JAC Advance Access originally published online on March 13, 2003
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Journal of Antimicrobial Chemotherapy (2003) 51, 1039-1041
© 2003 The British Society for Antimicrobial Chemotherapy
Correspondence |
Introduction of erm(C) into a linezolid- and methicillin-resistant Staphylococcus aureus does not restore linezolid susceptibility
1 Infectious Diseases–Crystal Run Healthcare, 155 Crystal Run Road, Middletown, NY 10941; Departments of 2 Medicine and 4 Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215; 3 Harvard Medical School, Boston, MA 02115; 5 Departments of Pathology and Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
Keywords: linezolid, macrolides, resistance, Staphylococcus aureus
Sir,
Oxazolidinones are a new class of compounds with activity against resistant Gram-positive organisms such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus spp. (VRE). However, resistance to linezolid, the only oxazolidinone available for clinical use, has emerged in enterococci1 as well as MRSA.2,3 In both reports of linezolid-resistant MRSA published to date, resistance was due to a mutation in the 23S rRNA at position 2576 in the peptidyl transferase centre of domain V.2,3 We found it interesting that both reported linezolid-resistant MRSA demonstrated susceptibility to erythromycin. Howe et al.4 observed that strains selected by passage in vitro sometimes lost resistance to erythromycin. Our study of serial isolates that were indistinguishable by pulsed-field gel electrophoresis (PFGE) obtained from a patient with prolonged MRSA bacteraemia showed that the loss of erythromycin resistance conferred by constitutive ribosomal methyltransferase activity occurred coincidentally with an increase in linezolid MIC from 0.5 to 2 mg/L (G. Sakoulas, H. S. Gold, R. C. Moellering, Jr & G. M. Eliopoulos, unpublished observation). We hypothesized that mutations conferring linezolid resistance might be incompatible with expression of macrolide resistance due to methylation at A2058. Therefore, we undertook a study to determine whether introduction of a methyltransferase gene into a linezolid-resistant MRSA might restore susceptibility to linezolid.
The absence of ribosomal methyltransferase erm genes conferring macrolide–lincosamide–streptogramin B (MLSB) resistance within S. aureus A7819, one of the linezolid-resistant MRSA isolates (MIC > 32 mg/L) that we recently characterized,2 was confirmed using PCR with degenerate erm primers according to published methods.5 S. aureus A7819 was made electrocompetent using ice-cold 10% glycerol. Plasmid pE194 containing inducible erm(C) was purified from S. aureus RN2442 (Wizard Plus Miniprep; Promega, Madison, WI, USA) and introduced via electroporation into S. aureus A7819 (100 
, 25 µF, 2.3 kV, time constant 2 s). Transformed cells were selected on brain–heart infusion agar plates containing erythromycin 20 mg/L. Authenticity of the clones derived from S. aureus A7819 was verified by PFGE. Inducible MLSB resistance was confirmed by measuring zone sizes around erythromycin and clindamycin discs on Mueller–Hinton agar plates and on plates supplemented with erythromycin 0.1 mg/L. The presence of erm(C) in the transformants was confirmed by PCR as above.5
Using NCCLS methods6 we determined MICs of linezolid on agar. We also determined MICs of linezolid in agar with erythromycin 0.1 mg/L to study the effect of methyltransferase induction on MIC of linezolid. Growth curves were performed in antibiotic-free Mueller–Hinton (II) broth (MHB), and MHB containing linezolid 10 mg/L or erythromycin 0.1 mg/L, or both. Twenty millilitre cultures were maintained in 250 mL Erlenmeyer flasks incubated at 35°C without agitation.
For six erythromycin-resistant transformants [erm(C)] of S. aureus A7819, growth in the presence or absence of an inducing concentration of erythromycin did not restore susceptibility to linezolid (MICs remained >32 mg/L). Growth curves showed that the erm(C) transformants did not demonstrate impaired growth in the presence of linezolid 10 mg/L and an inducing concentration of erythromycin (Figure 1).
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Our central observation is that linezolid resistance mediated by the G2576U 23S rRNA mutation in S. aureus is not incompatible with macrolide resistance due to methylation. We felt that exploring this possibility was important because incompatibility of these resistance mechanisms might have provided clinicians the option of concomitant administration of a macrolide with linezolid to prevent the development of oxazolidinone resistance. Furthermore, if resistance to oxazolidinones required loss of methyltransferase-mediated macrolide resistance, linezolid-resistant S. aureus isolates might have been rendered more vulnerable to killing by quinupristin–dalfopristin.
Despite these negative findings, we believe that additional study of the relationship between macrolide resistance and linezolid resistance among clinical isolates of staphylococci is warranted. The possible interaction of macrolide resistance mechanisms with ribosomal mutations conferring oxazolidinone resistance will be a subject of interesting future investigation.
Acknowledgements
We are grateful to Dr Richard P. Novick (Skirball Institute of Biomolecular Medicine, New York, NY, USA) for providing us with S. aureus RN2442 and to Dr Steve Brecher (West Roxbury VA Medical Center, West Roxbury, MA, USA) for providing the serial MRSA clinical isolates.
Footnotes
* Corresponding author. Tel: +1-845-703-6999 ext. 4233; Fax: +1-845-361-1156; E-mail: gsakoulas{at}crystalrunhealthcare.com 
References
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Prystowsky, J., Siddiqui, F., Chosay, J., Shinabarger, D. L., Millichap, J., Peterson, L. R. et al. (2001). Resistance to linezolid: characterization of mutations in rRNA and comparison of their occurrences in vancomycin-resistant enterococci. Antimicrobial Agents and Chemotherapy 45, 2154–6.
2 . Tsiodras, S., Gold, H. S., Sakoulas, G., Eliopoulos, G. M., Moellering, R. C., Jr & Ferraro, M. J. (2001). Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet 358, 207–8.[CrossRef][Web of Science][Medline]
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4 . Howe, R. A., Noel, A., Bowker, K. E., Enne, V. I., Walsh, T. R. & MacGowan, A. P. (2002). Emergence of linezolid (LIN) resistance in Staphylococcus aureus can be associated with loss of erythromycin (ERY) resistance. In Program and Abstracts of the Forty-second Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, USA, 2002. Abstract C1-1608, p. 75. American Society for Microbiology, Washington, DC, USA.
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6 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Testing for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
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