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Antimicrobial Agents and Chemotherapy, February 2008, p. 435-440, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.01074-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Telithromycin Resistance in Streptococcus pneumoniae Is Conferred by a Deletion in the Leader Sequence of erm(B) That Increases rRNA Methylation

Nicole Wolter,^1* Anthony M. Smith,¹ David J. Farrell,² John Blackman Northwood,² Stephen Douthwaite,³ and Keith P. Klugman^1,4

Respiratory and Meningeal Pathogens Research Unit, National Institute for Communicable Diseases, Medical Research Council and University of the Witwatersrand, Johannesburg, South Africa,¹ GR Micro Ltd., London, United Kingdom,² Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark,³ Hubert Department of Global Health, Rollins School of Public Health, and Division of Infectious Diseases, School of Medicine, Emory University, Atlanta, Georgia⁴

Received 15 August 2007/ Returned for modification 23 October 2007/ Accepted 15 November 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A telithromycin-resistant clinical isolate of Streptococcus pneumoniae (strain P1501016) has been found to contain a version of erm(B) that is altered by a 136-bp deletion in the leader sequence. By allele replacement mutagenesis, a second strain of S. pneumoniae (PC13) with a wild-type erm(B) gene was transformed to the telithromycin-resistant phenotype by introduction of the mutant erm(B) gene. Whereas the wild-type PC13 strain showed slight telithromycin resistance only after induction by erythromycin (telithromycin MIC increased from 0.06 to 0.5 µg/ml), the transformed PC13 strain is constitutively resistant (MIC of 16 µg/ml). Expression of erm(B) was quantified by real-time reverse transcription-PCR in the presence of erythromycin or telithromycin; erm(B) expression was significantly higher in the transformed PC13 strain than the wild-type strain. Furthermore, the transformed strain had significantly higher levels of ribosomal methylation in the absence as well as in the presence of the antibiotics. Growth studies showed that the transformed PC13 strain had a shorter lag phase than the wild-type strain in the presence of erythromycin. Telithromycin resistance is conclusively shown to be conferred by the mutant erm(B) gene that is expressed at a constitutively higher level than the inducible wild-type gene. Elevated erm(B) expression results in a higher level of rRNA methylation that presumably hinders telithromycin binding to the ribosome.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Macrolide resistance in Streptococcus pneumoniae is conferred by two predominant mechanisms: target-site modification and active drug efflux due to the acquisition of erm(B) and mef(A) genes, respectively, or a combination of these mechanisms (16). The erm(B) gene encodes an rRNA methylase that methylates nucleotide A2058 (Escherichia coli numbering) in 23S rRNA, resulting in the macrolide-lincosamide-streptogramin B (MLS_B) resistance phenotype (36). The mef(A) gene encodes an efflux pump and confers the M phenotype with resistance to 14- and 15-membered ring macrolides (29). In a small proportion of isolates, mutations in 23S rRNA and/or ribosomal proteins L4 and L22 were found to confer macrolide resistance (11, 30, 31).

Telithromycin is a ketolide antibiotic, a semisynthetic derivative of the 14-membered macrolide erythromycin A. Macrolides and ketolides bind close to the peptidyl transferase region of 23S rRNA and inhibit bacterial protein synthesis by blocking elongation of the peptide chain through the tunnel of the large ribosomal subunit (8, 41). Macrolides and ketolides also interfere with 50S ribosomal assembly (4). However despite common mechanisms of action, ketolides remain active against most macrolide-resistant strains expressing erm(B) and mef(A) (3, 14, 18). Ketolides bind to the ribosome with greater affinity than macrolides such as erythromycin (3, 7). The primary contact site of erythromycin and telithromycin is at nucleotide A2058 of domain V of 23S rRNA, and telithromycin possibly makes additional contacts within domain II of 23S rRNA (1, 7, 13, 39), although the exact site of the other contacts remains controversial (34).

Telithromycin resistance in S. pneumoniae remains rare. In laboratory-generated strains with reduced susceptibility to telithromycin, mutations have been observed in domains II and V of 23S rRNA, ribosomal protein L22, and erm(B) (2, 35). Mutations in these genes, as well as ribosomal protein L4, have also been found in clinical telithromycin-resistant isolates (12, 20, 21, 22, 31), and a combination of mutated genes can result in a higher telithromycin resistance than mutation of only one gene (10, 20). Although deletions in the erm(B) leader sequence are a common molecular change associated with telithromycin resistance in pneumococci (32, 35), the exact effects of these mutations on the expression of erm(B) and rRNA methylation have not previously been quantified using isogenic pneumococcal strains. In this study, a clinical isolate of S. pneumoniae identified as resistant to telithromycin and containing a large deletion mutation in the leader sequence of erm(B) was investigated to characterize the resistance mechanism.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial strains. A clinical isolate of S. pneumoniae (P1501016) identified as resistant to telithromycin (MIC of 8 µg/ml) was obtained from the PROTEKT surveillance study. Clinical patient information was not available, and therefore previous treatment with telithromycin could not be determined. A strain (PC13) representative of pneumococcal clone 13 (South Africa^19A) (17) was used as a recipient in allele replacement mutagenesis. PC13 was resistant to macrolides (erythromycin MIC [µg/ml] of >256 and clarithromycin MIC of >256) and clindamycin (MIC of >256 µg/ml) but susceptible to telithromycin (MIC of 0.06 µg/ml). Pneumococci were routinely cultured at 37°C in 5% CO₂ on Mueller-Hinton agar supplemented with 5% horse blood.

Phenotypic and genotypic characterization. MICs were determined by the agar dilution method and the Etest (AB Biodisk, Solna, Sweden). Agar dilution plates were prepared according to the CLSI guidelines pertaining to the general preparation of agar dilution plates (5). Plates contained 5% horse blood and were incubated in ambient air for 20 to 24 h. MICs were interpreted as susceptible, intermediately resistant, or resistant using CLSI breakpoints for broth microdilution (6). For telithromycin the breakpoints were 1 µg/ml for susceptible, 2 µg/ml for intermediate, and 4 µg/ml for resistant. S. pneumoniae ATCC 49619 was used as a reference strain. P1501016 was serotyped by the quellung reaction with antiserum from the Statens Serum Institut (Copenhagen, Denmark).

Chromosomal DNA was extracted from overnight pneumococcal cultures as previously described (26). A duplex PCR was used to identify the presence of the erm(B) and mef(A) genes (28). The transposon (Tn1545 or Tn917) carrying erm(B) was identified as previously described (19) using transposon-specific forward primers (Tn1545 primer ermBF, 5'-CTTAGAAGCAAACTTAAGAG-3'; Tn917 primer Tn917F, 5'-TGACGGTGACATCTCTC-3') and a common reverse primer (ermBM-R2, 5'-CTGTCTAATTCAATAGACGT-3'). Genes encoding ribosomal proteins L4 and L22 and all four alleles encoding 23S rRNA were amplified and sequenced according to previously described methods (11, 30).

The erm(B) gene was amplified using forward primer ermBF (5'-CTTAGAAGCAAACTTAAGAG-3') and reverse primer ermBR (5'-ATCGATACAAATTCCCCGTAG-3'). For each 50-µl reaction mixture, 3 µl of chromosomal DNA was added to a mixture containing 2.5 U of Taq DNA polymerase; 1x reaction buffer; 1.5 mM MgCl₂; 200 µM each of dATP, dCTP, dGTP, and dTTP; and 800 nM each of forward and reverse primer. Cycling parameters were 94°C for 2 min; 30 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 3 min; and 72°C for 5 min. Amplified products were purified with a QIAquick gel extraction kit (Qiagen, Surrey, United Kingdom). DNA sequencing was performed using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and an Applied Biosystems Model 310 automated DNA sequencer.

Allele replacement mutagenesis. PC13 was chosen as a recipient strain for allele replacement mutagenesis as it contains a wild-type copy of erm(B), which can be replaced by an exogenous copy by homologous recombination. The laboratory strain R6 does not contain an erm(B) gene, and attempts to introduce an erm(B) gene into R6 by means of electroporation and conjugation were unsuccessful. The wild-type erm(B) gene in PC13 is encoded on transposon Tn1545, and, when uninduced, the strain is susceptible to telithromycin (MIC of 0.06 µg/ml); the genes in PC13 encoding 23S rRNA and ribosomal proteins L4 and L22 were confirmed to be wild type. PC13 was made competent by culture in C medium (33), and allele replacement mutagenesis was performed as previously described (27). The erm(B) gene of strain P1501016 was used as donor DNA, and transformants were selected on Mueller-Hinton agar supplemented with 5% horse blood and containing telithromycin (0.5 µg/ml). MICs of the transformants were determined, and the presence of the mutant erm(B) gene was confirmed by sequencing. In addition, the genes encoding 23S rRNA, L4, and L22 were resequenced.

Induction assays. Telithromycin MICs for P1501016, PC13, and the PC13 transformant were determined by the agar dilution method as described above, in the absence and presence of erythromycin (0.25 µg/ml).

Relative quantification of erm(B) mRNA expression. P1501016, PC13, and the PC13 transformant were grown in tryptic soya broth (TSB) at 37°C in 5% CO₂ to an optical density at 600 nm (OD₆₀₀) of 0.2. Three cultures were grown for each strain in the absence of antibiotic and in the presence of telithromycin (at 0.008 µg/ml) or the presence of erythromycin (at 0.25 µg/ml). At the required OD, cultures were aliquoted and incubated with RNAProtect bacteria reagent (Qiagen) and centrifuged, and the pellet was stored at –70°C. Total RNA was extracted using an RNeasy Mini Kit for bacteria (Qiagen). RNA was reverse transcribed using a High-Capacity cDNA Archive Kit (Applied Biosystems), according to the manufacturer's instructions. The target gene [erm(B)] and the endogenous reference gene, glucose-6-phosphate dehydrogenase (gdh), were amplified separately from the same total RNA for each sample. For each PCR, 5 µl of cDNA was added to a 45-µl mixture containing 1x SYBR Green PCR Mastermix (Applied Biosystems) and 12.5 pmol each of forward primer and reverse primer (Applied Biosystems). The erm(B) cDNA was amplified using primers ermB747F (5'-AGGGTTGCTCTTGCACACTCA-3') and ermB806R (5'-CATTCCGCTGGCAGCTTAAG-3'). The cDNA of gdh, a housekeeping gene, was amplified using primers SPgdhF1 (5'-ATTCCGTGGTGTTCCTTTCTTTT-3') and SPgdhR1 (5'-TTCCTTTTTCAGTCAGTCGTTTAC-3'). Real-time PCR was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Relative standard curves were constructed for each gene, and the ratio of erm(B) to gdh was calculated for each sample.

Quantification of ribosomal dimethylation. P1501016, PC13, and the PC13 transformant were grown in 20 ml of TSB without antibiotic or with telithromycin or erythromycin, as described above. Cells were harvested by centrifugation at 1,500 x g for 15 min; pellets were resuspended in 1 ml of RNAProtect bacteria reagent (Qiagen) and incubated at room temperature for 5 min. Samples were centrifuged at 3,500 x g for 10 min at 4°C, and total RNA was extracted using a FastRNA Pro Blue Kit (QBiogene, Inc., CA) and FastPrep Instrument (QBiogene), for 40 s at a speed setting of 6.0.

Primer extension analysis was performed on the RNA as previously described (11, 25). Briefly, a 5' ³²P-labeled deoxynucleotide primer (5'-AGTAAAGCTCCATGGGGTC) complementary to the G2061-U2079 region of the streptococcal 23S rRNA was extended with 1 U of reverse transcriptase (Life Sciences) using a mixture of either 1 mM dTTP, 1 mM dCTP, and 5 mM ddGTP in the test samples or 1 mM each dTTP, dCTP, dATP, and dGTP in control samples; in both cases, 1.5 pmol of rRNA was used as the template. Extension reactions were performed in triplicate and were run on denaturing polyacrylamide gels alongside dideoxy sequencing reactions performed on an unmodified rRNA template from S. pneumoniae strain R6. Gels were autoradiographed and quantified by phosphorimager scanning (Typhoon model; Amersham).

Growth studies. Pneumococci were inoculated from glycerol stocks into TSB (1:100 dilution) and TSB containing erythromycin (1 µg/ml) and incubated at 37°C in 5% CO₂. Growth was monitored by measurement of the OD₆₀₀ at intervals of 30 min, and doubling times during the exponential phase of growth were calculated.

Statistical analysis. Statistical differences between means were calculated using the paired t test with P values interpreted at the 95% confidence level.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The S. pneumoniae isolate P1501016 is serotype 23F, highly resistant to macrolides (erythromycin MIC, >256 µg/ml; clarithromycin MIC, >256 µg/ml) and clindamycin (MIC, >256 µg/ml) and resistant to telithromycin (MIC, 8 µg/ml). P1501016 is mef(A) negative and erm(B) positive, with the latter gene located in the transposon Tn1545. The four alleles encoding 23S rRNA and ribosomal proteins L4 and L22 have unchanged wild-type sequences. However, the erm(B) operon has a 136-bp deletion that removed the last three codons of the leader and the entire Shine-Dalgarno sequence 2 immediately proximal to the erm(B) cistron (Fig. 1A). The putative mutant Erm(B) protein therefore consists of the remaining portion of the leader peptide fused in frame with the full-length Erm(B) sequence. The mutant Erm(B) is translated from Shine-Dalgarno sequence 1 and is extended by 24 amino acids at its N-terminal compared to the wild-type protein (Fig. 1B).

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FIG. 1. (A) Diagram representing a wild-type erm(B) gene and the mutant erm(B) gene of isolate P1501016. SD, Shine-Dalgarno sequence. (B) Amino acid sequence alignment of the wild-type Erm(B) protein of telithromycin-susceptible strain PC13 with the putative mutant Erm(B) protein of isolate P1501016. The arrow indicates the break between the control (leader) peptide and structural protein in the wild-type protein. Asterisks indicate conserved residues.

Allele replacement mutagenesis was performed to determine whether the deletion in erm(B) was responsible for the observed telithromycin resistance. After transformation of PC13 with the mutant erm(B) from P1501016, the minimal concentration of telithromycin required to inhibit growth was 16 µg/ml (Table 1). The erm(B) gene of the transformants was confirmed to contain a deletion mutation identical to that of P1501016; genes encoding 23S rRNA, L4, and L22 were shown to have remained unchanged. The MIC of telithromycin for PC13 increased from 0.06 µg/ml to 0.5 µg/ml in the presence of erythromycin, whereas the MICs of telithromycin for P1501016 and the PC13 transformant remained unaltered by the presence of erythromycin (Table 1).

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TABLE 1. Phenotypic and genotypic results of P1501016, PC13, and the PC13 transformant

The relative expression of erm(B) compared to the control gene gdh is shown in Fig. 2. In the absence of antibiotic, baseline relative expression of erm(B) in the clinical telithromycin-resistant isolate P1501016 (mean ± standard error of the mean, 2.54 ± 0.36) was significantly higher than PC13 (0.96 ± 0.08) (P = 0.02). When the mutant erm(B) was introduced into PC13, in the absence of antibiotic there was a small but insignificant increase in the relative expression of erm(B) between PC13 (0.96 ± 0.08) and the PC13 transformant (1.52 ± 0.30) (P = 0.12). Telithromycin exposure did not significantly increase the expression of erm(B) relative to the baseline without antibiotic, but the expression was significantly higher in the PC13 transformant (1.31 ± 0.22) than in PC13 (0.66 ± 0.09) in the presence of telithromycin (P = 0.02). In contrast, in the presence of erythromycin significant increases in erm(B) expression were found (P < 0.05), and expression was significantly higher in the PC13 transformant (4.98 ± 0.97) than in PC13 (2.57 ± 0.49) (P = 0.04).

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FIG. 2. Relative expression of erm(B), determined by quantitative real-time reverse transcription-PCR, in isolates P1501016, PC13, and the PC13 transformant. Cultures were grown in either the absence of antibiotic, presence of telithromycin (0.008 µg/ml), or presence of erythromycin (0.25 µg/ml). Error bars represent the standard error of the mean (n = 4).

rRNA dimethylation (Fig. 3) was low (4% ± 0.9%) in the wild-type PC13 strain in the absence of antibiotic but increased to 29% (± 1.5%) (P = 0.007) in the presence of telithromycin and further to 46% (± 1.3%) in the presence of erythromycin (P = 0.002 compared to baseline; P = 0.001 compared to telithromycin). Ribosomal dimethylation was high in the telithromycin-resistant clinical strain (51% ± 0.6%) and the PC13 transformant (52% ± 0.9%) in the absence of antibiotic and was further increased in the presence of telithromycin to 66% (± 2%) and 64% (± 2%), respectively, or in the presence of erythromycin to 68% (± 1.5%) and 72% (± 2.9%), respectively. Ribosomal dimethylation was significantly higher in the PC13 transformant in comparison with wild-type PC13 in the absence of antibiotic (P < 0.001) and in the presence of telithromycin (P = 0.025) or erythromycin (P = 0.01).

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FIG. 3. Percentage of dimethylation at position A2058 of 23S rRNA of P1501016, PC13, and the PC13 transformant. Cultures were grown in either the absence of antibiotic, presence of telithromycin (0.008 µg/ml), or presence of erythromycin (0.25 µg/ml). Error bars represent the standard error of the mean (n = 3).

Growth curves are shown in Fig. 4A (absence of erythromycin) and B (presence of erythromycin at 1 µg/ml). Mass doubling times during the exponential phase of growth in the absence of erythromycin were less for the clinical strain than the wild-type or mutant PC13 strains: P1501016, 35.5 min; PC13, 47.9 min; PC13 transformant, 48.9 min. Erythromycin extended the lag phase of growth for the wild-type PC13 strain, which was longer than for the PC13 transformant. Mass doubling times during the exponential phase of growth in the presence of erythromycin were as follows: for P1501016, 43.3 min; for PC13, 53 min; and for the PC13 transformant, 52.5 min.

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FIG. 4. Growth curves at 37°C of isolates P1501016, PC13, and the PC13 transformant in the absence (A) and presence (B) of erythromycin (1 µg/ml).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, a telithromycin-resistant clinical isolate of S. pneumoniae was identified and found to contain a 136-bp deletion in the leader sequence of the erm(B) gene, resulting in a putative mutant Erm(B) that is longer than the wild-type protein (Fig. 1). By allele replacement mutagenesis of telithromycin-susceptible PC13 with the mutant erm(B) gene from isolate P1501016, the mutant gene was shown to confer telithromycin resistance. Mutations in 23S rRNA and ribosomal proteins L4 and L22 have been associated with telithromycin resistance (12, 20, 21, 22, 31), and high-level telithromycin resistance is conferred by the combination of erm(B) with a mutation of ₆₉GTG₇₁ to TPS in ribosomal protein L4 (38). The strains used in this study were shown to have no changes in their 23S rRNA, L4, or L22 sequences.

The S. pneumoniae strain PC13 contains a wild-type erm(B) gene, and the MIC of telithromycin for this strain increases in the presence of the inducer, erythromycin. The increased resistance is consistent with the increase in rRNA methylation that occurs in the presence of telithromycin and erythromycin. These observations fit with previous reports that in the pneumococcus the erm(B) gene is predominantly inducibly expressed (24). In contrast, basal levels of ribosomal methylation were high in the clinical strain and in the PC13 transformant containing the mutant erm(B) gene. These levels increased further in the presence of telithromycin and erythromycin and indicate that the mutant erm(B) has retained an inducible component despite being expressed in a predominantly constitutive manner. Previous studies have described a pneumococcal strain with a large deletion in the erm gene displaying constitutive macrolide-lincosamide-streptogramin B resistance (24).

The difference in expression levels of the wild-type and mutant erm(B) genes was correlated to the growth curves. In the absence of erythromycin, the growth curves of PC13 and the PC13 transformant were almost identical. Both strains were able to grow equally well as ribosomal methylation was not required, and therefore the level at which it occurred did not influence growth (Fig. 4A). However, in the presence of erythromycin, the lag phase of PC13 was considerably longer than that of the PC13 transformant. This is explained by the high basal levels of methylation in the transformant. As induction is not required for high levels of erm(B) expression in the transformant, it requires a shorter period of time to achieve sufficiently high levels of methylation to overcome the effects of erythromycin. On the other hand, the wild-type PC13 strain requires a longer period of time for induction of erm(B) expression and to achieve sufficiently high levels of ribosomal methylation to be able to resist erythromycin action. Once this methylation level has been reached, the culture can enter the exponential phase and replicate normally, as reflected by the similar mass doubling times of PC13 and the PC13 transformant during the exponential phase of growth in erythromycin (Fig. 4B).

The constitutive expression of erm(B) as a result of the deletion in the leader sequence of this gene resulted in significantly higher levels of ribosomal methylation and conferred resistance to telithromycin. Studies have shown that low levels of ribosomal methylation by erm genes do not confer telithromycin resistance; however, higher levels do confer resistance. In pneumococcal strains with various levels of methylation, strains with higher methylation levels had higher ketolide MICs (40). Monomethylation of A2058 in E. coli ribosomes confers resistance to erythromycin but not telithromycin; however, dimethylation of this site confers high-level resistance to erythromycin and telithromycin (15). In Streptococcus pyogenes the degree of ribosomal methylation was found to correlate with resistance to ketolides, with a reduction in ketolide susceptibility being observed with an increase in the proportion of dimethylated ribosomes (9).

The levels of erm(B) mRNA transcription were not significantly different between wild-type PC13 and the PC13 transformant in the absence of antibiotic, although distinct variations in these levels were observed in the presence of erythromycin (Fig. 2). This indicates some degree of induction at the transcriptional level in all of the strains. The main mechanism of erm induction, however, is generally accepted to occur posttranscriptionally by translational attenuation (37). For the PC13 strain, the marked increases in rRNA methylation in the presence of antibiotics clearly indicate that the translational attenuation mechanism was operational here (Fig. 3). It should be noted that not only erythromycin but also telithromycin induced expression of the wild-type erm(B) gene, and ketolides have previously been noted to be weak inducers of erm(B) (23, 40). As telithromycin remains an effective antimicrobial against the majority of inducible pneumococcal strains (18, 23), it seems that insufficient levels of ribosomal methylation are induced to confer ketolide resistance. We also note that the levels of rRNA methylation in the constitutive erm(B) strains, which are already relatively high in the absence of drug, are increased further by the presence of either erythromycin or telithromycin. This indicates that the constitutive erm(B) strains are not fully induced in the absence of drug.

In conclusion, we show that telithromycin resistance is conferred by a mutant erm(B) gene with a deletion in its leader sequence. The deletion interferes with translational attenuation of the erm(B) mRNA, allowing constitutive expression, and results in higher basal levels of rRNA methylation. Increased rRNA methylation within the ribosomal binding site hinders telithromycin binding and confers resistance. Despite the "constitutive" classification of the erm(B) mutants, the addition of erythromycin and telithromycin further stimulates erm(B) expression. Telithromycin is noted to be an inducer of both wild-type and mutant erm(B) sequences, albeit to a lesser extent than erythromycin.

 
This research was supported by grants from the Medical Research Council, the National Institute for Communicable Diseases, and the University of the Witwatersrand, South Africa. Support from the Danish Research Agency (FNU-Rammebevilling 21-04-0520) and the Nucleic Acid Center of the Danish Grundforskningsfond (to S.D.) is gratefully acknowledged. The PROTEKT study is financially supported by sanofi-aventis.

D.J.F. has received research grants and consultancy fees from sanofi-aventis related to telithromycin research, publications, and presentations.

Lykke Haastrup Hansen is thanked for excellent technical assistance.

 
* Corresponding author. Mailing address: Respiratory and Meningeal Pathogens Research Unit, National Institute for Communicable Diseases, Private Bag X4, Sandringham 2131, South Africa. Phone: 27 11 555 0352. Fax: 27 11 555 0437. E-mail: nicolew{at}nicd.ac.za

Published ahead of print on 3 December 2007.

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Antimicrobial Agents and Chemotherapy, February 2008, p. 435-440, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.01074-07
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