GR Micro Limited, 79 William Road, London NW1 3ER, UK
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Abstract |
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Introduction |
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Macrolide resistance in streptococci occurs predominantly by two mechanisms: target modification or efflux. In target modification, a specific adenine residue on the 23S rRNA (A2058; Escherichia coli numbering) is methylated by rRNA erm methylase [erm(B) or erm(A) subclass erm(TR)].2 We have used the designation erm(A) subclass erm(TR) to distinguish between erm(A) and this gene, previously known as erm(TR) before the recent nomenclature changes.3 This methylation is thought to lead to a conformational change in the ribosome resulting in decreased binding of macrolide, lincosamide and streptogramin B antimicrobials (MLSB phenotype).3 erm(A) (TR variant), although common in S. pyogenes, has only recently been reported in S. pneumoniae.4 erm(C), encoding a third methylase, has only been reported in Staphylococcus aureus. Target modification may also occur by base substitutions in 23S rRNA and genes that encode the L4 and L22 riboproteins.5 The phenotype of some of these mutations may be identical to those seen with methylases. On the other hand, a mutation at a different site on the 23S rRNA can result in a new phenotype. For example, a recently described 23S rRNA A2062C mutation produced resistance to the 16-membered macrolides and streptogramin B only.6
The mef(A) gene product for both S. pneumoniae and S. pyogenes mediates macrolide efflux. Typically, isolates with mef(A) have low macrolide MICs (e.g. 24 mg/L for S. pneumoniae and 18 mg/L for S. pyogenes) and retain susceptibility to clindamycin (the so-called M-phenotype),7 although erythromycin MICs of up to 32 mg/L have been reported.8 Constitutive MLSB resistance due to methylases in both species ranges from 16 to >512 mg/L, while inducible MLSB resistance ranges from 2 to 512 mg/L in S. pneumoniae7 and from 1 to 64 mg/L in S. pyogenes.9 The acquisition of both a methylase and an efflux mechanism in the same strain has been described for both S. pyogenes10 and S. pneumoniae.11 As a result of overlapping MIC ranges, extrapolation of genotype from phenotype, therefore, may not always be reliable, and the detection of dual and/or unknown mechanisms may be masked by a predominant phenotype.
Sutcliffe et al.12 have developed a multiplex PCR system using agarose gel analysis for the detection of macrolide resistance mechanisms in these two species. However, gel detection is laborious and therefore not amenable to rapid screening of large numbers of isolates. In addition, the use of a probe detection system is, in general, more specific than band sizing in agarose. The aim of our study was to develop a rapid, accurate, standardized multiplex PCR assay with microwell-format oligonucleotide probe detection, to support large-scale screening of isolates obtained from surveillance studies.
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Materials and methods |
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Control strains.
The control strains used in this study were: RN1389 [S. aureus, erm(A)], 02C1061 [S. pyogenes, erm(B)], JH2-2 [Enterococcus faecalis, erm(B)], RN4220 [S. aureus, erm(C)], 02C1110 [S. pyogenes, erm(A) subclass erm(TR)], 02J1175 [S. pneumoniae, mef(A)]. These strains were kindly provided by Dr Joyce Sutcliffe, Pfizer Global Research and Development, Groton, CT, USA.
S. pneumoniae.
Eighty-three clinical isolates of macrolide-resistant S. pneumoniae were tested. These isolates were obtained from patients with community-acquired lower respiratory tract infection from 27 centres geographically spread throughout Great Britain and Ireland during the 19971998 cold season (November to April).
S. pyogenes.
Fifty-six clinical isolates of macrolide-resistant S. pyogenes were tested. These isolates were pathogens from various anatomical sites (e.g. throat, blood, skin and soft tissue), collected from 25 geographically separated centres in Great Britain and Ireland in 1999.
Preparation of isolates for testing
All isolates were subcultured from storage (heavy inoculum in horse serum at 70°C) on to horse blood agar overnight at 35°C in 5% CO2. A 1 µL plastic disposable loop was used to sample the area of confluent growth on the plate. The loopful of organism was transferred to a sterile 0.5 mL tube containing 100 µL of nuclease-free H2O (Sigma, Poole, UK). Each tube was capped and vortexed well before being heated at 99°C for 5 min. After incubation, tubes were centrifuged using a Genofuge 16M (Techne, Cambridge, UK) at 11 300g for 3 min. For the two S. aureus control strains (RN1389 and RN4220), a 0.5 McFarland suspension of the isolate was prepared, followed by centrifugation (11 300g for 3 min) of 1 mL of the suspension. The supernatant was discarded and the pellet resuspended in 100 µL of cell lysis solution [250 U lysozyme/mL, 25 U lysostaphin, 10 mM EDTA, 10 mM Tris (pH 8.0)]. After incubation for 30 min at 37°C, the specimens were boiled for 10 min and then diluted by adding 900 µL of RNase- and DNase-free sterile distilled water (Sigma). Prepared specimens were used immediately for testing.
For the investigation of strains containing double mechanisms, no mechanisms or when the genotype did not match the phenotype, five single colonies were subcultured and tested individually to reconfirm genotype.
PCR methods
Oligonucleotides for the macrolide rapid cycle multiplex PCR with probe detection were designed using GenBank sequence data (accession numbers Y17294, U70055, U83667, AF002716, X52632, U83557) and are listed in the Table. They were synthesized commercially by Interactiva (Ulm, Germany). For the PCR, 10 µL of treated specimen was added to 40 µL of a master mix (made immediately before use) containing 5 µL of 10x PCR buffer, 200 mM of each nucleotide (dATP, dCTP, dGTP, dTTP) (Hybaid, Ashford, UK), 25 pmol of each primer (see Table
), 2.0 U Platinum Taq polymerase (Life Technologies, Paisley, UK) per reaction and 1.5 mM MgCl2 (Life Technologies). Amplification was carried out in a Perkin-Elmer 9700 GeneAmp thermal cycler (Applied Biosystems, Warrington, UK) with the following parameters: 2 min at 95°C followed by 37 cycles of: 94°C 15 s, 52°C 15 s, 72°C 15 s, with the last cycle concluding with a reaction for 7 min at 72°C.
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Gel-based detection of PCR products.
The gel-based multiplex PCR was a modification of a previously described method.12 Modifications were as follows: a universal bacterial 16S rRNA PCR was included in each multiplex, where the 241 bp product was used to confirm amplification and, therefore, presence of bacterial DNA. Primers used were as described previously.13 The bacterial 16S primers (5'-GGAGGAAGGTGGGGATGACG-3' and 5'-ATG GTGTGACGGGCGGTGTG-3') were added to each multiplex reaction at a concentration of 1 pmol per reaction.2 The genes detected in each multiplex were changed to reflect the expected genotype. Multiplex I targeted erm(B), mef(A) and 16S rRNA. Multiplex II targeted erm(A), erm(C) and 16S rRNA. Multiplex III targeted erm(A) subclass erm(TR) and 16S rRNA.
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Results and discussion |
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All negative controls and wells containing probes for non-target genes (excluding the exception above) produced absorbance values (A450/620) of <0.20. All positive results produced absorbance values (A450/620) >0.5. Based on this information, which was consistent after five separate assays, a negative cut-off was set at (A450/620) 0.2, and a positive cut-off at (A450/620) 0.5. Results >0.2 but <0.5 were defined as equivocal and the test repeated.
For the 83 clinical isolates of macrolide-resistant S. pneumoniae tested, 51 (61.4%) were positive for mef(A), 29 (34.9%) for erm(B), two (2.4%) for double mechanisms mef(A) + erm(B) and one (1.2%) was negative for all mechanisms tested. Data from all but two of the isolates tested using the probe method were in agreement with the modified reference PCR method (97.6% agreement).12 The two discrepant strains were the two strains with combined mef(A) and erm(B) mechanisms. With these two strains only erm(B) and not mef(A) was detected by the gel detection method. Therefore, the probe detection method was better able to characterize isolates with two mechanisms of macrolide resistance than the modified gel detection method.
For the 56 clinical isolates of macrolide-resistant S. pyogenes tested, 33 (58.9%) were positive for erm(A) subclass erm(TR), 18 (32.1%) for mef(A) and five (8.9%) for erm(B). One hundred per cent agreement was obtained with the modified reference PCR method.12
The method reported in this study is a rapid cycle (c. 100 strains per day can be genotyped) multiplex PCR (amplifies all five genes in the same reaction) with a probe/ microwell detection format that has been purpose designed to perform standardized testing of large numbers of isolates. In addition, the use of an oligonucleotide probe adds specificity to the detection, which increases the reliability of the results. The method was validated against a modification of the method of Sutcliffe et al.12 and a high correlation was observed using clinical isolates of macrolideresistant S. pneumoniae (97.6%) and S. pyogenes (100%). The probe detection method was better able to characterize isolates with two mechanisms of macrolide resistance than the modified gel method. This assay should allow cost-effective, rapid, high-throughput and standardized detection of macrolide resistance mechanisms in S. pneumoniae and S. pyogenes for use in large global surveillance studies that are likely to result in high numbers (typically thousands) of macrolide-resistant strains being isolated.
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Acknowledgements |
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Notes |
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References |
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Received 29 May 2001; returned 2 July 2001; revised 16 July 2001; accepted 25 July 2001