Resistance to macrolides in clinical isolates of Streptococcus pyogenes due to ribosomal mutations

Brigitte Malbruny1, Kensuke Nagai2, Maëlle Coquemont1, Bülent Bozdogan2, Arjana Tambic Andrasevic3, Helena Hupkova4, Roland Leclercq1,* and Peter C. Appelbaum2

1Service de Microbiologie, Hôpital Côte de Nacre, UFR de Médecine, Av. Côte de Nacre, 14033 Caen, France; 2Department of Pathology, Hershey Medical Center, Hershey, PA 17033, USA; 3Department of Clinical Microbiology, University Hospital for Infectious Diseases ‘Dr F. Mihaljevic’, 10000 Zagreb, Croatia; 4St Cyril and Metod Hospital, 85107 Bratislava, Slovakia

Received 4 October 2001; returned 2 February 2002; revised 21 February 2002; accepted 26 February 2002.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 resistance 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 mutations can be found in macrolide-resistant S. pyogenes.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although penicillins are still the first-line treatment for Streptococcus pyogenes (group A streptococcus) pharyngitis, macrolides are often the recommended substitute when these antimicrobials fail or in cases of patient intolerance to these drugs. In addition, short-course therapy with certain macrolides, such as azithromycin, has been reported to be effective for eradicating oropharyngeal S. pyogenes.1 Although the incidence of resistance to macrolides in S. pyogenes was low in the past, high incidences have now been reported from several countries including Finland, Italy, Korea, Spain and Thailand.26 Epidemiological surveys have shown that the acquisition of erm(B) and erm(A) genes, encoding ribosomal methylases, and of mef(A) genes, encoding efflux proteins, accounted for resistance in nearly all strains.58 In this report, we show that ribosomal mutations affecting sites involved directly or indirectly in ribosomal binding of macrolides may also be a cause of resistance to macrolides and related antibiotics in S. pyogenes.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

A large number of clinical isolates of S. pyogenes strains resistant to macrolides collected during a multicentre European study were studied for the presence of erm(B) and mef(A) genes.9 S. pyogenes strains 237 and 544, isolated in Slovakia and Croatia from the ear of a 1-year-old child suffering from otitis, and from a throat sample from a 20-year-old patient, respectively, did not contain either gene and were studied further. Erythromycin-susceptible clinical isolates of S. pyogenes, UCN5 and 11, were used as controls for PCR and MIC studies.

MIC determination

MICs of azithromycin, clindamycin, dalfopristin, erythromycin, quinupristin, quinupristin–dalfopristin, spiramycin and telithromycin were determined by the agar dilution method with Mueller–Hinton medium supplemented with 5% defibrinated sheep blood.10 Agar plates were incubated overnight at 37°C in ambient air.

Detection of erythromycin resistance genes

The erythromycin-resistant isolates were screened for the erythromycin resistance genes erm(A), erm(A) subclass erm(TR), erm(B), erm(C), mef(A) and msr(A) by PCR amplification, as described previously.11 Streptococcus pneumoniae HM28 containing the erm(B) gene, Staphylococcus aureus HM1054/R containing erm(C), S. aureus HM1051 containing erm(A), S. pyogenes UCN1 containing erm(TR), Staphylococcus epidermidis HM1053 containing msr(A) and S. pneumoniae O2J1175 containing mef(A), were used as controls in PCR experiments.

Detection of mutations in the ribosomal target of macrolides

Nucleotide sequences of 23S rRNA and L4 and L22 ribosomal proteins in Escherichia coli were obtained from The Institute for Genomic Research website (www.tigr.org) and homologues were detected in S. pyogenes by using BLAST software (www.ncbi.nlm.nih.gov/Microb_blast).12 Specific oligonucleotide primers were then designed. We amplified a portion of the rrl gene for domain II, from nucleotide 580 to 852 (E. coli numbering) with primers 5'-CGGCGATTACGATATGATGC-3' and 5'-CTCTAATGTCGACGCTAGCC-3', and two overlapping fragments of domain V of 23S rRNA (nucleotides 1990–2405 and 2331–2769) with the two pairs of primers 5'-CTGTCTCAACGAGAGACTC-3' and 5'-GGAACCACCGGATCACTAAG-3', and 5'-GTATAAGGGAGCTTGACTG-3' and 5'-GGGTTTCACACTTAGATG-3'. The entire L22 (rplV) and L4 (rplD) genes were amplified, using pairs of primers 5'-GCTGACGACAAGAAAACACG-3' and 5'-GCCGACACGCATACCAATTG-3', and 5'-CAAGTCAGGAGTTAAAGCTGC-3' and 5'-CAACTTCGAAAGTGTATTTGCC-3', respectively. The three amplified rrl fragments (two for domain V and one for domain II) included bases critical for erythromycin resistance (G2057, A2058, A2062, G2505, C2611, A752 and A754). Amplicons were obtained for erythromycin-susceptible and -resistant strains and were subsequently analysed by single-strand conformation polymorphism (SSCP) as follows. Aliquots of 20 µL H2O containing 20 ng of PCR product were mixed with 20 µL of denaturant solution (95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, 20 mM EDTA). The mixture was heated for 10 min at 100°C and cooled on ice, and the single-strand PCR product was then separated by non-denaturing PAGE (10% acrylamide 29–bisacrylamide 1 in Tris-borate/EDTA buffer) by using the vertical slab gel unit model SE 400 (Hoefer Scientific Instruments, San Francisco, CA, USA). The gel was run for 12–15 h at 200 V at 4°C. Bands were then visualized by ethidium bromide staining. Fragments with migration profiles differing from those of erythromycin-susceptible strains were sequenced by the dRhodamine dye terminator method with an Abi Prism 377 sequencer (Perkin-Elmer Corp., Norwalk, CT, USA). The oligonucleotides used for PCR were also used as primers for DNA sequencing.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Macrolide resistance phenotypes

MICs of macrolides and related antibiotics for S. pyogenes strains are shown in Table 1. Strain 237 was borderline susceptible to erythromycin, resistant to azithromycin and highly resistant to spiramycin (a 16-membered macrolide). The MIC of telithromycin for strain 237 was low, although it was three- to seven-fold higher than the MIC for susceptible strains. Activities of clindamycin, dalfopristin (A-type streptogramin), quinupristin (B-type streptogramin) and quinupristin–dalfopristin were similar to those against control strains. This phenotype of resistance to macrolides with susceptibility to clindamycin and streptogramins could be defined as an M phenotype. S. pyogenes 544 was resistant to azithromycin but remained susceptible to erythromycin despite a nearly five-fold increase in MIC, and to spiramycin. Again, telithromycin was active. In contrast to strain 237, S. pyogenes 544 was resistant to clindamycin and had an increased quinupristin MIC, although synergy between the two streptogramins was maintained. This phenotype could be defined as an MLSB phenotype.


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Table 1.  MICs of macrolides and related antibiotics for S. pyogenes strains
 
Macrolide resistance genotypes

PCR experiments showed that no known acquired resistance gene could be amplified from DNA of the azithromycin-resistant strains. Recent literature data has suggested that macrolide resistance could result from mutations of ribosomal structures, including domains II and V of 23S rRNA, and ribosomal proteins L4 and L22, in a variety of microorganisms, including S. pneumoniae.1317 These ribosomal structures are involved directly or indirectly in macrolide binding. To explore this hypothesis, we carried out analysis of the DNA sequences encoding these RNA sequences or proteins. Amplicons were obtained from azithromycin-susceptible and -resistant strains and were analysed by SSCP. By this technique, a single base pair change produces a migration profile different from that visualized for the wild-type DNA.18 We have confirmed previously the sensitivity of this technique, by the detection of a single mutation in sequences of 23S rRNA and rplD genes in mutants of S. pneumoniae.13 Migration profiles for S. pyogenes strains showed differences in the domain V amplicon of strain 544 and in the rplD amplicon of strain 237. Sequencing of the fragments in both directions revealed a C2611U mutation in domain V of 23S rRNA in S. pyogenes 544. In S. pneumoniae containing four copies of the rrl genes, the SSCP technique allowed us to distinguish a single wild-type copy from three mutated copies, on the basis of heterogeneous electrophoretic profiles.13 S. pyogenes has six copies of the rrl gene and the homogeneity of the electrophoretic profile of the amplicon from strain 544 indicated that all copies were mutated.15

S. pyogenes 237 harboured an insertion of six nucleotides in the sequence of the rplD gene. This insertion would result in an insertion of the two amino acids K and G after position 69. This insertion occurred in a highly conserved fragment of the L4 protein, 63KPWRQKGTGRAR74.17


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the two strains of azithromycin-resistant S. pyogenes studied, which lacked erm and mef(A) genes, resistance was associated with mutations of domain V of 23S rRNA at position C2611 and the L4 protein, respectively. In the absence of other mutations in the bases critical for erythromycin resistance in 23S rRNA and in the two proteins L4 and L22, the mutations identified explained macrolide resistance in our strains, which is a similar finding to that made with other bacterial species.1315,17 C2611 is a residue that pairs with G2057 in the secondary structure of 23S rRNA, and the C2611U mutation results in a disruption in the rRNA structure at the end of the stem preceding the single-stranded portion of the peptidyl transferase region containing A2058 and A2059.19 So far, the C2611U mutation has been characterized in erythromycin-resistant laboratory mutants of E. coli and a C2611A/G mutation in laboratory mutants of S. pneumoniae.1315,20 In S. pneumoniae, C2611 mutations increased the MICs of 14- and 15-membered macrolides, telithromycin, lincosamides and streptogramin B.13,14 In S. pyogenes, this mutation yielded cross-resistance between azithromycin, clindamycin and streptogramin B (MLSB resistance). The MIC differed according to the macrolide tested. The strains were categorized as resistant to azithromycin but susceptible to erythromycin. The low MIC of telithromycin could be explained by the ability of this ketolide to bind domain II of 23S rRNA as an alternative to mutated domain V, which no longer binds macrolides.21 Mutations in the L4 protein of S. pyogenes strains and of S. pneumoniae selected in vitro and in vivo were clustered in an identical conserved region (Figure 1).14 This region is located in a segment of the L4 protein joining two {alpha}-helices, designated {alpha}3 and {alpha}2, which is essential for the binding of this protein to 23S rRNA.22,23 In E. coli, L4 mutations perturb the three-dimensional structure of 23S rRNA at multiple sites and, hypothetically therefore, could prevent macrolide binding by affecting the opening of the nascent peptide exit tunnel.24,25 In S. pyogenes, the mutation resulted in cross-resistance between the macrolides tested with elevated MICs of spiramycin. To the best of our knowledge, mutations in 23S rRNA have never been reported in clinical isolates or laboratory mutants of S. pyogenes, although a few S. pyogenes strains with L4 mutations were reported in a recent study on the efficacy of azithromycin in the treatment of pharyngitis.26 This concomitant report of mutational resistance might indicate that this new type of resistance is emerging, or alternatively, that its prevalence has been underestimated so far. The impact of the resistance on the efficacy of therapy with macrolides, and on clinical outcome might vary according to the macrolide used, since, as has already been pointed out, marked differences exist between MICs of macrolides according both to the site of mutation and to the type of substitution. The clinical impact could not be analysed in our study as data on antibiotic therapy and clinical outcome in the patients infected with strains 237 and 544 were not available. If mutational resistance increases in frequency, its routine detection might be difficult and would not be achieved by testing with erythromycin only. Indeed, in the case of S. pyogenes 544, testing with erythromycin only would have indicated that the strain was susceptible to macrolides, if the result had been extrapolated to all 14- and 15- membered macrolides. Ideally, the specific macrolide used in the patient should be tested. Finally, the contribution of 23S rRNA and L4 protein mutations to macrolide resistance in clinical isolates containing acquired genes of resistance to these antimicrobials should be assessed.



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Figure 1. Comparison of deduced amino acid sequences of the conserved portion of the L4 protein from S. pyogenes and S. pneumoniae strains. S. pyogenes M1 and S. pneumoniae TIGR4 are susceptible to macrolides.12,14 The other strains are resistant to macrolides. A portion of the L4 sequence, from amino acid 63 to amino acid 74, of S. pyogenes M1, S. pyogenes 237, S. pneumoniae TIGR4 (www.tigr.org), S. pneumoniae BM4418, S. pneumoniae 4Az and S. pneumoniae 5Az were aligned.12,14,17 Insertions or deletions are indicated in bold.

 


    Footnotes
 
* Corresponding author. Tel: +33-2-31-06-45-72; Fax: +33-2-31-06-45-73; E-mail: leclercq-r{at}chu-caen.fr Back


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 Materials and methods
 Results
 Discussion
 References
 
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