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Antimicrobial Agents and Chemotherapy, December 2002, p. 3750-3755, Vol. 46, No. 12
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.12.3750-3755.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Novel Efflux System in Inducibly Erythromycin-Resistant Strains of Streptococcus pyogenes

Eleonora Giovanetti, Andrea Brenciani, Roberto Burioni, and Pietro Emanuele Varaldo*

Department of Microbiology and Biomedical Sciences, University of Ancona Medical School, 60131 Ancona, Italy

Received 17 June 2002/ Returned for modification 24 July 2002/ Accepted 20 August 2002

Streptococcus pyogenes strains inducibly resistant (iMLS phenotype) to macrolide, lincosamide, and streptogramin B (MLS) antibiotics can be subdivided into three phenotypes: iMLS-A, iMLS-B, and iMLS-C. This study focused on inducibly erythromycin-resistant S. pyogenes strains of the iMLS-B and iMLS-C types, which are very similar and virtually indistinguishable in a number of phenotypic and genotypic features but differ clearly in their degree of resistance to MLS antibiotics (high in the iMLS-B type and low in the iMLS-C type). As expected, the iMLS-B and iMLS-C test strains had the erm(A) methylase gene; the iMLS-A and the constitutively resistant (cMLS) isolates had the erm(B) methylase gene; and a control M isolate had the mef(A) efflux gene. mre(A) and msr(A), i.e., other macrolide efflux genes described in gram-positive cocci, were not detected in any test strain. With a radiolabeled erythromycin method for determination of the intracellular accumulation of the drug in the absence or presence of an efflux pump inhibitor, active efflux of erythromycin was observed in the iMLS-B isolates as well as in the M isolate, whereas no efflux was demonstrated in the iMLS-C isolates. By the triple-disk (erythromycin plus clindamycin and josamycin) test, performed both in normal test medium and in the same medium supplemented with the efflux pump inhibitor, under the latter conditions iMLS-B and iMLS-C strains were no longer distinguishable, all exhibiting an iMLS-C phenotype. In conjugation experiments with an iMLS-B isolate as the donor and a Rifr Fusr derivative of an iMLS-C isolate as the recipient, transconjugants which shared the iMLS-B type of the donor under all respects, including the presence of an efflux pump, were obtained. These results indicate the existence of a novel, transferable efflux system, not associated with mef(A) or with other known macrolide efflux genes, that is peculiar to iMLS-B strains. Whereas the low-level resistance of iMLS-C strains to MLS antibiotics is apparently due to erm(A)-encoded methylase activity, the high-level resistance of iMLS-B strains appears to depend on the same methylase activity plus the new efflux system.

* Corresponding author. Mailing address: Department of Microbiology and Biomedical Sciences, University of Ancona Medical School, Via Ranieri, Monte d'Ago, 60131 Ancona, Italy. Phone: 39 071 2204694. Fax: 39 071 2204693. E-mail: pe.varaldo{at}unian.it.

Antimicrobial Agents and Chemotherapy, December 2002, p. 3750-3755, Vol. 46, No. 12
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.12.3750-3755.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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