance in a Clinical Isolate of Streptococcus pneumoniae

Abstract

A rare clinical isolate of Streptococcus pneumoniae, highly resistant to telithromycin, contained erm(B) with a truncated leader peptide and a mutant ribosomal protein L4. By transformation of susceptible strains, this study shows that high-level telithromycin resistance is conferred by erm(B), wild type or mutant, in combination with a ₆₉GTG₇₁-to-TPS mutation in ribosomal protein L4.

Telithromycin (TEL), the first ketolide approved for clinical use, is a semisynthetic derivative of the macrolide erythromycin A. Modifications of the 14-membered macrolactone ring include a replacement of the l-cladinose sugar at position 3 with a ketone group and a carbamate extension at position C11-C12. Telithromycin and the macrolide antibiotics bind to the peptidyl transferase region of the large ribosomal subunit and inhibit protein synthesis by blocking the peptide exit tunnel (7, 33). Macrolides and ketolides also interfere with assembly of the 50S ribosomal subunit (3). The C11-C12 carbamate extension of telithromycin enables it to bind to A752 in domain II of 23S rRNA, in addition to the primary binding site of erythromycin, A2058, in domain V (7). As a result, telithromycin has a stronger binding affinity for the ribosome and therefore can overcome common macrolide resistance mechanisms including target modification and drug efflux (2, 13). The former is directed by a methylase, encoded by the erm(B) gene, which methylates a specific adenine residue (A2058) in domain V of the 23S rRNA to block macrolide binding. This process results in high-level resistance to macrolides, lincosamides (clindamycin), and streptogramin B, referred to as the MLS_B phenotype (32). Mutations in the 23S rRNA and ribosomal proteins, involved in translation, that interrupt macrolide binding have also been described (10, 27, 28). The mef gene mediates active drug efflux that leads to low-level resistance to macrolides only (M phenotype) (26).

Pneumococcal resistance to telithromycin remains rare. In laboratory-generated telithromycin-resistant mutants, mutations were shown to occur in the erm(B) upstream region (31). Clinical isolates with reduced susceptibility to telithromycin have shown mutations to occur in erm(B) (29) and ribosomal proteins L4 and L22 (11, 17, 18, 28). Tait-Kamradt et al. (29) described a highly resistant clinical isolate of Streptococcus pneumoniae, BSF11524, isolated from the conjunctiva of a 1-year-old boy in Canada in 1996 and submitted to the Canadian Bacterial Surveillance Network as part of an ongoing pneumococcal resistance surveillance program. This occurred several years prior to the approval of telithromycin in Canada. No further telithromycin-resistant isolates have been detected by the same network (15, 20). The isolate was found to contain mutations in erm(B) and ribosomal protein L4. In this study we investigate BSF11524 further, to establish the mechanism of resistance in this rare isolate.

Pneumococci were routinely cultured at 37°C in 5% CO₂ on Mueller-Hinton agar supplemented with 5% horse blood. MICs were determined by the agar dilution method according to CLSI guidelines (4) and the Etest (AB Biodisk, Solna, Sweden). CLSI breakpoints were used (5). For telithromycin breakpoints were ≤1 μg/ml for susceptibility, 2 μg/ml for intermediacy, and ≥4 μg/ml for resistance. Serotyping was performed by the Quellung reaction with antisera from the Statens Serum Institut (Copenhagen, Denmark). Chromosomal DNA was extracted as previously described (23). PCR-based methods were used to screen for erm(B) and mef(A) (25). Genes encoding L4 and L22 and all four alleles encoding 23S rRNA were amplified according to previously described methods (10, 27). The erm(B) gene was amplified using forward primer ermBF (5′-CTTAGAAGCAAACTTAAGAG-3′) and reverse primer ermBR (5′-ATCGATACAAATTCCCCGTAG-3′). Amplified products were purified with the QIAquick gel extraction kit (QIAGEN Ltd., Surrey, United Kingdom). DNA sequencing was performed using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and an Applied Biosystems Model 310 automated DNA sequencer.

BSF11524 is serotype 19A and is highly resistant to erythromycin (MIC, >256 μg/ml), clindamycin (MIC, >256 μg/ml), and telithromycin (MIC, >256 μg/ml). It is resistant to tetracycline (MIC, 12 μg/ml) and penicillin (MIC, 16 μg/ml) but susceptible to chloramphenicol (MIC, 2 μg/ml). It was confirmed, as described previously (29), to be erm(B) positive and mef(A) negative. The erm(B) gene contained an adenine base insertion in the control peptide creating a stop codon and resulting in the truncation of the control peptide to 10 amino acids. In addition, three mutations were found in erm(B): I75T, S100N, and H118R. Ribosomal protein L4 was found to contain the following mutations: E13Q, S20N, E30Q, ₆₉GTG₇₁ to TPS, V88I, G98A, A128S, and S130E. Ribosomal protein L22 and all four alleles of 23S rRNA were wild type.

In order to confirm the role of the mutations in conferring telithromycin resistance, transformations were carried out. Two pneumococcal strains were used for transformation studies: an unencapsulated laboratory strain (R6), susceptible to all antibiotics, and a strain (PC13) representative of pneumococcal clone 13 (South Africa^19A) (16). PC13 was used as a recipient strain for transformation studies due to the fact that R6 does not contain an erm(B) gene for homologous recombination. Attempts to introduce a wild-type erm(B) gene into R6 by means of electroporation and conjugation were unsuccessful. The PC13 strain was selected based on its containing a wild-type erm(B) gene and being susceptible to telithromycin (MIC, 0.06 μg/ml). The elevated telithromycin MIC of PC13 in comparison with those of R6 and other fully susceptible strains is due to the presence of the erm(B) gene (6, 12). The genes encoding 23S rRNA and ribosomal proteins L4 and L22 in PC13 were confirmed to be wild type. R6 and PC13 were made competent by culture in C medium (30), and transformation was performed as previously described (24). Transformants were selected on Mueller-Hinton agar supplemented with 5% horse blood and containing erythromycin (1 μg/ml) for R6 and TEL (0.5 μg/ml) for PC13. MICs of the transformants were determined, and the presence of mutations was confirmed by sequencing.

PC13 (TEL MIC, 0.06 μg/ml) was transformed with the mutant erm(B) gene of BSF11524. PC13^ermB transformants had a telithromycin MIC of 1 μg/ml (Table 1). PC13 was transformed with the mutant L4 gene of BSF11524. PC13^L4 transformants had a TEL MIC of >256 μg/ml (Table 1). The L4 genes of 10 PC13^L4 transformants were sequenced and were found to contain various combinations of the mutations in the L4 gene of BSF11524; however, only the ₆₉GTG₇₁-to-TPS mutation occurred in all transformants. This mutation occurs in a highly conserved region (₆₃KPWRQKGTGRAR₇₄) of L4. In order to confirm that this mutation alone confers telithromycin resistance, a fragment of the L4 gene (L4Fr) containing only this mutation was used to transform PC13. PC13^L4Fr transformants had a telithromycin MIC of >256 μg/ml. The S20N mutation in L4 of BSF11524 has been associated with resistance to macrolides (21) and has been identified in telithromycin-nonsusceptible strains (1); therefore, PC13 was transformed with a mutated L4 gene containing only the S20N mutation. Transformants were not selected in the presence of telithromycin. The role of the L4 mutations in the absence of an erm(B) gene was investigated by transforming R6 (TEL MIC, 0.015 μg/ml) with the full-length L4 gene of BSF11524 and the L4Fr fragment containing only the ₆₉GTG₇₁-to-TPS mutation. R6^L4 and R6^L4Fr transformants had telithromycin MICs of 0.12 μg/ml and erythromycin MICs of >256 μg/ml (Table 1).

TABLE 1.

Phenotypic and genotypic results of pneumococcal transformations

Strain	erm(B)a	L4b	MIC (μg/ml) of drugc:
			TEL	ERY	AZM	CLR	CLI	TET	CHL
BSF11524	mt	mt	>256	>256	>256	>256	>256	12	2
PC13 transformantsd
PC13	wt	wt	0.06	>256	>256	>256	>256	12	12
PC13ermB	mt	wt	1	>256	>256	>256	>256	8	12
PC13L4	wt	mt	>256	>256	>256	>256	>256	12	12
PC13L4Fr	wt	69GTG71 to TPS	>256	>256	>256	>256	>256	8	12
R6 transformantse
R6		wt	0.015	0.094	0.25	0.06	0.06	0.094	1.5
R6L4		mt	0.12	>256	>256	24	0.125	0.094	2
R6L4Fr		69GTG71 to TPS	0.12	>256	>256	24	0.094	0.06	2

^awt, wild-type erm(B) gene; mt, mutant erm(B) gene of BSF11524.

^bwt, wild-type L4 gene; mt, mutant L4 gene of BSF11524.

^cERY, erythromycin; AZM, azithromycin; CLR, clarithromycin; CLI, clindamycin; TET, tetracycline; CHL, chloramphenicol.

^dPC13 transformed with the mutant erm(B) gene, the mutant L4 gene, or with a gene fragment (L4Fr) housing the ₆₉GTG₇₁-to-TPS mutation of L4 from BSF11524.

^eR6 transformed with the mutant L4 from BSF11524 or the L4Fr gene fragment.

The mutant erm(B) gene of BSF11524 reduces the susceptibility of PC13 to telithromycin; however, it does not confer the high-level resistance observed in the isolate. The reduced telithromycin susceptibility of the PC13^ermB transformants in comparison with PC13 may be due to increased dimethylation of A2058 in 23S rRNA as a result of the truncated control peptide in erm(B) (8, 14). The full-length L4 gene and the fragment of L4 containing the ₆₉GTG₇₁-to-TPS mutation (L4Fr) conferred high-level telithromycin resistance on PC13. The L4 gene and L4Fr fragment reduced the susceptibility of R6 to telithromycin; however, they did not confer resistance as for PC13. It is therefore highly likely to be the combination of erm(B) with the ₆₉GTG₇₁-to-TPS mutation of L4 that confers high-level telithromycin resistance.

The ₆₉GTG₇₁-to-TPS mutation of L4 has been previously described (19, 28), and as shown here, when not combined with erm(B) it confers high-level erythromycin resistance but confers only reduced susceptibility to telithromycin. Erythromycin and telithromycin share a common binding site; however, telithromycin forms a tighter bond with the ribosome due to an additional interaction with A752 in domain II of 23S rRNA (7). The ₆₉GTG₇₁-to-TPS mutation in L4 may therefore destabilize the binding of telithromycin; however, it does not block it completely, as for erythromycin. Higher levels of resistance to telithromycin appear to be a result of a combination of mutations. Faccone et al. (9) described a clinical isolate with a telithromycin MIC of 256 μg/ml with an A2058T mutation in 23S rRNA and a deletion in L22. A combination of an A2058G mutation in 23S rRNA and an RTAHIT insertion in L22 resulted in a telithromycin MIC of 16 μg/ml (17). In addition, a telithromycin-resistant isolate with a MIC of 8 μg/ml was found to contain an erm(B) gene, an S20N mutation in L4, and a number of mutations in 23S rRNA (22). A highly resistant laboratory-generated strain (MIC, >32 μg/ml) contained a 210-bp deletion in the erm(B) upstream region together with a K94Q mutation in riboprotein L22 (31).

Growth studies were performed by inoculating tryptone soy broth with glycerol stocks (1:100 dilution) and monitoring turbidity at 600 nm every 30 min. Statistical differences between mass doubling times were calculated using the unpaired t test with P values interpreted at the 95% confidence level. Mass doubling times (minutes, mean ± standard error of the mean) during the exponential phase of growth were as follows: BSF11524, 40.7 ± 2.05; PC13, 42.05 ± 0.45; PC13^ermB, 41.6 ± 0.9; PC13^L4Fr, 40.7 ± 0.4; R6, 48.3 ± 0.05 and R6^L4Fr, 47.3 ± 0.65. There was no significant difference between the doubling times of PC13 and PC13^ermB (P = 0.71) or PC13 and PC13^L4Fr (P = 0.16). The doubling times of R6 and R6^L4Fr were not significantly different (P = 0.37). The mutations in erm(B) of BSF11524 and the ₆₉GTG₇₁-to-TPS mutation of ribosomal protein L4 do not appear to be associated with a fitness cost.

In this study, high-level telithromycin resistance was shown to be conferred by an erm(B) gene in combination with a ₆₉GTG₇₁-to-TPS mutation in a highly conserved region of ribosomal protein L4. The rarity of the emergence of such a resistant phenotype, despite the availability and worldwide use of telithromycin since 2002, may be because of the need for both the erm gene and mutations in ribosomal genes. Its failure to disseminate may in part be due to the lack of selective pressure, since its appearance predated the approval and use of telithromycin in Canada by several years.