JAC Advance Access originally published online on January 23, 2006
Journal of Antimicrobial Chemotherapy 2006 57(3):443-449; doi:10.1093/jac/dki490
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Molecular epidemiology of macrolide resistance in ß-haemolytic streptococci of Lancefield groups A, B, C and G and evidence for a new mef element in group G streptococci that carries allelic variants of mef and msr(D)
Department of Medical Microbiology, University of Aberdeen School of Medicine, Polwarth Buildings, Foresterhill, Aberdeen AB25 2ZD, UK
* Corresponding author. Tel: +44-1224-553786; Fax: +44-1224-552692; E-mail: h.mckenzie{at}abdn.ac.uk
Received 1 September 2005; returned 25 September 2005; revised 16 December 2005; accepted 20 December 2005
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Abstract |
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Objectives: To study the molecular mechanisms of erythromycin resistance in ß-haemolytic streptococci of Lancefield groups A, B, C and G.
Methods: Erythromycin-resistant clinical isolates from North East Scotland were collected over 2 years. Resistance phenotypes were determined by disc diffusion and MICs by Etest. Resistance genes mef, msr(D), erm(B) and erm(TR) were identified by PCR and mef and msr(D) were sequenced.
Results: Erythromycin resistance prevalence was 1.9% in group A streptococci (31 of 1625), 4.3% in group B (53 of 1233), 3.8% in group C (18 of 479) and 6.2% in group G (64 of 1034). The numbers of resistant isolates available were 26, 42, 9 and 52 in each group respectively. The majority of resistant isolates in groups A (57.7%, 15 of 26), B (88.1%, 37 of 42) and G (90.4%, 47 of 52) were MLSB. The contribution of M phenotype was significant in groups C (77.8%, 7 of 9) and A (42.3%, 11 of 26). Group A isolates carried mef(A) and group B carried mef(E) exclusively. A mef sequence distinct from mef(A) and mef(E) was identified in group G and was associated with a new msr(D) sequence. These sequence variants appear to be part of a new genetic element that is inserted in the comEC gene. A bimodal distribution of erythromycin MICs was noted in erm(TR) isolates.
Conclusions: The results demonstrate significant differences in the mechanisms of macrolide resistance amongst different Lancefield groups in the same geographical area. New sequences show that resistance mechanisms are still evolving.
Keywords: M phenotype , MLSB phenotype , erm(B) , erm(TR)
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Introduction |
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There have been many reports of increases in macrolide resistance in streptococci over the last few years. There are two common mechanisms of macrolide resistance, the prevalence of which differs in different geographical areas. Thus macrolide resistance in group A streptococci in central Italy increased from 9% in 1992 to 53% in 1997, and this was entirely due to an increase in prevalence of strains of the MLSB phenotype.1 In such strains, methylase genes of the erm family confer resistance to macrolides, lincosamides and streptogramin B by enzymatic modification of the ribosomal target site of these antibiotics.2 There are several different sequence variations in the erm gene, with erm(A) subclass erm(TR) and erm(B) as the most common in streptococci.3 Resistance to clindamycin in such isolates may be expressed constitutively (cMLSB) or inducibly (iMLSB). In a study of invasive isolates of Streptococcus pneumoniae in Atlanta, USA, Gay et al.4 found that macrolide resistance increased from 16% in 1994 to 32% in 1999, and that this increase was due to increased prevalence of isolates of the M phenotype. In M phenotype strains, resistance is due to an efflux mechanism and is associated with the presence of the mef gene. There are two main sequence variants of the mef gene in streptococci with 90% sequence identity. The mef(A) sequence was originally described in Streptococcus pyogenes5 and mef(E) in S. pneumoniae,6 but it is now known that these sequences are not species specific and it was recommended that both should be designated as mef(A).3 However, different mef sequences appear to be transferred on different genetic elements and inserted at different sites in the host genome. The mef(A) sequence can be associated with a variety of elements including Tn1207.1, Tn1207.3 and a larger 58.8 kb chimeric element,7–9 which all appear to be prophage associated in S. pyogenes.9,10 In S. pyogenes, the mef(A) element is inserted in the comEC competence gene in tetracycline-susceptible isolates but not in tetracycline-resistant isolates.11 The mef(E) sequence has been associated with the genetic element mega in S. pneumoniae12 but mega can also be associated with the tetracycline resistance gene tet(M) in the larger composite element Tn2009.13 In S. pneumoniae, the mef(A) element is inserted in the celB competence gene and such isolates cannot be transformed, while mef(E)-carrying isolates are still transformable.14 These differences have led to the suggestion that the differentiation between mef(A) and mef(E) gene sequences should be retained and that they should be referred to as subclass mef(A) or mef(E).14,15 A further difference between mef(A) and mef(E) elements is the sequence of the gene downstream from mef which is homologous to the staphylococcal msr(A) gene and has been designated as msr(D).16 This gene encodes an ATP binding protein that confers macrolide resistance independently of mef.16
We have previously shown that the M phenotype is responsible for 76% of macrolide resistance among clinical isolates of S. pneumoniae in North East Scotland and this is largely due to the predominance of a serotype 14 clone, characterized as ST9 by multi-locus sequence typing.17 This clone is an important cause of invasive disease and contains the subclass mef(A) gene sequence originally described in S. pyogenes. In order to establish whether this gene was responsible for macrolide resistance across a range of streptococcal species in our area, we have now undertaken an epidemiological survey of macrolide resistance in clinical isolates of ß-haemolytic streptococci of Lancefield groups A, B, C and G. Macrolide resistance due to both M and MLSB phenotypes has been reported previously in ß-haemolytic streptococci of Lancefield groups A18–21 and B.21–26 There have been fewer studies of macrolide resistance in groups C and G, but Kataja et al.27 suggested that the M phenotype was common in group C streptococci and that the MLSB phenotype predominated in group G isolates. However, M phenotype resistance in group G streptococci has been reported in North America,28 Hong Kong29 and Korea.21
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Bacterial isolates
All bacterial isolates were cultured from clinical specimens submitted to the routine diagnostic laboratories of the Department of Medical Microbiology, University of Aberdeen, UK. Over a 2 year period, from 2000 to 2001, erythromycin-resistant isolates of ß-haemolytic streptococci of Lancefield groups A, B, C and G were collected. Specimens were from a wide range of non-sterile sites except for one group A isolate from blood. ß-Haemolysis was identified on blood agar and grouping was carried out by latex agglutination using a Streptococcal grouping kit (Oxoid Ltd, Basingstoke, UK). Erythromycin resistance was detected in the routine diagnostic laboratory by disc diffusion assay following the guidelines of the NCCLS.30,31 Details of all streptococci isolated during the study period, including total number of isolates from each Lancefield group and their antibiotic susceptibilities, were obtained from the computer database of our diagnostic laboratory. Duplicate isolates from the same episode of infection in any one patient were counted only once. Isolates were stored at –70°C in ProtectTM (TSC Ltd, Heywood, UK) and recovered when required by culture on blood agar plates at 37°C. Erythromycin resistance phenotype, M or MLSB, was determined subsequently by disc diffusion assay with adjacent erythromycin and clindamycin discs.32 Erythromycin MICs were determined in the presence of 5% CO2 by Etest (AB BIODISK, Solna, Sweden) according to the manufacturer's recommendations. S. pneumoniae strain ATCC 49619 was tested simultaneously as a quality control and had MIC values within the manufacturer's recommended range.
DNA extraction, gene amplification and sequencing
Cells were harvested from one confluent blood agar plate. Chromosomal DNA was extracted from cell suspensions with a DNA extraction kit for Gram-positive bacteria (Puregene, Gentra Systems, Minneapolis, MN, USA) or by the method of Pitcher et al.33 with the following modification. Mutanolysin (50 U, Sigma-Aldrich, Poole, UK) was added to a freshly prepared lysozyme solution in TE buffer to resuspend bacterial cells and this increased the DNA yield especially in group G isolates.
All isolates were initially screened for the presence of mef genes with primers MEF 57F and MEF 402R as described by Sutcliffe et al.34 (Table 1). PCR conditions for mef amplification were as described by Amezaga et al.17 To obtain the complete sequence of the new mef variant in group G streptococci, a 1265 bp fragment was amplified with primers MEF 57F and MEF 1321R; sequencing of this fragment was carried out with the two amplification primers and another two internal primers as shown in Table 1. To complete the sequence at the 5'end of the new mef variant, a 457 bp overlapping fragment was amplified and sequenced with primers MEFus 61F and MEF 396R. The msr(D) gene in mef(A) and mef(E) isolates was routinely amplified and sequenced with primers MSR 201F and MSR 908R.17 To sequence the new msr(D) variant and flanking regions, a 1724 bp fragment that also incorporated the new mef was amplified with primers MEF 523F and MSR 908R; sequencing of this fragment was carried out with the two amplification primers and an internal primer as shown in Table 1. To complete the sequence at the 3' end of the new msr(D) variant, a 1420 bp fragment was amplified with primers MSR 461F and ORF6 R and sequenced with the two amplification primers and another two internal primers as shown in Table 1. The insertion site of the element carrying the new mef and msr(D) variants was amplified and sequenced with primers COMEC 1485F and MEF 969R (Table 1). Amplification of the erm(B) gene was as before.17 Primers to amplify the erm(TR) gene, 5'-AAACAGAAAAACCCGAAAA (forward) and 5'-AGCAAATCCCCTCTCTAC (reverse), were based on the sequence of this gene in S. pyogenes (GenBank accession number AF002716 [GenBank] ). PCR conditions were as for mef amplification but with longer annealing (1 min), elongation (1 min 30 s) and final elongation (10 min) times. All M phenotype and selected MLSB isolates of group G were confirmed as Streptococcus dysgalactiae subspecies equisimilis by sequencing of the 16S rRNA genes.35
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Standard PCR reactions contained 100 ng of genomic DNA, forward and reverse primers (250 nM), MgCl2 (1.5 mM), dNTPs (200 µM, Amersham Pharmacia Biotech UK Ltd, Little Chalfont, UK) and Taq polymerase (5 U) plus buffer (Bioline, London, UK). For long PCR reactions the Roche Expand Long Template PCR System (Roche Diagnostics UK, Lewes, UK) was used following the manufacturer's instructions. PCR was performed on a GeneAmp® PCR System 9700 Thermocycler (AB Applied Biosystems, Warrington, UK) and PCR products were detected by electrophoresis on agarose gels followed by staining with ethidium bromide and UV transillumination. PCR products were purified with Centricon C100 columns (Amicon, Millipore UK Ltd, Watford, UK) or a Hybaid Purification kit (Hybaid Ltd, Ashford, UK). Sequencing reactions were carried out with the BigDye Terminator Cycle Sequencing kit (AB Applied Biosystems) and sequencing products were run on an ABI 377 Automated DNA Sequencer. Sequences were analysed with the SeqEd 1.0.3 DNA analysis program (AB Applied Biosystems).
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Erythromycin resistance phenotypes in ß-haemolytic streptococci of Lancefield groups A, B, C and G
Over the 2 year study period, 4371 ß-haemolytic streptococci of Lancefield groups A, B, C and G were tested for antibiotic susceptibility; 1625 were group A, 1233 group B, 479 group C and 1034 group G. The prevalence of erythromycin resistance was highest in group G (6.2%, 64 of 1034), followed by groups B (4.3%, 53 of 1233) and C (3.8%, 18 of 479). The lowest prevalence of erythromycin resistance was 1.9% in group A (31 of 1625). The numbers of isolates available in each group for further study were 26 (group A), 42 (group B), 9 (group C) and 52 (group G). The distribution of erythromycin resistance phenotypes, M or MLSB, in the available isolates of all Lancefield groups studied is shown in Table 2. The MLSB phenotype predominated in groups A (57.7%, 15 of 26), B (88.1%, 37 of 42) and G (90.4%, 47 of 52). The M phenotype was identified in all Lancefield groups studied but was most frequent in groups C (77.8%, 7 of 9) and A (42.3%, 11 of 26). Among isolates with the MLSB phenotype, cMLSB resistance was predominant in groups A (60.0%, 9 of 15), B (59.5%, 22 of 37) and C (100%, 2 of 2). In contrast, the majority of MLSB isolates in group G (85.1%, 40 of 47) were iMLSB (Table 2).
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Erythromycin resistance genes in ß-haemolytic streptococci of Lancefield groups A, B, C and G and evidence for a new element that carries mef and msr(D) variant sequences in group G
The majority of cMLSB isolates in group A (88.9%, 8 of 9) and group B (86.4%, 19 of 22) carried the methylase gene erm(B). In all Lancefield groups studied, iMLSB isolates carried the erm(TR) subclass gene, with the exception of two isolates that did not produce amplification products in PCR reactions for any of the erythromycin resistance genes tested (Table 2).
M phenotype isolates of all Lancefield groups were mef positive by PCR, except for one isolate of group C that did not produce amplification products in PCR reactions for any of the erythromycin resistance genes tested. All mef amplicons were sequenced to reveal whether isolates carried the mef(A) sequence, originally described in S. pyogenes (GenBank accession number U70055 [GenBank] ), or the mef(E) sequence, originally described in S. pneumoniae (GenBank accession number U83667 [GenBank] ). All M phenotype isolates of group A streptococci (11 isolates) carried the mef(A) sequence and all M phenotype isolates of group B (5 isolates) carried the mef(E) sequence (Table 2). In group C, five out of seven M phenotype isolates carried mef(E) and one carried mef(A). One MLSB isolate of group C carried mef(E) in addition to erm(B) (Table 2).
Of the five M phenotype isolates in group G, one carried mef(A) and another one carried mef(E). The remaining three isolates carried a mef sequence which was 88.0 and 89.7% identical to mef(A) and mef(E) respectively. The complete sequence of the allelic mef variant found in group G streptococci was submitted to GenBank with accession number AJ617704. The variant mef gene found in this study showed 99.75% identity with a sequence recently reported in an M phenotype group G streptococci isolate in Hong Kong29 (GenBank accession number AY355405 [GenBank] ). The three group G isolates that carried the new mef sequence also carried a variant msr(D) sequence which was 91.6 and 91.5% identical to the sequence of the corresponding gene found in the genetic elements that carry mef(A) and mef(E) respectively. The msr(D) sequences in mef(A) and mef(E) elements are 98% identical to each other. The complete sequence of the variant msr(D) gene has been submitted to GenBank with accession number AM084232.
Since other mef-containing elements in S. pyogenes have been shown to be inserted in the comEC gene,8,9,11 long PCR reactions with primers that targeted the comEC and mef genes were performed in group G isolates (Table 1). Amplification products were obtained in the three isolates with the variant mef and in the isolate with mef(A), but no products were obtained in the isolate with mef(E). Sequencing with the amplification primers confirmed that the genetic element that carried the new mef and msr(D) sequences in group G isolates was inserted downstream from the adenine in position 1679 of the comEC gene (see GenBank AM168138 [GenBank] ). Similar results were obtained with the group G isolate that carried the original mef(A) sequence (data not shown). The amplification fragment from comEC to mef was fully sequenced in one of the three isolates that carried the variant mef and msr(D) genes (Table 1). A 6374 bp sequence which encompasses the left junction of the new element inserted in comEC, together with orfs 1–3, mef, msr(D) and a partial sequence of orf6 has been submitted to GenBank with accession number AM168138 [GenBank] . In all Lancefield groups studied, isolates that carried the original mef(A) or mef(E) sequences also carried the msr(D) sequence previously associated with their corresponding genetic element.7,12
Erythromycin MICs for ß-haemolytic streptococci of Lancefield groups A, B, C and G
The erythromycin MICs for isolates with different resistance genes are shown by Lancefield group in Table 3. In all Lancefield groups investigated, isolates that carried the erm(B) gene had high erythromycin MIC values (>256 mg/L). Isolates that carried the erm(TR) methylase had a much wider range of MICs (Table 3) and these were distributed bimodally (Figure 1). The erm(TR) isolates with high MICs comprised both cMLSB (2 isolates of group B and 3 of group G) and iMLSB phenotypes (1 isolate of groups A and B and 8 of group G). M phenotype isolates that carried mef genes had erythromycin MICs similar to the lower range of erm(TR) MICs (Table 3; Figure 1).
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Our results show that the predominant mechanism of macrolide resistance varies considerably amongst different streptococcal Lancefield groups in the same geographical area. Thus the M phenotype, which we have previously shown to be the most important form of macrolide resistance in pneumococci in our area (76%),17 was numerically significant only in group A streptococci, where it accounted for 42.3% of resistance. This is comparable to reports of 46.7% M phenotype resistance in group A streptococci in Boston, USA19 and 46.6%36 and 52.5%37 in Italy. There are contrasting figures for the contribution of the M phenotype to erythromycin resistance in group A streptococci in other parts of Europe, with reports of 90% in Spain38 and 93.9% in Germany,39 whereas in France the MLSB phenotype is responsible for 73.6% of resistance.18 The mef(A) gene subclass mef(A), originally described in group A streptococci,5 was found in all our M phenotype group A isolates. Most studies of macrolide resistance in group A have detected the presence of the mef(A) gene by PCR, but few have confirmed the sequence of the PCR products. We are aware of only one report of the mef(E) sequence in a group A isolate40 and in our study group A was the only group in which M phenotype isolates contained no mef sequences other than mef(A).
In contrast to group A streptococci, all our M phenotype isolates of group B carried the subclass mef(E) sequence originally described in S. pneumoniae.6 Arpin et al.41 also reported mef(E) to be predominant in group B isolates in France and subclass mef(A) was only found in 1 of 18 M phenotype isolates. The contribution of M phenotype to resistance in our group B isolates (11.9%) was comparable to that of 12.5% in Canada23 but lower than the 35% found in blood isolates in the USA.25 Reports from other parts of Europe show the contribution of M phenotype to resistance in group B to be <10%.22,24,26 In this study, the M phenotype was the predominant resistance mechanism in group C (66.6%, 6 of 9), lower than the 95% (20 of 21) reported in Finland.27 The mef(E) subclass sequence was more common in this group.
Kataja et al.27 found that 31 of 32 erythromycin-resistant group G isolates collected in Finland between 1992 and 1995 were of the MLSB phenotype, but they did not characterize the remaining isolate. Our finding of five M phenotypes amongst 52 erythromycin-resistant isolates is, to our knowledge, the first confirmed report of M phenotype resistance in group G streptococci in Europe. Small numbers of M phenotype group G isolates have recently been reported in Hong Kong29 and in South Korea.21 We have confirmed in three of our five isolates a mef sequence distinct from mef(A) or mef(E) but closely related to a sequence previously described in Hong Kong isolates by Woo et al.29 In association with this mef sequence, we have described a new msr(D) sequence which is distinct from the variants of this gene found in the previously described elements that carry mef(A) or mef(E). The exact role of the msr(D) gene in macrolide resistance is not yet clear, but there is evidence that it does contribute to resistance independently of mef.16 We have also shown that the element that carries the new mef and msr(D) sequences in group G streptococci is inserted in the competence gene comEC, similar to elements that carry mef(A) in S. pyogenes.8,9 In S. pneumoniae the mef(A) element Tn1207.1 is also inserted in a competence gene, celB.
All iMLSB isolates in our collection carried the erm(TR) subclass gene and the association between inducibility and erm(TR) has been noted before.37 The erm(TR) gene, and therefore inducibility, was much more prevalent in group G streptococci (82.7%) than in other groups (A, 26.7%; B, 38.1%; C, 0%). The observation that MICs were bimodal in isolates of groups A, B and G that carried erm(TR) is interesting and suggests either differences in the expression of erm(TR) or alternatively the presence in high MIC isolates of another co-existing resistance mechanism. Giovanetti et al.37,42 found evidence of a hitherto unrecognized efflux mechanism in iMLSB isolates of group A streptococci that carried erm(TR) and had high erythromycin MICs. The bimodal distribution of MICs for erm(TR)-containing isolates in our survey could reflect the existence of a similar mechanism in isolates with high MICs and further work is required to clarify this. Doktor and Shortridge43 have recently correlated differences in the upstream attenuator region of erm(TR) with macrolide resistance in S. pyogenes. No mef gene was detected in our erm(TR) isolates with high MICs and other resistance mechanisms which have been demonstrated in streptococci such as mutations in genes for 23S rRNA or ribosomal proteins44 were not sought in the current study.
We have previously shown that the mef(A) gene makes an important contribution to erythromycin resistance in S. pneumoniae isolates in clinical isolates from our diagnostic laboratory17 and in the present study we set out to determine whether this gene was also spreading through different Lancefield groups of ß-haemolytic streptococci. Our data show that, not only is the distribution of M and MLSB phenotypes different in different Lancefield groups, the erm and mef gene sequences associated with these phenotypes differ in different groups. Thus it appears that clonal expansion within Lancefield groups may make a bigger contribution to the evolution of macrolide resistance in ß-haemolytic streptococci than horizontal transfer of resistance genes between groups. The recent emergence in Hong Kong, and now in the UK, of M phenotype strains of group G streptococci that carry new mef and msr(D) sequences indicates that macrolide resistance amongst streptococci continues to evolve and continued surveillance is required to ensure that this process is monitored.
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None to declare.
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Acknowledgements |
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We acknowledge the technical assistance of Linda Ford and Julia Skinner. We are also grateful to our colleagues in the diagnostic section of our laboratory for their collaboration. We especially thank David Stuart for extensive searches of the diagnostic laboratory database. M. R. A. was supported by funds from the School of Medicine, University of Aberdeen.
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References |
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