mef(A), mef(E) and a new mef allele in macrolide-resistant Streptococcus spp. isolates from Norway

Maria Sangvik1, Pia Littauer1, Gunnar Skov Simonsen1,2, Arnfinn Sundsfjord1,2,* and Kristin Hegstad Dahl1

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


* Corresponding author. Tel: +47-77-64-62-02; Fax: +47-77-64-53-50; E-mail: arnfinns{at}fagmed.uit.no

Received 22 June 2005; returned 27 July 2005; revised 12 August 2005; accepted 17 August 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Objectives: To type mef genes in a nationwide collection of clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes as well as pharyngeal carrier strains of viridans streptococci in Norway.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Resistance to macrolides based on an efflux system was established in 1996 by Sutcliffe and coworkers.1 The presence of macrolide efflux (mef) genes is characterized by a low to moderate level of resistance to 14- and 15-membered macrolides, but susceptibility to 16-membered macrolides, lincosamides and streptogramin B antimicrobials. This is referred to as the M-type resistance, which is discriminated from the MLSB-phenotype conferring high-level resistance to all macrolides, lincosamides and streptogramin B.2 The macrolide efflux pump belongs to the Major Facilitator Superfamily (MFS) of transporters.3 Several different mef genes have been described in different microbial species.4,5 Originally, mef(A) was described in Streptococcus pyogenes whereas mef(E) was found in Streptococcus pneumoniae. The two mef genes show 90% DNA identity and were regarded as a single gene class designated mef(A) by Roberts et al.5 However, even though mef(A) and mef(E) are 90% identical at the nucleotide level, they are characterized by major differences,2 and in this study, recent nomenclature recommendations have been used.4 The genetic elements carrying mef(A) and mef(E) were first characterized in S. pneumoniae. mef(A) has been shown to be part of a 7.2 kb defective transposon (Tn1207.1).6 The mef(E)-containing 5.5 kb mega (macrolide efflux genetic assembly) element was recently demonstrated to be integrated into a new composite element, Tn2009 of ~23.5 kb.7 In S. pyogenes, the Tn1207.1 element has been identified as part of a ~52 kb conjugative transposon (Tn1207.3)8 and a 58.8 kb chimeric element resulting from the chromosomal insertion of Tn1207.1 into a prophage.9 Moreover, a recent study observed a novel mef(A)-tet(O)-element of ~60 kb in an Italian S. pyogenes isolate.10,11 According to Giovanetti et al.,12 all mef(A)-containing elements of S. pyogenes seem to be associated with prophages. The genetic support of the mef(E) gene in S. pyogenes has to our knowledge not been described.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Bacterial isolates

Erythromycin-resistant (MIC ≥ 1 mg/L) clinical isolates of S. pneumoniae (n = 36) and S. pyogenes (n = 12) from NORM 2001–2 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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Table 1. Distribution of mef genes in a number of Streptococcus spp.

 
Bacterial identification

For S. pneumoniae isolates, identification was based on colony morphology, Gram staining, catalase reaction, {alpha}-haemolysis on Mueller–Hinton agar plates supplemented with 5% (v/v) sheep blood, optochin susceptibility (Rosco, Taastrup, Denmark), Pneumokit (bioMérieux, Marcy l'Étoile, France) and bile solubility (Sigma–Aldrich 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 Mueller–Hinton 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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Table 2. Oligonucleotide primers used in this study

 
DNA sequence analysis

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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
BamHI restriction analyses and sequence typing of mef amplicons revealed differences between the three groups of streptococci (Table 1). In S. pneumoniae, mef(A) was detected in 23 out of 36 M-type strains. This was due to the clonal spread of ST9 which constituted 22 of 23 mef(A)-positive strains.14 Both mef(A) and mef(E) have been frequently observed in S. pneumoniae isolates.2,24,31 Previous studies have also reported that most S. pneumoniae isolates carrying mef(A) appear to be clonally related.2,32 mef(E) seemed to be a more widely distributed mef class within the Norwegian S. pneumoniae population, as it was found in 13 isolates with nine different sequence types. These results indicate that the genetic element(s) involved in the spread of mef(E) is more successful in the evolution of a diverse M-type S. pneumoniae population compared with mef(A). The mechanisms behind this observation were not addressed in this study, but might be due to an accessible mef(E) resistance pool within phylogenetically related species in the pharyngeal flora represented by the mef(E)-containing group of viridans streptococci. mef(E) was detected in 18 out of 20 viridans strains by BamHI restriction analysis of mef amplicons and confirmed by sequence analysis in all 20 strains, representing four different species (S. parasanguis, S. mitis, S. salivarius and S. oralis). Both mef(A) and mef(E) have previously been found in viridans streptococci. Although the material gathered is limited, mef(E) appears to be the predominant mef allele in these studies.33,34 The mef distribution in S. pyogenes was more complex. mef(A) was found in nine strains, all ST39, thus representing clonal spread. mef(E) was found in only one strain of ST46, and this strain possessed the erm(TR) gene as well. These results are in line with most previous publications, where the mef(A) allele is by far the most prevalent.35,36 In addition, a new mef allele was detected in two different STs (STs 3 and 205) with different emm types (emm types 33 and stNS1033) of S. pyogenes.

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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Figure 1. Multiple sequence alignment of diverse amino acid sequences corresponding to the mef alleles. Both termini are in the cytoplasm. Putative TM segments derived from hydropathy plots are indicated in grey for the new mef allele, while the conserved motif in the cytoplasmic loop between TM2 and TM3 is framed. The encoded protein from AJ557257 is unlikely to encode an actual macrolide efflux pump, as it lacks 29 amino-terminal residues and 66 carboxy-terminal residues.4 The amino acid sequence from AAEK0100016 is annotated as a putative macrolide efflux protein. Charged amino acids in the TMs are in bold face.

 
Membrane-embedded charged amino acids are often functionally significant, and in tetracycline exporters, the charged residues in the transmembrane regions are highly conserved.37,40 In the Escherichia coli multidrug transporter MdfA, a single membrane-embedded negative charge was reported as critical for recognition of positively charged drugs.41 Four of the putative membrane-embedded charged amino acids in the new mef allele from this study were also conserved in the other six mef genes in the alignment. There were three acidic residues (Asp-81, Asp-174 and Glu-261) and one basic residue (Arg-112). The conserved arginine in TM4 has been proposed to be responsible for proton transfer in MFS,38 or substrate translocation in drug/H+ antiporters.37

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.


    Conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
The mef(A) and mef(E) genes were both found in Norwegian erythromycin-resistant Streptococcus spp. of different origins. In S. pneumoniae, the predominance of mef(A) appeared to be due to the clonal spread of ST9. mef(E) was found in isolates with nine different S. pneumoniae STs and four different viridans species indicating a widespread and ecologically successful reservoir of this mef class. The mef gene distribution in S. pyogenes was more diverse, including a new mef allele in two different STs. The new allele displayed high sequence identity to mef genes previously found in group G streptococci, indicating a possible transfer of macrolide resistance genes between group A and group G streptococci. The number of mef-variants will probably increase as more isolates are sequenced, and care should be taken when selecting primers for amplification of mef genes.


    Acknowledgements
 
We thank Professors Bruna Facinelli and Don Clewell for kindly providing reference strains S. pyogenes m46 and E. faecalis DS16, respectively. Thanks also go to Manuela Krämer for excellent technical assistance. This study was funded by Helse Nord (Medical Research Foundation in North Norway).


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
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