JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2008, p. 546–550 Vol. 46, No. 2 0095-1137/08/$08.000 doi:10.1128/JCM.01925-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved. Differences in Potential for Selection of Clindamycin-Resistant Mutants Between Inducible erm(A) and erm(C) Staphylococcus aureus Genes  Claire Daurel, 1,2 Corinne Huet, 2,3 Anne Dhalluin, 2,4 Miche `le Bes, 5 Jerome Etienne, 5 and Roland Leclercq 1,2 * Service de Microbiologie, CHU Coˆte de Nacre, 1 and EA 2128 Interactions hoˆte et microorganismes des e ´pithe ´liums 2 and UFR Sciences Pharmaceutiques, 4 Universite ´ de Caen Basse-Normandie, 14033 Caen cedex, CH Louis Pasteur, Cherbourg, 50100, 3 and INSERM U851, Centre National de Re ´fe ´rence des Staphylocoques, Faculte ´ Laennec, Universite ´ Lyon 1, 69008 Lyon, 5 France Received 28 September 2007/Returned for modification 17 November 2007/Accepted 2 December 2007 In staphylococci, inducible macrolide-lincosamide-streptogramin B (MLS B ) resistance is conferred by the erm(C) or erm(A) gene. This phenotype is characterized by the erythromycin-clindamycin “D-zone” test. Although clindamycin appears active in vitro, exposure of MLS B -inducible Staphylococcus aureus to this antibiotic may result in the selection of clindamycin-resistant mutants, either in vitro or in vivo. We have compared the frequencies of mutation to clindamycin resistance for 28 isolates of S. aureus inducibly resistant to erythromycin and bearing the erm(C) (n  18) or erm(A) (n  10) gene. Seven isolates susceptible to erythromycin or bearing the msr(A) gene (efflux) were used as controls. The frequencies of mutation to clindamycin resistance for the erm(A) isolates (mean  standard deviation, 3.4  10 8  2.4  10 8 ) were only slightly higher than those for the controls (1.1  10 8  6.4  10 9 ). By contrast, erm(C) isolates displayed a mean frequency of mutation to clindamycin resistance (4.7  10 7  5.5  10 7 ) 14-fold higher than that of the S. aureus isolates with erm(A). The difference was also observed, although to a lower extent, when erm(C) and erm(A) were cloned into S. aureus RN4220. We conclude that erm(C) and erm(A) have different genetic potentials for selection of clindamycin-resistant mutants. By the disk diffusion method, erm(C) and erm(A) isolates could be distinguished on the basis of high- and low-level resistance to oleandomycin, respectively. Clindamycin is an alternative drug for infections due to Staphylococcus aureus in case of intolerance to penicillins or resistance to methicillin. Furthermore, clindamycin represents an attractive option for several reasons. First, clindamycin is available in both intravenous and oral formulations. Second, the drug has a remarkable distribution into skin and skin struc- tures. Third, community-acquired methicillin-resistant S. aureus (CA-MRSA), which has rapidly emerged in recent years as a cause of skin and soft-tissue infections, is frequently suscepti- ble to several antibiotics, including clindamycin (12, 19). Fi- nally, it has been shown that clindamycin inhibits the produc- tion of toxins and virulence factors in gram-positive organisms through inhibition of protein synthesis (7). However, resistance to clindamycin, which is not rare, limits the use of this antibiotic in therapy. Two primary mechanisms result in resistance to macrolide antibiotics in staphylococci: macrolide efflux, controlled by the msr(A) gene, and modifi- cation of the drug-binding site on the ribosome, controlled by erm (erythromycin ribosome methylation) genes (9). The efflux mechanism yields inducible resistance to 14-membered (eryth- romycin, clarithromycin, roxithromycin) and 15-membered (azithromycin) macrolides and type B streptogramins but not to lincosamides (clindamycin and lincomycin). Ribosomal methylation confers cross-resistance to macrolides, lincos- amides, and type B streptogramins, the so-called MLS B phe- notype. In staphylococci, erm(A) or erm(C) is responsible for this cross-resistance phenotype by controlling the methylation of the 23S rRNA binding site of adenosine 2058 (A2058) (Escherichia coli numbering). Methylation results in impaired binding of the three drug classes that share this residue as a common binding site. MLS B resistance can be expressed either constitutively or inducibly. Strains with constitutive resistance express cross- resistance to MLS B antibiotics. Strains with inducible MLS B resistance (MLS B i) display in vitro resistance to 14- and 15- membered macrolides, which are inducer antibiotics, while appearing susceptible to lincosamides and type B strepto- gramins, which are not inducers. In the absence of an inducer, the inactivity of the mRNA is due to the structure of its 5 end, which comprises a leader peptide and a set of inverted repeats that form a hairpin structure which sequesters the initiation sequences (ribosome binding site and initiation codon) for the methylase by base pairing. According to the model of posttran- scriptional regulation, induction arises through binding of an inducer macrolide to a ribosome during translation of the leader peptide, leading to destabilization of the hairpin struc- ture, exposure of the initiation sequences to the ribosome, and translation of the Erm methylase (2). Although clindamycin is not an inducer, exposure of MLS B - inducible S. aureus to this antibiotic may result in cross-MLS B resistance, either in vitro or in vivo. This is due to the selection of preexisting constitutive erm mutants (4, 6, 17). Whether in laboratory strains or in clinical isolates, deletion of the entire attenuator, point mutations, or tandem duplications in the attenuator yield constitutive expression of resistance by de- * Corresponding author. Mailing address: CHU de Caen, Service de Microbiologie, Avenue Coˆte de Nacre, 14033 Caen Cedex, France. Phone: (33) 02 31 06 45 72. Fax: (33) 02 31 06 45 73. E-mail: leclercq-r @chu-caen.fr.  Published ahead of print on 12 December 2007. 546 on May 31, 2015 by guest http://jcm.asm.org/ Downloaded from