Journal of Antimicrobial Chemotherapy (2001) 48, 541-544
© 2001 The British Society for Antimicrobial Chemotherapy
Brief report |
Detection of macrolide resistance mechanisms in Streptococcus pneumoniae and Streptococcus pyogenes using a multiplex rapid cycle PCR with microwell-format probe hybridization
GR Micro Limited, 7–9 William Road, London NW1 3ER, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
In this study, a multiplex rapid cycle PCR with microwell-format probe hybridization method was developed to perform high-volume screening for macrolide resistance determinants in isolates of Streptococcus pneumoniae and Streptococcus pyogenes. The method was then utilized to determine the distribution of macrolide resistance mechanisms in recent isolates of S. pneumoniae and S. pyogenes from Great Britain and Ireland. For 83 strains of macrolide resistant S. pneumoniae tested, 51 (61.4%) were positive for mef(A), 29 (34.9%) erm(B), two (2.4%) double mechanisms mef(A) + erm(B), and one (1.2%) negative for all mechanisms tested. For 56 strains of macrolide-resistant S. pyogenes tested, 33 (58.9%) were positive for erm(A) subclass erm(TR), 18 (32.1%) mef(A) and five (8.9%) erm(B).
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
Rapidly increasing antimicrobial resistance in Streptococcus pneumoniae and Streptococcus pyogenes is of major clinical concern. Global spread of resistance has been reported, spanning different classes of antimicrobials, principally the penicillins, cephalosporins and macrolides.1 The rapid emergence of ß-lactam-resistant S. pneumoniae has resulted in a clinical need for substitute antimicrobials, particularly the macrolides, to be used as empirical therapy. Penicillin resistance has not been described in S. pyogenes. However, in patients with ß-lactam hypersusceptibility, or treatment failure, macrolides are a suitable substitute. Macrolides also provide cover for the so-called atypical bacterial pathogens Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila.
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. 2–4 mg/L for S. pneumoniae and 1–8 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.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
Bacteria
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 1997–1998 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.
|
Probe-based detection of PCR products.. Detection of amplified products was carried out using a modification of the PCR DIG ELISA kit (Roche Diagnostics Ltd, Lewes, UK). Unless specified, the reagents described in the following methodology were those present in the PCR DIG ELISA kit. Twenty microlitres of denaturation solution (kit reagent 1a) was added to each streptavidin-coated microwell. Five microlitres of each amplified specimen was added to each of two wells. The microwell plate was gently mixed and allowed to sit at room temperature for 10 min. During this time, probes (20 nmol/mL) were freshly prepared for addition to the microwells. Probes (see Table
) were diluted 1/200 in hybridization buffer (kit reagent 2), gently mixed by inversion and placed in a 45°C incubator for at least 30 min before the next step. Two hundred microlitres of diluted probe were added to the appropriate microwells, the plate covered with a plastic plate cover and incubated at 45°C on a heating block for 30 min. During hybridization, fresh conjugate was prepared by diluting the anti-DIG-POD 1/100 with conjugate dilution buffer (kit reagent 4). At 30 min, the plate was washed four times using an automated EIA plate washer (Multiwash II; Jencons-PLS, Leighton Buzzard, UK) using PCR ELISA buffer. One hundred microlitres of the prepared conjugate was added to each well, the plate covered and incubated at 37°C for 30 min. The plate was then washed four times with the plate washer. One hundred microlitres of 3,3',5,5'-tetramethyl-benzidine (TMB) liquid substrate system for ELISA (Sigma) was added to each well. The reaction was stopped exactly 5 min after the addition of the substrate by adding 100 µL of 10% H2SO4. Absorbances [450 nm with a reference reading at 620 nm (A450/620)] using a Dynex MRX plate reader (Jencons-PLS) were read within 1 h after the reaction had been stopped. 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.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
A 100% correlation was observed between the in-house method and the modified reference method12 when testing the control strains. A cross reaction was observed between the probe for the erm(A) subclass erm(TR) gene and the erm(A) target, but the reverse did not occur, thus still allowing differentiation between the two genes when both probes were used. We feel that despite the fact that the two genes have been re-classified into the same designation recently,3 there is merit in reconsidering the distinction. First, as there is only 83% homology between the two genes' nucleotide sequence and an 81% homology in amino acid sequence,3 this readily allows both genes to be differentiated, as we have shown in this study. Secondly, this distinction has important epidemiological consequences because the two genes appear to be both genus and species specific. erm(A) has been found only in staphylococci,2 whereas erm(TR) is limited to S. pyogenes and has only recently been found in S. pneumoniae.4 Thirdly, if other researchers assume that all erm(A) genes are identical it is possible that the gene previously known as erm(TR) may be missed. As a compromise we have chosen to use the nomenclature erm(A) and erm(A) subclass erm(TR) to distinguish between the two genes in this paper.
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.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
We are grateful to Dr Joyce Sutcliffe, Pfizer Global Research and Development, Groton, CT, USA for providing the control strains used in this study.
|
Notes |
|---|
* Corresponding aurthor: Tel: +44-20-7388-7320; Fax: +44-20-7388-7324; E-mail: d.farrell{at}grmicro.co.uk
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
1 . Felmingham, D., Washington, J. & the Alexander Project Group. (1999). Trends in the antimicrobial susceptibility of bacterial respiratory tract pathogens—findings of the Alexander Project 1992–1996. Journal of Chemotherapy 11, Suppl. 1, 5–21.
2 . Weisblum, B. (1995). Erythromycin resistance by ribosome modification. Antimicrobial Agents and Chemotherapy 39, 577–85.[Web of Science][Medline]
3
.
Roberts, M. C., Sutcliffe, J., Courvalin, P., Bogo Jensen, L., Rood, J. & Seppala, H. (1999). Nomenclature for macrolide and macrolide-lincosamide-streptogrammin B resistance determinants. Antimicrobial Agents and Chemotherapy 43, 2823–30.
4
.
Syrogiannopoulos, G. A., Grivea, I. N., Tait-Kamradt, A., Katopodis, G. D., Beratis, N. G., Sutcliffe, J. et al. (2001). Identification of an erm(A) erythromycin resistance methylase gene in Streptococcus pneumoniae isolated in Greece. Antimicrobial Agents and Chemotherapy 45, 342–4.
5
.
Tait-Kamradt, A., Davies, T. A., Cronan, M., Jacobs, M. R., Appelbaum, P. C. & Sutcliffe, J. (2000). Mutations in 23S rRNA and ribosomal protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage. Antimicrobial Agents and Chemotherapy 44, 2118–25.
6
.
Depardieu, F. & Courvalin, P. (2001). Mutation in 23S rRNA responsible for resistance to 16-membered macrolides and streptogramins in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 45, 319–23.
7
.
Descheemaeker, P., Chapelle, S., Lammens, C., Hauchecorne, M., Wijdooghe, M., Vandamme, P. et al. (2000). Macrolide resistance and erythromycin resistance determinants among Belgian Streptococcus pyogenes and Streptococcus pneumoniae isolates. Journal of Antimicrobial Chemotherapy 45, 167–73.
8 . Hoban, D. J., Zhanel, G. G., Wierzbowski, A. & Karlowsky, J. A. (2000). Incidence of mef(A) and erm(B) among macrolide resistant Streptococcus pneumoniae (SPN) isolated in Canada during 1998 and 1999. In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 2000. Abstract 2149, p. 177. American Society for Microbiology, Washington, DC.
9
.
Kataja, J., Huovinen, P., Skurnik, M., The Finnish Study Group for Antimicrobial Resistance & Seppälä, H. (1999). Erythromycin resistance genes in group A Streptococci in Finland. Antimicrobial Agents and Chemotherapy 43, 48–52.
10
.
Bingen, E., Fitoussi, F., Doit, C., Cohen, R., Tanna, A., George, R. et al. (2000). Resistance to macrolides in Streptococcus pyogenes in France in pediatric patients. Antimicrobial Agents and Chemotherapy 44, 1453–7.
11 . Corso, A., Severina, E. P., Petruk, V. F., Mauriz, Y. R. & Tomasz, A. (1998). Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates causing respiratory disease in the United States. Microbial Drug Resistance 4, 325–37.
12 . Sutcliffe, J., Grebe, T., Tait-Kamradt, A. & Wondrack, L. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 2562–6.[Abstract]
13 . Martineau, F., Picard, F. J., Roy, P. H., Ouellette, M. & Bergeron, M. G. (1996). Species-specific and ubiquitous DNA-based assays for rapid identification of Staphylococcus aureus. Journal of Clinical Microbiology 34, 2888–93.[Abstract]
Received 29 May 2001; returned 2 July 2001; revised 16 July 2001; accepted 25 July 2001
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Blackman Northwood, M. Del Grosso, L. R. Cossins, M. D. Coley, R. Creti, A. Pantosti, and D. J. Farrell Characterization of Macrolide Efflux Pump mef Subclasses Detected in Clinical Isolates of Streptococcus pyogenes Isolated between 1999 and 2005 Antimicrob. Agents Chemother., May 1, 2009; 53(5): 1921 - 1925. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Farrell, T. M. File, and S. G. Jenkins Prevalence and Antibacterial Susceptibility of mef(A)-Positive Macrolide-Resistant Streptococcus pneumoniae over 4 Years (2000 to 2004) of the PROTEKT US Study J. Clin. Microbiol., February 1, 2007; 45(2): 290 - 293. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zeng, F. Kong, H. Wang, A. Darbar, and G. L. Gilbert Simultaneous Detection of Nine Antibiotic Resistance-Related Genes in Streptococcus agalactiae Using Multiplex PCR and Reverse Line Blot Hybridization Assay Antimicrob. Agents Chemother., January 1, 2006; 50(1): 204 - 209. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Davignon, E. A. Walter, K. M. Mueller, C. P. Barrozo, D. A. Stenger, B. Lin, and on behalf of the Epidemic Outbreak Surveillance Co Use of Resequencing Oligonucleotide Microarrays for Identification of Streptococcus pyogenes and Associated Antibiotic Resistance Determinants J. Clin. Microbiol., November 1, 2005; 43(11): 5690 - 5695. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. N. Twagira, J. J. Wade, I. Eltringham, and M. Smith Development of a real-time PCR assay on the Roche Light-Cycler for the detection of erm and mef erythromycin resistance genes in {beta}-haemolytic streptococci J. Antimicrob. Chemother., October 1, 2005; 56(4): 793 - 794. [Full Text] [PDF]
|
|
|
|
|
|
C. H. W. Klaassen and J. W. Mouton Molecular Detection of the Macrolide Efflux Gene: To Discriminate or Not To Discriminate between mef(A) and mef(E) Antimicrob. Agents Chemother., April 1, 2005; 49(4): 1271 - 1278. [Full Text] [PDF]
|
|
|
|
|
|
S. D. Brown, D. J. Farrell, and I. Morrissey Prevalence and Molecular Analysis of Macrolide and Fluoroquinolone Resistance among Isolates of Streptococcus pneumoniae Collected during the 2000-2001 PROTEKT US Study J. Clin. Microbiol., November 1, 2004; 42(11): 4980 - 4987. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D J Farrell, S G Jenkins, and R R Reinert Global distribution of Streptococcus pneumoniae serotypes isolated from paediatric patients during 1999-2000 and the in vitro efficacy of telithromycin and comparators J. Med. Microbiol., November 1, 2004; 53(11): 1109 - 1117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Farrell, I. Morrissey, S. Bakker, S. Buckridge, and D. Felmingham In Vitro Activities of Telithromycin, Linezolid, and Quinupristin-Dalfopristin against Streptococcus pneumoniae with Macrolide Resistance Due to Ribosomal Mutations Antimicrob. Agents Chemother., August 1, 2004; 48(8): 3169 - 3171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Villedieu, M. L. Diaz-Torres, A. P. Roberts, N. Hunt, R. McNab, D. A. Spratt, M. Wilson, and P. Mullany Genetic Basis of Erythromycin Resistance in Oral Bacteria Antimicrob. Agents Chemother., June 1, 2004; 48(6): 2298 - 2301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Farrell, I. Morrissey, S. Bakker, L. Morris, S. Buckridge, and D. Felmingham Molecular Epidemiology of Multiresistant Streptococcus pneumoniae with Both erm(B)- and mef(A)-Mediated Macrolide Resistance J. Clin. Microbiol., February 1, 2004; 42(2): 764 - 768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. N. Jones, D. J. Farrell, and I. Morrissey Quinupristin-Dalfopristin Resistance in Streptococcus pneumoniae: Novel L22 Ribosomal Protein Mutation in Two Clinical Isolates from the SENTRY Antimicrobial Surveillance Program Antimicrob. Agents Chemother., August 1, 2003; 47(8): 2696 - 2698. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Farrell, S. Douthwaite, I. Morrissey, S. Bakker, J. Poehlsgaard, L. Jakobsen, and D. Felmingham Macrolide Resistance by Ribosomal Mutation in Clinical Isolates of Streptococcus pneumoniae from the PROTEKT 1999-2000 Study Antimicrob. Agents Chemother., June 1, 2003; 47(6): 1777 - 1783. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zampaloni, P. Cappelletti, M. Prenna, L. A. Vitali, and S. Ripa emm Gene Distribution among Erythromycin-Resistant and -Susceptible Italian Isolates of Streptococcus pyogenes J. Clin. Microbiol., March 1, 2003; 41(3): 1307 - 1310. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Liu and S. Douthwaite Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy PNAS, November 12, 2002; 99(23): 14658 - 14663. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||