1 Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology, University Hospital of North Norway (UNN) and Department of Microbiology and Virology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø, Norway; 2 Division of Infectious Disease Control, Norwegian Institute of Public Health, Pb 4404 Nydalen, N-0403 Oslo, Norway
Received 22 June 2005; returned 27 July 2005; revised 12 August 2005; accepted 17 August 2005
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Abstract |
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Methods: Erythromycin-resistant mef-positive multilocus sequence-typed (MLST) clinical isolates of S. pneumoniae (n = 36) and S. pyogenes (n = 12) from the National Surveillance Program for Antimicrobial Resistance (NORM) as well as viridans streptococci (n = 20) from healthy adults were included. PCR-amplified mef genes were initially discriminated by BamHI digestion. Selected mef genes from representatives of different sequence types (STs) of S. pneumoniae (n = 11) and S. pyogenes (n = 4), and viridans group streptococcal species (n = 8) were typed by sequencing and their strains examined for co-resistances. Hydropathy plots of different mef-encoded proteins were performed.
Results: A predominance of mef(A) was detected in S. pneumoniae (23/36) and S. pyogenes (9/12) due to the clonal spread of ST9 and ST39, respectively. mef(E) was the most widely distributed mef determinant occurring in nine different STs of S. pneumoniae and in four different viridans species. A new mef allele was identified in two STs of S. pyogenes.
Conclusions: mef(E) is the most widely distributed mef determinant in Norwegian clinical strains of S. pneumoniae and pharyngeal carrier strains of various viridans streptococci. However, mef(A) is more prevalent in S. pneumoniae and S. pyogenes due to clonal spread. A new mef allele was found in two different STs of S. pyogenes.
Keywords: macrolide efflux , erythromycin , M-type resistance , Major Facilitator Superfamily
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Introduction |
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Novel mef variants have recently been described in group G streptococci, with 90% and 88% DNA sequence identity to mef(E) and mef(A), respectively.4,13 These alleles have not yet been associated with any mobile genetic elements.
As mef(A) and mef(E) are known to disseminate differently,4 the aim of this study was to identify and type mef genes in M-type erythromycin-resistant multilocus sequence-typed (MLST) clinical strains of S. pneumoniae and S. pyogenes from the National Surveillance Program for Antimicrobial Resistance (NORM). Moreover, pharyngeal carrier isolates of M-type erythromycin-resistant viridans streptococci from healthy adults were included.
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Materials and methods |
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Erythromycin-resistant (MIC 1 mg/L) clinical isolates of S. pneumoniae (n = 36) and S. pyogenes (n = 12) from NORM 20012 and various erythromycin-resistant pharyngeal carrier isolates of viridans streptococci (n = 20) from healthy Norwegian adults in 2004 (Littauer P, Haldorsen BC, Simonsen GS, Sundsfjord A, unpublished results) were examined by mef PCR and restriction enzyme analysis of the amplicon. The mef-carrying clinical isolates of S. pneumoniae have already been partially described.14 An overview of the bacterial isolates is given in Table 1. Representative isolates from all sequence types (STs) of S. pneumoniae (n = 11) and S. pyogenes (n = 4) were selected for mef sequence typing and additional analyses. All of the 20 isolates of viridans streptococci were mef sequence typed, and eight strains were selected for further analysis on the basis of species, mef type and susceptibility to tetracycline.
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For S. pneumoniae isolates, identification was based on colony morphology, Gram staining, catalase reaction, -haemolysis on MuellerHinton agar plates supplemented with 5% (v/v) sheep blood, optochin susceptibility (Rosco, Taastrup, Denmark), Pneumokit (bioMérieux, Marcy l'Étoile, France) and bile solubility (SigmaAldrich Chemie GmbH, Steinheim, Germany). In addition, the isolates were serotyped by the capsular swelling test using specific antisera (Statens Serum Institut, Copenhagen, Denmark) and typed by MLST.15 Identification of S. pyogenes isolates was based on ß-haemolysis, bacitracin susceptibility (Rosco) on blood agar plates, and confirmed by the Oxoid streptococcal grouping kit (Oxoid, Hampshire, UK) and a pyrrolidonyl-arylamidase test (PYR) (Dalynn Biologicals, Calgary, Canada), in addition to emm-typing16 and MLST.17 The viridans group streptococci were identified by colony morphology, Gram staining, the rapid ID 32 Strep API system (bioMérieux), and sequencing of the sodA gene.18
Antimicrobial susceptibility tests
MICs were determined by Etest on MuellerHinton agar supplemented with 5% sheep blood (S. pneumoniae and S. pyogenes) or horse blood (viridans group streptococci). The antimicrobials tested were erythromycin, clindamycin, tetracycline, penicillin G and chloramphenicol. S. pneumoniae ATCC 49619 was included for quality control. Susceptibility results were categorized according to CLSI (formerly NCCLS) breakpoints.19
Genetic analyses of resistance genes
Primers are given in Table 2. Established methods were used to detect tet(M)20 and tet(O)21 genes. The positive controls were Enterococcus faecalis DS16 [tet(M)]22 and S. pyogenes m46 [tet(O)].10 The previously described mef consensus primers23 were used. The 346 bp amplicon was digested with the BamHI restriction enzyme (New England BioLabs), which has no restriction site in mef(E) and one in mef(A), resulting in two fragments of 281 and 65 bp.24
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The PCR products from the selected isolates were sequenced using primers as previously described.23 The mef genes displaying differences from the mef(A) and mef(E) consensus sequences (U70055 and U83667, respectively), were fully sequenced using primers designed in this study (Table 2). Mef 4 F and Mel R primers were based on GenBank accession no. AF227520 (Tn1207.1) and AF274302 (the mega-element). The primers Mef 2 F, Mef 1 R and Mef 2 R were designed using GenBank accession no. U83667 and U70055, while the Mef 3 F primer was based on a provisional sequence of the mef gene of TUH 36-54. A semi-nested PCR was performed on the PCR product from the primers Mef 4 F and Mel R in order to obtain sufficient DNA for sequence typing of TUH36-54 and 02-59. This second PCR included the primers Mef 4 F and Mef 1 R. Sequencing was carried out bi-directionally, using an ABI Prism 377 DNA Sequencer or an ABI prism 3100 Genetic Analyzer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) and 3'-dye-labelled terminators. Sequence similarity search was performed using the BLAST algorithm at the National Center for Biotechnology Information of the National Library of Medicine (Bethesda, MD, USA) [http://www.ncbi.nlm.nih.gov (date last accessed 11 August 2005)]. The nucleotide and amino acid sequence comparisons were performed by multiple sequence alignment using the CLUSTAL W program.25 BioEdit [http://www.mbio.ncsu.edu/BioEdit/bioedit.html (date last accessed 11 August 2005)] was used to present data from the alignment. The mef sequences were from Streptococcus dysgalactiae (GenBank accession no. AY355406 and AY355410),13 Bacteroides ovatus (GenBank accession no. AJ557257)26 and Bacillus cereus (GenBank accession no. AAEK01000016),27 in addition to the mef(A) and mef(E) consensus sequences.
Hydropathy plots
Prediction of transmembrane (TM) regions of MFS proteins was performed using the bioinformatical resources MEMSAT28 and the TMHMM29,30 algorithms, to reveal whether conserved motifs could be found in mef genes of diverse origins.
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Results and discussion |
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The mef variant (GenBank accession no. DQ016305) found in two S. pyogenes isolates displayed 97% identity on the nucleotide level to the mef allele found in S. dysgalactiae (GenBank accession no. AY355406), and it was 89% and 88% identical with the mef(E) gene of S. pneumoniae (GenBank accession no. U83667) and the mef(A) gene of S. pyogenes (GenBank accession no. U70055), respectively. On the amino acid level, the new mef allele also displayed 97% sequence identity to the S. dysgalactiae mef gene, and 89% and 87% identity to mef(E) and mef(A), respectively. The primers designed for mef(A) and mef(E)23 detected the new mef variant, even though there were two mismatches in the forward primer (Mef A F) and one mismatch in the reverse primer (Mef A R). The two mismatches to Mef A F affected the 5'-end of the primer whereas the single Mef A R mismatch affected the 3'-side, but not the very end. The increasing number of known mef alleles should be taken into consideration when designing primers for detection of macrolide resistance determinants. Moreover, when restriction fragment length polymorphism is used to distinguish between mef(A) and mef(E) genes, one should be aware that several variants of a gene can give identical restriction patterns, e.g. the new mef allele found in S. pyogenes passed as a mef(E) in the BamHI digestion.
Concerning the TM predictions of the new mef variant, it was not possible to interpret unambiguously the number of transmembrane segments from the individual hydropathy plots. However, MEMSAT28 predicted 12 membrane-spanning regions and also gave the correct sidedness compared with that previously described for MFS. The loop between the putative TM2 (transmembrane segment 2) and TM3 is the most conserved part of the alignment (Figure 1), with six out of seven identical amino acids for all seven sequences. If we include the four amino acids upstream of this sequence, the motif is 65GVXXDRXDRKK75. A conserved sequence motif in the cytoplasmic loop between TM2 and TM3 has also been found in tetracycline/H+ antiporters (62GXXXDRXGRR71),37 and in various antibiotic or antiseptic resistance-conferring proteins, including TetB, TetL and QacA.38 The Asp-66 has previously been demonstrated to be part of a highly conserved sequence motif postulated to act as an entrance gate for a substrate.39
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All mef(A)-positive isolates were found to be susceptible to both tetracycline and chloramphenicol, whereas 8 out of 16 mef(E)-positive isolates were tetracycline-resistant and possessed the tet(M)-gene (data not shown). Two out of these eight tetracycline-resistant isolates also displayed increased MICs to chloramphenicol. Interestingly, the two isolates possessing the new mef allele were also resistant to chloramphenicol and tetracycline, and were tet(M) positive. This finding strongly indicates genetically linked resistance determinants for erythromycin, tetracycline and chloramphenicol, since the observation was made in two genetic lineages of S. pyogenes. A recent report on mef(A)-containing elements in S. pyogenes10 concluded that the mef(A) gene is carried on a different chromosomal genetic element if the isolates are resistant to tetracycline than if the isolates are susceptible to tetracycline. In our case, it is not known which element the new mef variant is a part of, if any. Italian isolates of S. pyogenes with efflux-mediated macrolide resistance have been demonstrated to possess the tet(O) gene, linked to mef in a newly discovered mobile element.11 This gene, however, was not found among any of the Norwegian isolates that were examined (n = 23), indicating that other genetic elements are responsible for the erythromycin-tetracycline resistance in Norwegian streptococcal strains.
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Conclusions |
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Acknowledgements |
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