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Antimicrobial Agents and Chemotherapy, February 2006, p. 817-818, Vol. 50, No. 2
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.2.817-818.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Streptococcus pyogenes Isolates with High-Level Macrolide Resistance and Reduced Susceptibility to Telithromycin Associated with 23S rRNA Mutations

David J. Farrell,¹ Jemma Shackcloth,¹ Karen A. Barbadora,² and Michael D. Green^2*

GR Micro Limited, London, United Kingdom,¹ Division of Pediatric Allergy, Immunology, and Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania²

Received 31 August 2005/ Returned for modification 28 September 2005/ Accepted 9 November 2005

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Seven high-level macrolide-resistant Streptococcus pyogenes isolates had reduced activity to telithromycin but were negative for methylation and efflux genes. All were of the constitutive phenotype, were clonally related (emm type 12 and MLST type 36), and had identical dual mutations (A2058G and U2166C) in domain V of 23S rRNA.

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Macrolide resistance in isolates of Streptococcus pyogenes, or group A streptococcus (GAS), collected from across the United States is around 6% and stable (1). However, in southwestern Pennsylvania, a sudden increase (3.7 to 35%) found during a two-month period in 2002 has raised concern that macrolide-resistant GAS may be more common than is obvious from overall yearly surveillance data (5). Telithromycin is the first ketolide, a new class of antibacterial agents, and has been reported to be active against macrolide-resistant GAS expressing the efflux gene mef(A) and the inducible methylase gene erm(A) subclass erm(TR) but not active against most isolates expressing the methylase gene erm(B) (3).

In a recent (September 2002 to May 2003) large GAS surveillance study, a total of 2,797 isolates of GAS were collected from nine sites in the United States (6). Seven GAS isolates with high-level macrolide resistance had reduced susceptibility to telithromycin but were negative for mef(A), erm(A) subclass erm(TR), and erm(B). The seven isolates were sent to GR Micro Limited (London, United Kingdom) for the determination of erm(B), mef(A), and erm(A) subclass erm(TR) status by the method of Shackcloth et al. (8).

At GR Micro Limited, oligonucleotide primers for regions of interest in the six copies of the gene encoding 23S rRNA were designed from the complete S. pyogenes genome and are shown in Table 1. Forward primer SPY23SF is common to all six alleles. Long PCR for a 4-kb region of each of the six 23S gene copies was carried out in a 50-µl reaction mix containing 1 µM of each primer, 1 µl Elongase (Invitrogen), 200 µM deoxynucleoside triphosphates, 2% dimethyl sulfoxide, and 5 µl of extracted DNA template. Amplification conditions were 2 min of denaturation at 94°C, 15 cycles of 30 s at 92°C, hybridization at 48°C for 30 s, and an extension step of 70°C for 5 min, followed by 18 similar cycles where the extension time was increased by 15 s every cycle to a final extension time of 9.5 min.

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TABLE 1. 23S rRNA amplification and sequencing oligonucleotides for S. pyogenes

Subsequently, nested PCR for the domain II and domain V regions of the 23S gene was carried out using primers common to all six gene copies (Table 1). The reaction mixture (25 µl) contained 1 µM of each primer, 1 U platinum Taq (Invitrogen), 200 µM deoxynucleoside triphosphates, 2.5 mM MgCl₂, and 5 µl long PCR product diluted 1:100 in water. Amplification conditions were 94°C for 2 min, followed by 25 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 7 min.

After purification using shrimp alkaline phosphatase-exonuclease I treatment, PCR products were sequenced with an ABI Big Dye Terminator (version 3.1) cycle sequencing ready reaction kit and an ABI PRISM 3100 sequencer according to the manufacturer's instructions. PCR products were sequenced using four primers as described in Table 1. Investigation of L4 and L22 riboprotein gene mutations was performed as previously described (4).

The genetic relatedness of the isolates was investigated by field inversion gel electrophoresis (FIGE) after the digestion of bacterial DNA prepared in agarose plugs by ApaI using previously described methods (7). A comparison of banding patterns was performed by visual inspection, and the interpretation of clonal relatedness was based on guidelines proposed by Tenover et al. (9). The emm typing was determined by B. Beall at the Respiratory Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia. Types/subtypes were determined using the CDC database (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). Multilocus sequence typing (MLST) was performed as previously described (2). Sequence types were determined using the MLST database (http://www.mlst.net).

All seven isolates were negative for all macrolide resistance genes by all methods used. The date and site of isolation, erythromycin and telithromycin MICs, molecular epidemiological parameters, and 23S sequence mutations are shown in the Table 2. Mutations were identified in multiple copies of the 23S rRNA at A2058 (Escherichia coli numbering) and U2166 (S. pyogenes numbering corresponding to A2163 in E. coli) in each of the seven evaluated isolates.

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TABLE 2. Phenotypic and genotypic data for the seven strains of Streptococcus pyogenes described in this study

All seven isolates were designated as highly clonally related by emm typing, MLST, and similar 23S rRNA mutation patterns. By MLST convention, these isolates were designated clonal complex 36 (CC36). These seven isolates were recovered from four of the nine sites participating in the surveillance project. Three of these seven had different FIGE patterns (including two isolates from a single center) than the remaining four strains. Three of the seven isolates came from a single center; these isolates were recovered in October 2002, December 2002, and March 2003. Of interest, the last isolate recovered in March 2003 differed from the other two in FIGE pattern as well as in results of emm subtyping. If one applies the most strict definition of clonality possible from this study, that is that isolates are strongly related by all methods tested, the clone expressing FIGE pattern B, emm 12.0, MLST 36 with the identical 23S rRNA mutation patterns, isolates of this clone were recovered from two different centers between October 2002 and May 2003. Taken together, these results appear to support both limited spread of the resistant clone between centers and the emergence of the same resistant mutations in strains that differ from each other by at least two different molecular typing methods.

 
This publication made use of the Multi Locus Sequence Typing website (http://www.mlst.net) developed by Man-Suen Chan and David Aanensen and funded by the Wellcome Trust. Sanofi-Aventis is acknowledged for their financial support of the study.

 
* Corresponding author. Mailing address: Medical and Molecular Microbiology, GR Micro Limited, 7-9 William Road, London, NW1 3ER, United Kingdom. Phone: 44 (0)20 73887320. Fax: 44 (0) 20 73887324. E-mail: D.Farrell{at}grmicro.co.uk.

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2. Enright, M. C., B. G. Spratt, A. Kalia, J. H. Cross, and D. E. Bessen. 2001. Multilocus sequence typing of Streptococcus pyogenes and the relationships between emm type and clone. Infect. Immun. 69:2416-2427.[Abstract/Free Full Text]
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3. Farrell, D. J., I. Morrissey, S. Bakker, and D. Felmingham. 2002. Molecular characterisation of macrolide resistance mechanisms among Streptococcus pneumoniae and Streptococcus pyogenes isolated from the PROTEKT 1999-2000 study. J. Antimicrob. Chemother. 50(Suppl. S1):39-47.[Abstract]
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4. Farrell, D. J., S. Douthwaite, I. Morrissey, S. Bakker, J. Poehlsgaard, L. Jakobsen, and D. Felmingham. 2003. Macrolide resistance by ribosomal mutation in clinical isolates of Streptococcus pneumoniae from the PROTEKT 1999-2000 study. Antimicrob. Agents Chemother. 47:1777-1783.[Abstract/Free Full Text]
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5. Green, M., J. M. Martin, K. A. Barbadora, B. Beall, and E. R. Wald. 2004. Reemergence of macrolide resistance in pharyngeal isolates of group A streptococci in southwestern Pennsylvania. Antimicrob. Agents Chemother. 478:473-476.
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6. Green, M., C. Allen, J. Bradley, B. Dashefsky, J. R. Gilsdorf, M. J. Marcon, G. E. Schutze, C. Smith, E. Walter, J. M. Martin, K. A. Edwards, K. A. Barbadora, R. M. Rumbaugh, and E. R. Wald. 2005. In vitro activity of telithromycin in macrolide-susceptible and macrolide-resistant pharyngeal isolates of group A streptococci in the United States. Antimicrob. Agents Chemother. 49:2487-2489.[Abstract/Free Full Text]
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7. Martin, J. M., E. R. Wald, and M. Green. 1998. Field inversion gel electrophoresis as a typing system for group A streptococcus. J. Infect. Dis. 177:504-507.[Medline]
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8. Shackcloth, J., L. Williams, and D. J. Farrell. 2004. Streptococcus pneumoniae and Streptococcus pyogenes isolated from a paediatric population in Great Britain and Ireland: the in vitro activity of telithromcycin versus comparators. J. Infect. 48:229-235.[CrossRef][Medline]
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9. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.[Free Full Text]

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Antimicrobial Agents and Chemotherapy, February 2006, p. 817-818, Vol. 50, No. 2
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.2.817-818.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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