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Peter C Appelbaum - One of the best experts on this subject based on the ideXlab platform.
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Development of macrolide resistance by Ribosomal Protein L4 mutation in Streptococcus pyogenes during miocamycin treatment of an eight-year-old Greek child with tonsillopharyngitis.
Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases, 2003Co-Authors: Bulent Bozdogan, Peter C Appelbaum, Lois M. Ednie, Ioanna N. Grivea, George A. SyrogiannopoulosAbstract:Streptococcus pyogenes isolates with the same pulsed-field patterns were recovered from the throat cultures of a child with tonsillopharyngitis before and after treatment with miocamycin, a 16-membered macrolide. The initial isolate was macrolide-susceptible, but the isolates after the treatment were resistant to 14 and 15-membered macrolides and had two amino acid (65WR66) deletions in Ribosomal Protein L4.
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in vitro selection of resistance in haemophilus influenzae by amoxicillin clavulanate cefpodoxime cefprozil azithromycin and clarithromycin
Antimicrobial Agents and Chemotherapy, 2002Co-Authors: Catherine Clark, Bonifacio Dewasse, Michael R. Jacobs, Bulent Bozdogan, Mihaela Peric, Peter C AppelbaumAbstract:Abilities of amoxicillin-clavulanate, cefpodoxime, cefprozil, azithromycin, and clarithromycin to select resistant mutants of Haemophilus influenzae were tested by multistep and single-step methodologies. For multistep studies, 10 random strains were tested: 5 of these were β-lactamase positive. After 50 daily subcultures in amoxicillin-clavulanate, MICs did not increase more than fourfold. However, cefprozil MICs increased eightfold for one strain. Clarithromycin and azithromycin gave a >4-fold increase in 8 and 10 strains after 14 to 46 and 20 to 50 days, respectively. Mutants selected by clarithromycin and azithromycin were associated with mutations in 23S rRNA and Ribosomal Proteins L4 and L22. Three mutants selected by clarithromycin or azithromycin had alterations in Ribosomal Protein L4, while five had alterations in Ribosomal Protein L22. Two mutants selected by azithromycin had mutations in the gene encoding 23S rRNA: one at position 2058 and the other at position 2059 ( Escherichia coli numbering), with replacement of A by G. One clone selected by clarithromycin became hypersusceptible to macrolides. In single-step studies azithromycin and clarithromycin had the highest mutation rates, while amoxicillin-clavulanate had the lowest. All resistant clones were identical to parents as observed by pulsed-field gel electrophoresis. The MICs of azithromycin for azithromycin-resistant clones were 16 to >128 μg/ml, and those of clarithromycin for clarithromycin-resistant clones were 32 to >128 μg/ml in multistep studies. For strains selected by azithromycin, the MICs of clarithromycin were high and vice versa. After 50 daily subcultures in the presence of drugs, MICs of amoxicillin-clavulanate and cefpodoxime against H. influenzae did not rise more than fourfold, in contrast to cefprozil, azithromycin, and clarithromycin, whose MICs rose to variable degrees.
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in vitro selection of resistance in haemophilus influenzae by amoxicillin clavulanate cefpodoxime cefprozil azithromycin and clarithromycin
Antimicrobial Agents and Chemotherapy, 2002Co-Authors: Catherine Clark, Bonifacio Dewasse, Michael R. Jacobs, Bulent Bozdogan, Mihaela Peric, Peter C AppelbaumAbstract:Abilities of amoxicillin-clavulanate, cefpodoxime, cefprozil, azithromycin, and clarithromycin to select resistant mutants of Haemophilus influenzae were tested by multistep and single-step methodologies. For multistep studies, 10 random strains were tested: 5 of these were beta-lactamase positive. After 50 daily subcultures in amoxicillin-clavulanate, MICs did not increase more than fourfold. However, cefprozil MICs increased eightfold for one strain. Clarithromycin and azithromycin gave a >4-fold increase in 8 and 10 strains after 14 to 46 and 20 to 50 days, respectively. Mutants selected by clarithromycin and azithromycin were associated with mutations in 23S rRNA and Ribosomal Proteins L4 and L22. Three mutants selected by clarithromycin or azithromycin had alterations in Ribosomal Protein L4, while five had alterations in Ribosomal Protein L22. Two mutants selected by azithromycin had mutations in the gene encoding 23S rRNA: one at position 2058 and the other at position 2059 (Escherichia coli numbering), with replacement of A by G. One clone selected by clarithromycin became hypersusceptible to macrolides. In single-step studies azithromycin and clarithromycin had the highest mutation rates, while amoxicillin-clavulanate had the lowest. All resistant clones were identical to parents as observed by pulsed-field gel electrophoresis. The MICs of azithromycin for azithromycin-resistant clones were 16 to >128 micro g/ml, and those of clarithromycin for clarithromycin-resistant clones were 32 to >128 micro g/ml in multistep studies. For strains selected by azithromycin, the MICs of clarithromycin were high and vice versa. After 50 daily subcultures in the presence of drugs, MICs of amoxicillin-clavulanate and cefpodoxime against H. influenzae did not rise more than fourfold, in contrast to cefprozil, azithromycin, and clarithromycin, whose MICs rose to variable degrees.
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Resistance to macrolides in clinical isolates of Streptococcus pyogenes due to Ribosomal mutations
The Journal of antimicrobial chemotherapy, 2002Co-Authors: Brigitte Malbruny, Bulent Bozdogan, Kensuke Nagai, Maëlle Coquemont, Arjana Tambić Andrašević, Helena Hupkova, Roland Leclercq, Peter C AppelbaumAbstract:Objective: Two clinical strains of Streptococcus pyogenes, 237 and 544, one isolated in Slovakia and the other in Croatia, that were resistant to azithromycin (MIC 8 and 2 mg/L, respectively) but susceptible to erythromycin (MIC 0.5 and 0.12 mg/L, respectively) did not contain any gene known to confer macrolide resistance by Ribosomal modification (erm gene) or efflux (mef(A) and msr(A) genes). The aim of the study was to determine the mechanisms of macrolide resist- ance in both strains. Methods: Portions of genes encoding Ribosomal Proteins L22 and L4, and 23S rRNA (domains II and V) in the two macrolide-resistant strains and in control strains susceptible to macrolides, were analysed by PCR and single-strand conformational polymorphism, to screen for mutations. The DNA sequences of amplicons from resistant strains that differed from those of susceptible strains, in terms of their electrophoretic migration profiles, were determined. Results: S. pyogenes 237 displayed a KG insertion after position 69 in Ribosomal Protein L4. S. pyogenes 544 contained a C2611U mutation in domain V of 23S rRNA. Conclusion: Mutations at a similar position in Ribosomal Protein L4 and 23S rRNA have been reported previously in macrolide-resistant pneumococci. This report shows that similar muta- tions can be found in macrolide-resistant S. pyogenes.
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two new mechanisms of macrolide resistance in clinical strains of streptococcus pneumoniae from eastern europe and north america
Antimicrobial Agents and Chemotherapy, 2000Co-Authors: A Taitkamradt, Peter C Appelbaum, Michael R. Jacobs, Todd A Davies, Florence Depardieu, Patrice Courvalin, J W Petitpas, L Wondrack, A Walker, Joyce A SutcliffeAbstract:Resistance to macrolides in pneumococci is generally mediated by methylation of 23S rRNA via erm(B) methylase which can confer a macrolide (M)-, lincosamide (L)-, and streptogramin B (SB)-resistant (MLSB) phenotype or by drug efflux via mef(A) which confers resistance to 14- and 15-membered macrolides only. We studied 20 strains with unusual ML or MSB phenotypes which did not harbor erm(B) or mef(A). The strains had been isolated from patients in Eastern Europe and North America from 1992 to 1998. These isolates were found to contain mutations in genes for either 23S rRNA or Ribosomal Proteins. Three strains from the United States with an ML phenotype, each representing a different clone, were characterized as having an A2059G (Escherichia coli numbering) change in three of the four 23S rRNA alleles. Susceptibility to macrolides and lincosamides decreased as the number of alleles in isogenic strains containing A2059G increased. Sixteen MSB strains from Eastern Europe were found to contain a 3-amino-acid substitution (69GTG71 to TPS) in a highly conserved region of the Ribosomal Protein L4 (63KPWRQKGTGRAR74). These strains formed several distinct clonal types. The single MSB strain from Canada contained a 6-amino-acid L4 insertion (69GTGREKGTGRAR), which impacted growth rate and also conferred a 500-fold increase in MIC on the ketolide telithromycin. These macrolide resistance mechanisms from clinical isolates are similar to those recently described for laboratory-derived mutants.
Lasse Lindahl - One of the best experts on this subject based on the ideXlab platform.
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RNA-structural mimicry in Escherichia coli Ribosomal Protein L4-dependent regulation of the S10 operon.
The Journal of biological chemistry, 2003Co-Authors: Ulrich Stelzl, Janice M. Zengel, Marina Tovbina, T. Walker, Knud H. Nierhaus, Lasse Lindahl, Dinshaw J. PatelAbstract:Ribosomal Protein L4 regulates the 11-gene S10 operon in Escherichia coli by acting, in concert with transcription factor NusA, to cause premature transcription termination at a Rho-independent termination site in the leader sequence. This process presumably involves L4 interaction with the leader mRNA. Here, we report direct, specific, and independent binding of Ribosomal Protein L4 to the S10 mRNA leader in vitro. Most of the binding energy is contributed by a small hairpin structure within the leader region, but a 64-nucleotide sequence is required for the bona fide interaction. Binding to the S10 leader mRNA is competed by the 23 S rRNA L4 binding site. Although the secondary structures of the mRNA and rRNA binding sites appear different, phosphorothioate footprinting of the L4-RNA complexes reveals close structural similarity in three dimensions. Mutational analysis of the mRNA binding site is compatible with the structural model. In vitro binding of L4 induces structural changes of the S10 leader RNA, providing a first clue for how Protein L4 may provoke transcription termination.
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Assay of Transcription Termination by Ribosomal Protein L4
Methods in enzymology, 2003Co-Authors: Janice M. Zengel, Lasse LindahlAbstract:Publisher Summary This chapter discusses assaying of transcription termination by Ribosomal Protein L4. The chapter discusses the two approaches, reporter genes and in vitro transcription that characterize attenuation control of the S10 Ribosomal Protein operon in Escherichia coli (E. coli) and other bacteria. The use of reporter genes usually assumes that transcription rates can be deduced from the rate of accumulation of a Protein product. However, the outcome of the measurement depends not only on accumulation of transcript, but also on the rate of its translation. The mechanism of L4-mediated transcription control of the S10 operon by using a cell-free transcription system is discussed. The principal approaches used in the experimentation have included kinetic analysis of regulation, mutational analysis of the mRNA target, disruption of nascent RNA structures with oligonucleotides, and competition for L4 by addition of other RNAs. Furthermore, purification and restart of the transcription complex in the presence or absence of its ligands allow ordering of the NusA- and L4-dependent steps in the regulation. In vitro transcription studies shows that L4-mediated transcription termination is preceded by a NusA-stimulated pause of the RNA polymerase at the site of termination.
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A hairpin structure upstream of the terminator hairpin required for Ribosomal Protein L4-mediated attenuation control of the S10 operon of Escherichia coli.
Journal of bacteriology, 1996Co-Authors: Janice M. Zengel, Lasse LindahlAbstract:Ribosomal Protein L4 of Escherichia coli regulates transcription of the 11-gene S1O operon by promoting premature termination of transcription (attenuation) at a specific site within the 172-base untranslated leader. We have analyzed the roles of various domains of the leader RNA in this transcription control. Our results indicate that the first 60 bases of the leader, forming the three proximal hairpin structures, are not essential for in vivo L4-mediated attenuation control. However, a deletion removing the fourth hairpin, which is immediately upstream of the terminator hairpin, eliminates L4's effect on transcription. Base changes disrupting complementarity in the 6-bp stem of this hairpin also abolish L4 control, but compensatory base changes that restore complementarity also restore L4's effect. In vitro transcription studies confirm that this hairpin structure is necessary for L4's role in stimulating transcription termination by RNA polymerase.
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Domain I of 23S rRNA competes with a paused transcription complex for Ribosomal Protein L4 of Escherichia coli
Nucleic acids research, 1993Co-Authors: Janice M. Zengel, Lasse LindahlAbstract:Ribosomal Protein L4 of Escherichia coli regulates expression of its own eleven gene S10 operon both by inhibiting translation and by stimulating premature termination of transcription. Both regulatory processes presumably involve L4 recognition of the S10 leader RNA. To help define L4's regulatory target, we have investigated the Protein's cognate target on 23S rRNA. Binding of L4 to various fragments of the 23S rRNA was monitored by determining their ability to sequester L4 in an in vitro transcription system and thereby eliminate the Protein's effect on transcription. Using this approach we identified a region of about 110 bases within domain I of 23S rRNA which binds L4. A two base deletion within this region, close to the base to which L4 has been cross-linked in intact 50S subunits, eliminates L4 binding. These results also confirm the prediction of the autogenous control model, that L4 bound to its target on rRNA is not active in regulating transcription of the S10 operon.
Keith P. Klugman - One of the best experts on this subject based on the ideXlab platform.
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Mutations within the rplD Gene of Linezolid-Nonsusceptible Streptococcus pneumoniae Strains Isolated in the United States
Antimicrobial agents and chemotherapy, 2014Co-Authors: Wei Dong, Keith P. Klugman, Sopio Chochua, Lesley Mcgee, Delois Jackson, Jorge E. VidalAbstract:ABSTRACT Three invasive Streptococcus pneumoniae strains nonsusceptible to linezolid were isolated in the United States between 2001 and 2012 from the CDC9s Active Bacterial Core surveillance. Linezolid binds Ribosomal Proteins where structural changes within its target site may confer resistance. Our study identified mutations and deletions near the linezolid binding pocket of two of these strains within the rplD gene, which encodes Ribosomal Protein L4. Mutations in the 23S rRNA alleles or the rplV gene were not detected.
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High-Level Telithromycin Resistance in a Clinical Isolate of Streptococcus pneumoniae
Antimicrobial agents and chemotherapy, 2007Co-Authors: Nicole Wolter, Anthony M. Smith, Donald E. Low, Keith P. KlugmanAbstract: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.
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novel mechanism of resistance to oxazolidinones macrolides and chloramphenicol in Ribosomal Protein L4 of the pneumococcus
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: Nicole Wolter, Anthony M. Smith, Keith P. Klugman, David J Farrell, William Schaffner, Matthew D Moore, Cynthia G Whitney, James H JorgensenAbstract:Two clinical Streptococcus pneumoniae isolates, identified as resistant to macrolides and chloramphenicol and nonsusceptible to linezolid, were found to contain 6-bp deletions in the gene encoding riboProtein L4. The gene transformed susceptible strain R6 so that it exhibited such resistance, with the transformants also showing a fitness cost. We demonstrate a novel bacterial mechanism of resistance to chloramphenicol and nonsusceptibility to linezolid.
Markus C Wahl - One of the best experts on this subject based on the ideXlab platform.
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Expression, purification, crystallization and preliminary X-ray diffraction studies of bacterial and archaeal L4 Ribosomal Proteins.
Acta Crystallographica Section D Biological Crystallography, 2000Co-Authors: Michael Worbs, Markus C WahlAbstract:Ribosomal Protein L4 is implicated in the peptidyltransferase activity of the ribosome and in certain bacteria it regulates the transcription and translation of the 11-gene S10 operon. The genes for the L4 Ribosomal Proteins from the hyperthermophilic bacterium Thermotoga maritima and the halophilic archaeon Haloarcula marismortui have been PCR amplified from genomic DNA and cloned under the control of a T7 promoter to generate overexpressing Escherichia coli strains. For both Proteins, efficient purification procedures were developed to yield material suitable for crystallization trials. Crystals of T. maritima L4 were obtained in the orthorhombic space group P212121, with one molecule per asymmetric unit, diffracting to 1.7 A resolution with synchrotron radiation. Crystals of H. marismortui L4 belonged to the trigonal space group P3121 or P3221 and diffracted to 3.2 A resolution with a rotating-anode source, presumably containing three molecules per asymmetric unit. The results demonstrate that for certain halophilic Proteins the same purification and crystallization procedures can be employed as for conventional Proteins.
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crystal structure of Ribosomal Protein L4 shows rna binding sites for ribosome incorporation and feedback control of the s10 operon
The EMBO Journal, 2000Co-Authors: Michael Worbs, Robert Huber, Markus C WahlAbstract:Ribosomal Protein L4 resides near the peptidyl transferase center of the bacterial ribosome and may, together with rRNA and Proteins L2 and L3, actively participate in the catalysis of peptide bond formation. Escherichia coli L4 is also an autogenous feedback regulator of transcription and translation of the 11 gene S10 operon. The crystal structure of L4 from Thermotoga maritima at 1.7 A resolution shows the Protein with an alternating α/β fold and a large disordered loop region. Two separate binding sites for RNA are discernible. The N‐terminal site, responsible for binding to rRNA, consists of the disordered loop with flanking α‐helices. The C‐terminal site, a prime candidate for the interaction with the leader sequence of the S10 mRNA, involves two non‐consecutive α‐helices. The structure also suggests a C‐terminal Protein‐binding interface, through which L4 could be interacting with Protein components of the transcriptional and/or translational machineries.
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Crystal structure of Ribosomal Protein L4 shows RNA‐binding sites for ribosome incorporation and feedback control of the S10 operon
The EMBO journal, 2000Co-Authors: Michael Worbs, Robert Huber, Markus C WahlAbstract:Ribosomal Protein L4 resides near the peptidyl transferase center of the bacterial ribosome and may, together with rRNA and Proteins L2 and L3, actively participate in the catalysis of peptide bond formation. Escherichia coli L4 is also an autogenous feedback regulator of transcription and translation of the 11 gene S10 operon. The crystal structure of L4 from Thermotoga maritima at 1.7 A resolution shows the Protein with an alternating α/β fold and a large disordered loop region. Two separate binding sites for RNA are discernible. The N‐terminal site, responsible for binding to rRNA, consists of the disordered loop with flanking α‐helices. The C‐terminal site, a prime candidate for the interaction with the leader sequence of the S10 mRNA, involves two non‐consecutive α‐helices. The structure also suggests a C‐terminal Protein‐binding interface, through which L4 could be interacting with Protein components of the transcriptional and/or translational machineries.
Joyce A Sutcliffe - One of the best experts on this subject based on the ideXlab platform.
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resistance to macrolide antibiotics in public health pathogens
Cold Spring Harbor Perspectives in Medicine, 2016Co-Authors: Corey Fyfe, Trudy H Grossman, Kathy Kerstein, Joyce A SutcliffeAbstract:: Macrolide resistance mechanisms can be target-based with a change in a 23S Ribosomal RNA (rRNA) residue or a mutation in Ribosomal Protein L4 or L22 affecting the ribosome's interaction with the antibiotic. Alternatively, mono- or dimethylation of A2058 in domain V of the 23S rRNA by an acquired rRNA methyltransferase, the product of an erm (erythromycin ribosome methylation) gene, can interfere with antibiotic binding. Acquired genes encoding efflux pumps, most predominantly mef(A) + msr(D) in pneumococci/streptococci and msr(A/B) in staphylococci, also mediate resistance. Drug-inactivating mechanisms include phosphorylation of the 2'-hydroxyl of the amino sugar found at position C5 by phosphotransferases and hydrolysis of the macrocyclic lactone by esterases. These acquired genes are regulated by either translation or transcription attenuation, largely because cells are less fit when these genes, especially the rRNA methyltransferases, are highly induced or constitutively expressed. The induction of gene expression is cleverly tied to the mechanism of action of macrolides, relying on antibiotic-bound ribosomes stalled at specific sequences of nascent polypeptides to promote transcription or translation of downstream sequences.
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two new mechanisms of macrolide resistance in clinical strains of streptococcus pneumoniae from eastern europe and north america
Antimicrobial Agents and Chemotherapy, 2000Co-Authors: A Taitkamradt, Peter C Appelbaum, Michael R. Jacobs, Todd A Davies, Florence Depardieu, Patrice Courvalin, J W Petitpas, L Wondrack, A Walker, Joyce A SutcliffeAbstract:Resistance to macrolides in pneumococci is generally mediated by methylation of 23S rRNA via erm(B) methylase which can confer a macrolide (M)-, lincosamide (L)-, and streptogramin B (SB)-resistant (MLSB) phenotype or by drug efflux via mef(A) which confers resistance to 14- and 15-membered macrolides only. We studied 20 strains with unusual ML or MSB phenotypes which did not harbor erm(B) or mef(A). The strains had been isolated from patients in Eastern Europe and North America from 1992 to 1998. These isolates were found to contain mutations in genes for either 23S rRNA or Ribosomal Proteins. Three strains from the United States with an ML phenotype, each representing a different clone, were characterized as having an A2059G (Escherichia coli numbering) change in three of the four 23S rRNA alleles. Susceptibility to macrolides and lincosamides decreased as the number of alleles in isogenic strains containing A2059G increased. Sixteen MSB strains from Eastern Europe were found to contain a 3-amino-acid substitution (69GTG71 to TPS) in a highly conserved region of the Ribosomal Protein L4 (63KPWRQKGTGRAR74). These strains formed several distinct clonal types. The single MSB strain from Canada contained a 6-amino-acid L4 insertion (69GTGREKGTGRAR), which impacted growth rate and also conferred a 500-fold increase in MIC on the ketolide telithromycin. These macrolide resistance mechanisms from clinical isolates are similar to those recently described for laboratory-derived mutants.
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mutations in 23s rrna and Ribosomal Protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage
Antimicrobial Agents and Chemotherapy, 2000Co-Authors: A Taitkamradt, Peter C Appelbaum, Michael R. Jacobs, Todd A Davies, Melissa Cronan, Joyce A SutcliffeAbstract:The predominant forms of macrolide resistance in Streptococcus pneumoniae are mediated by mef(A), a gene encoding an efflux pump in the major facilitator superfamily, or by erm(B), an rRNA methylase. [Note that the mef(A) and mef(E) genes, originally named for the macrolide efflux determinants in Streptococcus pyogenes (6) and S. pneumoniae (44), respectively, have been classified into one group, mef(A) (32).] The prevalence of macrolide resistance varies geographically, being high in Japan (73%), Hong Kong (81.5%), France (47%), Italy (42%), and the United States (19 to 34%) (9; M. R. Jacobs, D. Felminghan, P. C. Appelbaum, and T. A. P. Group, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1044, 1999). In some countries, mef(A) predominates, while in others, erm(B) is the major resistance determinant (7, 19). Although the levels of prevalence of resistance to clarithromycin, erythromycin, and azithromycin are sometimes reported to be slightly different in surveillance studies, isolates containing either mef(A) or erm(B) should be regarded as coresistant to erythromycin, clarithromycin, and azithromycin regardless of the absolute MICs of these compounds (42; L. Brennan, J. Duignan, J. Petitpas, M. Anderson, W. Fu, J. Retsema, J. Rainville, D. Smyth, W. Su, and J. Sutcliffe, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-124, 1998). However, isolates containing mef(A) are susceptible to clindamycin and 16-membered macrolides while those containing erm(B) are resistant to these agents. Virtually all clinical isolates ofmacrolide-resistant S. pneumoniae that have been examined for macrolide resistance mechanisms have contained either mef(A) or erm(B), and occasional strains have contained both genes (7). In a previous study we described the selection of azithromycin-resistant mutants from several macrolide-susceptible clinical strains of S. pneumoniae containing neither mef(A) nor erm(B) (29). Examination of these passage-derived mutants by PCR for mef(A) and erm(B) sequences showed that neither determinant accounted for the resistance. In this study we investigated the resistance mechanisms of these mutants by testing for the presence of other known macrolide resistance determinants, including three genes that encode macrolide phosphorylases (22, 26, 27; J. Cheng, T. Grebe, L. Wondrack, P. Courvalin, and J. Sutcliffe, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 837, 1999; K. O'Hara, T. Kawabe, K. Taniguchi, A. Nakamura, and T. Sawai, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-67, 1997), two genes that encode macrolide esterases, erm(TR) [now reclassified as erm(A) (32)], a newly described methylase found in Streptococcus pyogenes (38), and msr(A), which encodes an ABC-type transporter (36). Failing to find genes previously characterized to confer macrolide resistance in these mutants, we reasoned that mutations in the 23S rRNA alleles and/or in the Ribosomal Proteins L4 and L22 might account for resistance. A mutation in the ribosome seemed likely based on the precedents that rRNA mutations do exist in other species of bacteria (17, 18, 23, 24, 34, 35, 41, 45, 48, 49) and that mutations in L4 or L22 exist in erythromycin-resistant laboratory-derived mutants of Escherichia coli and Bacillus spp. (5, 30, 37, 39, 46, 50, 51).
