1 Department of Medical Microbiology, Medical School, University of Edinburgh, Edinburgh EH8 9AG; 2 Aventis Pharma Ltd., Kings Hill, West Malling, Kent ME19 4 AH, UK
Received 29 November 2002; returned 22 January 2003; revised 8 May 2003; accepted 29 May 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: telithromycin, Streptococcus pneumoniae, resistance
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The two most regularly identified mechanisms of macrolide resistance in S. pneumoniae are target modification and efflux. Target modification causes resistance not only to macrolides but also to lincosamides and streptogramin B antibiotics and is associated with high-level macrolide resistance.2 erm genes cause methylation of the binding site of the MLSB antibiotics within the peptidyl transferase centre of the 23S rRNA. In S. pneumoniae, the erm(B) gene mediates methylation, although recently an erm(A) gene has also been associated with macrolide-resistant S. pneumoniae.3,4 Methylation can occur as monomethylation or dimethylation. It has recently been shown that variations in mono- or dimethylation lead to different resistance phenotypes. Erm(B) and Erm(A)(TR) are both A2058 dimethyltransferases and expression of resistance due to erm methylation can be inducible or constitutive.5
The inducible expression of MLSB resistance is putatively controlled at a post-transcriptional level by a regulatory region upstream from the erm gene.6 The exact mechanism or mechanisms of constitutive resistance have not been fully elucidated in S. pneumoniae. Constitutive expression of MLSB resistance in various bacteria has been associated with deletions or mutations in the regulatory region upstream from the erm gene.7 Two S. pneumoniae clinical isolates with deletions in the leader peptides of their erm(B) genes have been isolated, one each from Mexico and Canada, and both were ketolide-resistant. In these strains, however, there were also other mutations; both had three amino acid mutations in the Erm protein itself, and the Mexican strain also had mutations in the L4 riboprotein.8
The mef(A) genetic element was first described in 2000 as a chromosomal element designated Tn1207.1, 7244 base pairs in size.9 In 2001, Gay & Stephens described a 5.4 or 5.5 kb genetic element containing a mef(E) gene called MEGA.10 In MEGA, the open reading frame (ORF) sequence 3' of the mef(E) gene was designated mel. The mel ORF is also a homologue of msrA in staphylococci, which encodes an ATP-binding cassette to provide the energy for efflux. The mef(E) and mel are co-transcribed, which suggests that both are required for efflux.
Macrolide resistance in clinical isolates and laboratory-derived strains of S. pneumoniae have been linked to alterations of specific nucleotides in the 23S rRNA of the ribosomal subunit. Mutations at adenine 2058 are the most frequently identified ribosomal mutations associated with macrolide resistance in 23S rRNA.11 Specifically in S. pneumoniae, mutations have been located at nucleotides 2058, 2059, 2062 and 2611.1215 These ribosomal mutations were identified in clinical isolates, which did not contain the erm(B) or mef(A) genes. In addition to these mutation sites, a mutation in the hairpin 35 region of the domain II of the 23S rRNA has been associated with macrolide and ketolide resistance in a laboratory-derived mutant of S. pneumoniae.16 This laboratory-derived S. pneumoniae strain was found to have a deletion of one adenine in the series of four located at positions 749752 in the hairpin 35 of domain II.16
The ribosome consists not only of rRNA but also riboproteins, which interact with the rRNA to form the ribosomal subunits. Two such riboproteins are L4 and L22. Recently, mutations in both of these riboproteins have been associated with macrolide resistance in S. pneumoniae. Positively charged residues of both L4 and L22 interact with the negatively charged phosphate groups of the RNA forming nucleic acidprotein complexes.17,18 Erythromycin resistance mutation studies of these riboproteins implied that they also have interactions with the central loop of domain V of the rRNA. Gregory & Dahlberg also showed that these proteins have multiple contacts with rRNA in domains II, III and V of the rRNA.19
Mutations in the L4 riboprotein and L22, to a lesser extent, have been associated with macrolide and, on occasion, telithromycin resistance, either alone or in combination with 23S rRNA mutations or the presence of an erm(B) gene.4,1416 From the studies carried out to date, it appears that the L4 amino acid region from 67 to 72 is the hotspot for mutations conferring macrolide resistance.4,1416
Mutations in L22 associated with macrolide resistance have been mainly described in laboratory-derived strains of S. pneumoniae. In fact, only two types of L22 mutation have been identified in clinical isolates. They are a Gly-95 to Asp-95 amino acid change, and a six amino acid insertion (RTAHIT) at amino acid 109.12,20 Three strains selected in vitro on telithromycin were identified recently with two types of L22 mutation; one strain had a Gln-95 to Asp-95 mutation and the other an Ala-97 to Asp-97 change. These strains were not telithromycin-resistant but the mutation did cause the telithromycin MIC to increase 32-fold for the Gln-95 to Asp-95 mutant and 8- or 16-fold for the Ala-97 to Asp-97 mutants.21
The generation of telithromycin-resistant S. pneumoniae mutants to date has mainly used macrolide-susceptible S. pneumoniae to try to develop telithromycin-resistant mutants. Therefore, we decided to investigate telithromycin resistance development from S. pneumoniae, which were already macrolide resistant. The aim of this study was to investigate the development of telithromycin resistance in S. pneumoniae by generating telithromycin-resistant mutants in vitro, from strains of varying MLSB resistance profiles. Once the mutants were generated, the mechanisms by which these strains became telithromycin-resistant and finally the influence of erythromycin and clarithromycin on the development of telithromycin resistance were studied.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The three S. pneumoniae strains used as the parents for the step-wise selection of telithromycin-resistant mutants were 02J1175 [mef(A)+], 02J1095 [erm(B) +] and NCTC 13593, which is a macrolide-susceptible strain and does not contain either gene.
Mutation studies
Each of the three parent strains were inoculated into Todd Hewitt broth (Oxoid) and incubated in 5% CO2 overnight. Varying dilutions, from neat to 106, of the cultures were spread on Columbia agar (SigmaAldrich) plates supplemented with 5% defibrinated horse blood containing telithromycin at concentrations equal to or 2 x MIC for the strain. Plates with each concentration of telithromycin were inoculated in triplicate and a control with no antibiotic was also included. All plates were incubated in 5% CO2 for 48 h. The resulting mutants were purified by subculturing twice on plates with the selectant telithromycin concentration. The MICs of telithromycin against the mutants were tested at this point according to the BSAC guidelines.22 Successive generations of mutants were derived in the same way as the first generation. This process was repeated until high-level telithromycin resistance occurred or the MICs for successive generations remained constant. Serially subculturing the mutants on antibiotic-free medium for 10 generations and then retesting their telithromycin MICs was used to determine the stability of a representative of each generation.
MIC determination
MICs were determined on Columbia agar supplemented with 5% defibrinated horse blood and doubling dilutions of antibiotic according to the BSAC guidelines.22 All plates were incubated in 5% CO2 for 1618 h. The antibiotics were stored and prepared according to the manufacturers guidelines. Telithromycin was obtained from Aventis Pharma Ltd.
PCR conditions
S. pneumoniae strains were emulsified in 200 µL of Milli-Q water and boiled for 10 min in order to extract the total DNA. The supernatant was used as the DNA template in the PCR experiments. The PCR conditions and primers for the detection of erm(B) and mef(A) genes are described in Table 1 and are based on those previously devised by Sutcliffe et al. and Tait-Kamradt et al.23,24 The mef(A) gene reverse primer was designed using the primer design website Primer 3 at http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi in order to amplify the entire mef(A) gene. The three parents 02J1095, 02J1175 and NCTC 13593 and the selected generation representative mutants, shown in Table 2, were investigated for the presence of erm(B) or mef(A) genes.
|
|
The erm(B) upstream regions of the parent strain 02J1095 and mutants J I 1, J II 1, J II 4, J II 5, J II 6, J II 7, J II 8 and J II 9 were investigated by amplification by PCR. The forward primer was designed using the primer design website Primer 3 at http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi and the reverse primer was the same as the reverse primer used to amplify the erm(B) gene described in Table 1. The PCR mixture was the same as that of the erm(B) gene PCR except the MgCl2 concentration was 3 mM. The cycle parameters were the same as those of the erm(B) PCR.
In order to detect mutations in the peptidyl transferase region of the 23S rRNA, all four contigs of the domain V section were amplified using the four downstream primers and PCR conditions as published by Tait-Kamradt et al. and shown in Table 1.15 Problems were encountered in obtaining the sequence data for the nucleotide section from 2350 to 2650 of the DS 18 and DS 23 genes, as they were both 2000 nucleotides in size. As a result, primers were designed to amplify this inner portion of each of the genes. The PCR products of the downstream primers were purified and this product was then used as the DNA template for the inner PCR experiments. The primers are shown in Table 1. The inner primer PCR consisted of 94°C for 3 min, 35 cycles of 94°C for 1 min, 54°C for 1 min and 72°C for 1 min and a final step of 72°C for 10 min. The domain II portion of the 23S rRNA was amplified by using the domain II primers (Table 1) and conditions of Tait-Kamradt et al.15 The L4 and L22 genes were also amplified as previously published using the primers shown in Table 1.15
The PCR products were purified by the gel extraction method or directly using the Qiagen PCR product purification kit.
Sequencing
Sequencing of purified DNA was determined by the chain termination method.25 The PCR primers were also used as sequencing primers. Individual PCR fragments were set up in the Ready Reaction Format for fluorescence based on dideoxy cycle sequencing (PE Applied Systems, Warrington, UK).
Induction experiments
Disc diffusion: The strains 02J1095, J I 1, J II 1, J II 8 and the control strain NCTC 13593, described in Table 2, were each inoculated into 4.5 mL of sterile distilled water and adjusted to a turbidity equivalent to that of a 0.5 McFarland standard. Using a sterile swab, each strain was spread onto separate Columbia blood agar plates. Discs containing 15 µg of each of the following agentserythromycin, clarithromycin and telithromycinwere placed onto the plate approximately 2 cm apart. Each strain was tested in duplicate.
Induction assays: In order to investigate inducibility further, two induction assays were carried out.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Telithromycin-resistant mutants were created from the macrolide-resistant strains 02J1095 [erm(B) +] and 02J1175 [mef(A) +]. Selection of the macrolide-susceptible NCTC 13593 strain on telithromycin failed to produce telithromycin-resistant mutants (Table 2).
The MIC of 02J1095 and its mutants increased from 0.06 mg/L for the parent 02J1095 to >32 mg/L over two generations (Table 2). The first generation increase from 0.06 to 1 mg/L was a 16-fold increase; from first to second generation, the increase was greater than 32-fold. These increases were stable, such that when the mutants were serially subcultured 10 times, without the selective pressure of telithromycin, they still maintained the same telithromycin MIC.
The telithromycin MIC of the parent 02J1175 was 0.5 mg/L. The first generation mutants had telithromycin MICs between 1 and 4 mg/L (Table 2). The representative mutant, M I 2, selected to create the second generation had an MIC of 2 mg/L. This gave rise to a second generation with MICs between 2 and 4 mg/L. The third and fourth generations both had telithromycin MICs of 8 mg/L and further mutants with higher telithromycin MICs could not be obtained from them. The telithromycin MIC of the most resistant strain, fourth generation 02J1175 mutant, reverted from 8 to 1 mg/L after 10 passages in antibiotic-free medium. The MICs of M III 3 and M II 15 also decreased but only by one doubling dilution. The MIC of M I 2 decreased by two doubling dilutions. For the third generation strain, this means that its telithromycin resistance remained stable with an MIC of 4 mg/L. When the reverent strain of the fourth generation 02J1175 mutant was mutated on agar plates containing either 1 or 2 mg/L, all the resulting mutants returned to an MIC of 8 mg/L, the same as the fourth generation mutant. Therefore, in order for S. pneumoniae strains with a mef(A) gene to maintain their telithromycin MIC, selective pressure must be maintained. This has previously been noted by Davies et al.,26 when the telithromycin and macrolide MICs reverted back to baseline MICs (or close to) after 10 passages on antibiotic-free media. This is not true for those containing the erm(B) gene. Once resistance or an elevated telithromycin MIC has been achieved, it is stable with or without the selective pressure.
The NCTC 13593 strain, which was macrolide-susceptible, had a telithromycin MIC of 0.016 mg/L. This increased sequentially to 0.032, 0.12, 0.5 and 0.5 mg/L for the four mutant generations, respectively (Table 2). The final generation mutants were telithromycin-susceptible.
PCR and sequencing of erm(B) and mef(A)
A representative of each mutant generation was chosen for further molecular analysis. The 02J1095 parent and all mutants tested contained the erm(B) gene, determined by PCR amplification. In order to investigate the mechanism of telithromycin resistance, the erm(B) gene from each representative mutant was amplified by PCR and sequenced. The same procedure was carried out on 02J1175 and its representative mutants with the mef(A) gene (Table 2). No mutations were found in either the erm(B) or mef(A) genes. No erm(B) or mef(A) genes were present in the NCTC 13593 parent or its mutants. The strains only contained an erm(B) gene or a mef(A) gene, and no strains were positive for both genes.
The erm(B) upstream region of 02J1095 and the selected mutants, described in Table 2, was amplified by PCR in order to investigate the nucleotide region from the promoter to the erm(B) gene. Deletions in this region have been associated with a change from inducible to constitutive erythromycin A resistance.7,27 The corresponding bands of approximately 1000 base pairs of the erm(B) upstream region PCR products of 02J1095 and the selected mutants (Table 2) were visualized on the agarose gels. The J II 8 band however was just above the 750 bp band of the 1 kb ladder as shown in Figure 1.
|
|
|
Induction experiment results
On both plates, with 02J1095 and J I 1 (Figure 3), the zone of inhibition around telithromycin had two straight edges, one each from erythromycin and clarithromycin. The D-shaped zone indicates that the antimicrobial agent to the left of the D induces resistance. From these photographs (Figure 3), it can be seen that erythromycin and clarithromycin both induce telithromycin resistance. The growth of J II 1 to J II 9 was either up to the discs or within 1 mm of the discs and so were recorded as resistant to the three antimicrobial agents and, as such, had constitutive expression of the erm(B) gene.
|
The MICs of the mutant strains derived from growth of 02J1095 on erythromycin are shown in Table 4. The telithromycin MICs of the strains tested in the presence of erythromycin are shown in Table 5.
|
|
With regard to the second MIC test, the MICs of 02J1095 and all of the first generation mutants did increase. The telithromycin MICs of the already highly resistant second-generation strains increased or decreased regardless of the concentration of erythromycin. The telithromycin MIC of the parent 02J1095 increased from 0.06 to 1 mg/L or 2 mg/L and that of the first generation mutants increased gradually from 0.25 to 32 mg/L and that of the second generation mutants increased from 4 to >32 mg/L depending on the strain and the concentration of erythromycin. Therefore, as indicated by the disc diffusion test and the two induction tests, erythromycin does induce telithromycin resistance. However, the level of induction varies depending on the initial telithromycin MIC and does not increase beyond a telithromycin MIC of 64 mg/L.
Ribosomal mutations
Mutations in the domains II and V of the 23S rRNA have previously accounted for increases in MIC of both macrolides and telithromycin.12,14,15,2830 Therefore, it was plausible to speculate that changes in either or both of these regions could be responsible for the increase in telithromycin MIC. The region of 23S rRNA from nucleotide 1 to 1011, representing domain II, of the two most resistant strains J II 8 and M IV was amplified and sequenced. No heterogeneity in the nucleotide sequences of the hairpin 35 was observed. Thus no changes were present in any of the four alleles of the 23S rRNA.
All four genes of the domain V region of 23S rRNA were amplified and sequenced individually. Again the domain V sequences were identical to those of the TIGR 4 strain sequences.31 This indicates that no changes were present in the peptidyl transferase region of the 23S rRNA
The lack of mutation in the 23S rRNA suggested that changes could be present in L4 and L22 ribosomal proteins associated with the peptidyl transferase region. Changes in the L4 and L22 have been implicated in decreased susceptibility of S. pneumoniae to telithromycin and erythromycin.15,16,32 The L4 protein genes of 02J1095, J II 8 [erm(B) +], 02J1175 and M IV [mef(A) +] were amplified and sequenced. No changes, insertions or deletions occurred in either resistant strain in comparison to the susceptible TIGR 4 strain or the macrolide-resistant parent strains. Using primers described by Tait-Kamradt et al., the genes encoding the L22 protein of 02J1095, J I 1, J II 1, J II 4, J II 5, J II 6, J II 7, J II 8, J II 9, 02J1175 and M IV were amplified and sequenced.15 Again, there were no changes present in any of the strains tested except one strain, J II 8, in which a Lys-94 to Gln-94 change was determined (Figure 4). This change was due to an adenine to cytosine change. This is the first incidence of a Lys-94 to Gln-94 change in L22 being associated with telithromycin resistance.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of the erm(B) and mef(A) gene sequencing indicated that no changes in either gene were associated with telithromycin resistance. Therefore, whereas these genes are required for the selection of telithromycin resistance, they themselves do not change to facilitate telithromycin resistance.
Deletions in the erm gene attenuator region in Streptococcus pyogenes, S. pneumoniae and S. agalactiae have all been associated with constitutive expression of their Erm methylases. In S. pyogenes, deletions of 163 base pairs or six base pairs and a duplication of 101 base pairs in the erm(TR) upstream region resulted in an increase in clindamycin MIC from 1 to 128 mg/L when transformed into E. coli.33 The S. pyogenes strains with mutated attenuators were mutants, which had been selected on clindamycin, and the parent strain was inducibly intermediate to erythromycin and fully susceptible to clindamycin. Tait-Kamradt et al. described two S. pneumoniae clinical isolates with truncated erm(B) leader peptides of 15 and 19 amino acids.8 These two strains had telithromycin MICs of 1 and 256 mg/L, respectively and both also had changes in the gene itself, leading to three amino acid changes. The strain with the 256 mg/L telithromycin MIC also contained L4 riboprotein amino acid mutations of glycine, threonine and glycine at amino acids 69 to 71 to threonine, proline and serine, respectively.
The results of the PCR experiments of the erm(B) upstream region carried out with the parent 02J1095 and the mutants derived from it showed that the J II 8 strain had a large deletion of 210 bp in this section. This result is very similar to that found with the clindamycin-resistant S. pyogenes. Therefore, it is possible that a mechanism of resistance exists that alters the erm(B) attenuator of strains inducibly resistant to erythromycin in order to confer resistance to other antimicrobial agents within the same group, such as clindamycin and telithromycin. The results of Tait-Kamradt et al. are similar to the findings of this study in that the telithromycin-resistant strains both had truncated regions upstream from the erm(B) gene.8 The other mutants investigated in this study, which were also highly telithromycin-resistant, did not however have such a deletion. Therefore, whereas the mutation in the erm(B) attenuator may be at least partly responsible for telithromycin resistance in J II 8, this is not the case for the other five highly telithromycin-resistant strains.
The mutated nucleotides of the strains investigated at positions 292, 297 and 319 are the same nucleotides as those of plasmid pAM 77 from Streptococcus sanguis and the nucleotide change at position 318 was found previously in an oral streptococcus.7,27 These mutations were in the parent 02J1095 and all the mutants, except J II 8, and as such do not appear to be involved in the development of telithromycin resistance. It is, however, interesting to note the nucleotide changes at 318 and 319 are just at the end of the deletion in J II 8. Two previously reported S. pneumoniae isolates with the same 318 and 319 mutations were both inducibly erythromycin-resistant but sus-ceptible to the ketolide HMR 3004.27 The other single mutations in J II 1, J II 4, J II 7 and J II 9 have not previously been associated with changes in resistance patterns. But as each strain has a different mutation, it is unlikely that these mutations individually lead to telithromycin resistance.
The disc diffusion experiments indicated, from the D-shaped zones of inhibition around the telithromycin disc, that erythromycin and clarithromycin are both inducers of telithromycin resistance in the strains 02J1095 and J I 1. This finding was verified by two further induction studies with erythromycin at concentrations from 4 to 128 mg/L. There were variations in the increase in telithromycin MIC but the increase in telithromycin MICs in all strains caused a change from telithromycin-susceptible to non-susceptible or resistant. This is the first report of erythromycin induction of telithromycin resistance in S. pneumoniae.
The high-level telithromycin MICs for the mutants are not all caused by the same mutation in the erm(B) attenuator. Although the large deletion in J II 8 is such that it is probably part of the mechanism used by this strain to overcome telithromycin, the sizes of the upstream and downstream regions from the mef(A) genes suggest that no large nucleotide deletions have occurred. The presence of the mel genes in these strains confirms previous studies of the mef(A) operon, which suggested the presence of the mel gene downstream from the mef(A) gene. From this study, it can be seen that deletions of large numbers of nucleotides from either the upstream or downstream nucleotide regions of the mef(A) gene are not responsible for the increase in telithromycin MIC and resistance.
The main regions of interest to date for macrolide resistance in S. pneumoniae in the 23S rRNA are the domains II and V. The area of consequence in domain II is the hairpin 35. A deletion in one adenine in the series of four located at positions 749 to 752 resulted in a 500-fold increase in the telithromycin MIC for a S. pneumoniae strain. In this case, it became resistant to telithromycin (4 mg/L).16 Previously, a single point mutation (U754A) in a laboratory strain of E. coli resulted in the cells being resistant to telithromycin.30 For domain V there is less specificity with regard to one macrolide-resistant region. Nucleotide changes have been located at 2058, 2059 and 2611. There is also variation in the nucleotide changes for these sites.13,15,16,32 These two regions were the starting points of the search for answers to the increase in telithromycin MIC or resistance. However, as no nucleotide changes or deletions were identified in either region, the possibility of domain II or V providing the answers was ruled out.
The L4 protein has been associated with large increases in telithromycin MIC in S. pneumoniae. Insertion of six amino acids into a highly conserved area of ribosomal L4 protein (63KPWRQKGTGRAR74) has been associated with a 500-fold increase in the MIC of telithromycin.32 Once again, there were no changes in the nucleotide sequences and so the amino acid sequence of either J II 8 or M IV. Changes within the L22 protein amino acid sequence have also been reported as a cause of increased telithromycin MIC from 0.008 to 0.25 mg/L. This was due to three simultaneous amino acid mutations: alanine-93 to glutamic acid-93, proline-91 to serine-91 and glycine-83 to glutamic acid-83.16 The L22 protein binds primarily to the 23S rRNA. Mutations in this protein could change the conformation of 23S rRNA and thus affect ketolide binding.19 In this study, the only mutation that was found was in the L22 of J II 8, a highly telithromycin-resistant strain. The mutation did not occur in mutants of the same parent with MICs of 4 or 1 mg/L. The change at amino acid 94 from lysine to glutamine was between two previously documented changes: glycine-95 to aspartic acid-95 and alanine-93 to glutamic acid-93. The glycine-95 to aspartic acid-95 mutation was associated with increases in erythromycin MIC from 0.03 to 1 mg/L and 0.015 to 0.25 mg/L. The two strains were selected with erythromycin and roxithromycin, respectively.16 The alanine-93 to glutamic acid-93 mutation was in combination with two other changes as previously mentioned. The change from ionic lysine to the neutral glutamine near the 3' end of the protein could result in a conformational change of the protein and thus prevent telithromycin binding. The previously documented changes at amino acids 93 and 95 may also cause a conformational change in the L22 protein and thus prevent erythromycin binding.
No telithromycin resistance could be created from a macrolide-susceptible strain. Therefore although telithromycin has high activity against macrolide-resistant strains, if the strain is exposed to telithromycin in vitro, the activity of telithromycin will fall such that the strain becomes telithromycin-resistant within a few generations. The highly telithromycin-resistant strain J II 8 had two mutations, one in the erm(B) attenuator and the other in the L22 riboprotein. Whereas this strain had two mutations, none of the other strains had any mutations. Therefore, whereas in this strain these mutations are the most probable cause of telithromycin resistance, this is not the case with the other telithromycin-resistant strains.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Leclercq, R. & Courvalin, P. (1991). Bacterial resistance to macrolide, lincosamide, and streptogramin antibiotics by target modification. Antimicrobial Agents and Chemotherapy 35, 126772.[ISI][Medline]
3
.
Syrogiannopoulos, G. A., Ioanna, N. G., Tait-Kamradt, A. et al. (2001). Identification of an erm(A) erythromycin resistance methylase gene in Streptococcus pneumoniae isolated in Greece. Antimicrobial Agents and Chemotherapy 45, 3424.
4
.
Nagai, K., Appelbaum, P. C., Davies, T. A. et al. (2002). Susceptibilities to telithromycin and six other agents and prevalence of macrolide resistance due to L4 ribosomal protein mutation among 992 pneumococci from 10 central and eastern European countries. Antimicrobial Agents and Chemotherapy 46, 3717.
5
.
Liu, M. F. & Douthwaite, S. (2002). Activity of the ketolide telithromycin is refractory to erm monomethylation of bacterial rRNA. Antimicrobial Agents and Chemotherapy 46, 162933.
6
.
Weisblum, B. (1995). Insights into erythromycin action from studies of its activity as inducer of resistance. Antimicrobial Agents and Chemotherapy 39, 797805.
7
.
Rosato, A., Vicarini, H. & Leclercq, R. (1999). Inducible or constitutive expression of resistance in clinical isolates of streptococci and enterococci cross-resistant to erythromycin and lincomycin. Journal of Antimicrobial Chemotherapy 43, 55962.
8 . Tait-Kamradt, A. G., Reinart, R. R., Al-Lahham, A. et al. (2001). High-level ketolide-resistant streptococci. In Program and Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA, 2001. Abstract C1-1813, p. 101. American Society for Microbiology, Washington, DC, USA.
9
.
Santagati, M., Iannelli, F., Oggioni, M. R. et al. (2000). Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 44, 25857.
10 . Gay, K. & Stephens, D. S. (2001). Structure and dissemination of a chromosomal insertion element encoding macrolide efflux in Streptococcus pneumoniae. Journal of Infectious Diseases 184, 5665.[CrossRef][ISI][Medline]
11
.
Vester, B. & Douthwaite, S. (2001). Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrobial Agents and Chemotherapy 45, 112.
12 . Farrell, D. J., Morrissey, I., Bakker, S. et al. (2002). Macrolide resistance (Macr) by ribosomal mutation detected in clinical isolates of Streptococcus pneumoniae isolated from PROTEKT 2000. Clinical Microbiology and Infection 8, Suppl. 1, Abstract O318, p. 42.
13
.
Depardieu, F. & Courvalin, P. (2001). Mutation in 23S rRNA responsible for resistance to 16-membered macrolides and streptogramins in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 45, 31923.
14
.
Pihlajamäki, M., Kataja, J., Seppälä, H. et al. (2002). Ribosomal mutations in Streptococcus pneumoniae clinical isolates. Antimicrobial Agents and Chemotherapy 46, 6548.
15
.
Tait-Kamradt, A., Davies, T., Cronan, M. et al. (2000). Mutation in 23S rRNA and ribosomal protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage. Antimicrobial Agents and Chemotherapy 44, 211825.
16
.
Canu, A., Malbruny, B., Coquemont, M. et al. (2002). Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin and telithromycin in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 46, 12531.
17
.
Worbs, M., Huber, R. & Wahl, M. C. (2000). Crystal structure of ribosomal protein L4 shows RNA-binding sites for ribosome incorporation and feedback control of the S10 operon. EMBO Journal 19, 80718.
18 . Unge, J., Åberg, A., Al-Kharadaghi, S. et al. (1998). The crystal structure of ribosomal protein L22 from Thermus thermophilus: insights into the mechanism of erythromycin resistance. Structure 6, 157786.[ISI][Medline]
19 . Gregory, S. T. & Dahlberg, A. E. (1999). Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23S ribosomal RNA. Journal of Molecular Biology 289, 82734.[CrossRef][ISI][Medline]
20
.
Musher, D. M., Dowell, M. E., Shortridge, V. D. et al. (2002). Emergence of macrolide resistance during treatment of pneumococcal pneumonia. New England Journal of Medicine 346, 6301.
21 . Sutcliffe, J., Tait-Kamradt, A., Walker, A. et al. (2000). Macrolide resistance in pneumococci: analysis of resistant isolates obtained by passage with telithromycin. In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 2000. Abstract 1925, p. 117. American Society for Microbiology, Washington, DC, USA.
22 . British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D.
23 . Sutcliffe, J., Grebe, T., Tait-Kamradt, A. et al. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 25626.[Abstract]
24 . Tait-Kamradt, A., Clancy, J., Cronan, M. et al. (1997). mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 41, 22515.[Abstract]
25 . Sanger, F. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, USA 74, 54637.[Abstract]
26
.
Davies, T. A., Dewasse, B. E., Jacobs, M. R. et al. (2000). In vitro development of resistance to telithromycin (HMR 3647), four macrolides, clindamycin, and pristinamycin in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 44, 4147.
27
.
Rosato, A., Vicarini, H., Bonnefoy, A. et al. (1998). A new ketolide, HMR 3004, active against streptococci inducibly resistant to erythromycin. Antimicrobial Agents and Chemotherapy 42, 13926.
28
.
Weisblum, B. (1995). Erythromycin resistance by ribosome modification. Antimicrobial Agents and Chemotherapy 39, 57785.
29 . Douthwaite, S., Hansen, L. H. & Mauvais, P. (2000). Macrolide-ketolide inhibition of MLS-resistant ribosomes is improved by alternative drug interaction with domain II of 23S rRNA. Molecular Microbiology 36, 18393.[CrossRef][ISI][Medline]
30 . Xiong, L., Shah, S., Mauvais, P. et al. (1999). A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre. Molecular Microbiology 35, 6339.
31
.
Tettelin, H., Nelson, K. E., Paulsen, I. T. et al. (2001). Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293, 498506.
32
.
Tait-Kamradt, A., Davies, T., Appelbaum, P. C. et al. (2000). Two new mechanisms of macrolide resistance in clinical strains of Streptococcus pneumoniae from Eastern Europe and North America. Antimicrobial Agents and Chemotherapy 44, 3395401.
33
.
Fines, M., Gueudin, M., Ramon, A. et al. (2001). In vitro selection of resistance to clindamycin related to alterations in the attenuator of the erm(TR) gene of Streptococcus pyogenes UCN1 inducibly resistant to erythromycin. Journal of Antimicrobial Chemotherapy 48, 4116.