Reversion to susceptibility in a linezolid-resistant clinical isolate of Staphylococcus aureus

Venkata G. Meka1,*, Howard S. Gold1, Amy Cooke2, Lata Venkataraman1, George M. Eliopoulos1, Robert C. Moellering, Jr3 and Stephen G. Jenkins4

1 Division of Infectious Diseases,, and 3 Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA; 2 Department of Pharmacy, Carolinas Medical Center, Charlotte, NC; 4 Division of Microbiology and Immunology, Department of Pathology, Carolinas Medical Center, Charlotte, NC, USA

Received 23 June 2004; returned 1 July 2004; revised 9 August 2004; accepted 10 August 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: Linezolid resistance in rare isolates of Staphylococcus aureus has been associated with G2576T mutations in domain V of the 23S rRNA gene. We report the analysis of a clinical S. aureus isolate that developed linezolid resistance (MIC of linezolid of 12 mg/L) after a 25 day course of the drug. Sequencing identified G2576T mutations in four of the five copies of the 23S rRNA gene.

Methods: To examine the stability of this resistance, we serially passaged this original isolate 60 times over a 75 day period on antimicrobial-free medium.

Results: After 30 passages, the MIC of linezolid had decreased to 8 mg/L and only two of the five copies of the 23S rRNA gene contained the G2576T mutation. After 60 passages, the MIC of linezolid fell to 2 mg/L and only one of the five 23S rRNA gene copies contained the mutation. The original and two passaged staphylococci were indistinguishable by pulsed-field gel electrophoresis.

Conclusions: In the absence of antibiotic pressure, linezolid resistance was unstable in a clinical isolate that did not have all copies of the 23S rRNA gene mutated, although a single copy of mutant rDNA was maintained. Gene conversion was probably the mechanism for this reversion, using the wild-type 23S rRNA gene sequences to replace the G2576T mutation by homologous recombination.

Keywords: G2576T mutation , 23S rRNA gene , gene conversion


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Linezolid, an oxazolidinone antimicrobial, has activity against many drug-resistant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci.1 The drug binds to the domain V region of the 23S rRNA,2 and resistance in laboratory mutants has been associated with mutations in the central loop of the domain V region of the 23S rRNA gene.3,4

However, in nearly all bacteria, the 23S rRNA gene is present in multiple copies and strains of S. aureus have five or six copies.5 This may explain why resistance to linezolid is extremely rare in clinical isolates of staphylococci. To date, there have been only four reports of clinical S. aureus isolates with linezolid resistance associated with G2576T mutations in the domain V region of the 23S rRNA gene69 and one report with a mutation at a different locus in domain V.10

In this study, we examined another clinical S. aureus isolate that was found to be resistant to linezolid. Initially, the patient had been admitted for respiratory failure requiring a tracheostomy and was treated with linezolid for tracheobronchitis due to a linezolid-susceptible MRSA isolate (not available for subsequent testing). After 25 days of therapy with linezolid, a linezolid-resistant MRSA isolate was recovered from a tracheal specimen. Treatment was modified based on culture and susceptibility testing, and the patient responded clinically to vancomycin. No other linezolid-resistant staphylococci have been encountered by the clinical laboratory that identified this isolate. Sequencing of domain V of the 23S rRNA of this clinical isolate identified the G2576T mutation in four of the five copies. This provided an opportunity to study the stability of this resistant phenotype.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
MICs of linezolid were determined by broth macrodilution methods (according to NCCLS guidelines) and Etest methods (AB Biodisk, Solna, Sweden). The Etest was conducted with a suspension of overnight growth of the isolate equivalent to a 0.5 McFarland standard in 0.85% NaCl. The entire surface of a 90 mm Mueller–Hinton agar plate was streaked three times, with the plate rotated 90° with each application. The linezolid Etest strip was then applied and the plate incubated for 24 h in ambient air. Susceptibility to erythromycin was determined by disc diffusion methods (according to NCCLS guidelines).

To identify gene mutations associated with resistance, individual copies of the 23S rRNA genes were amplified using primer pairs, rrn1–rrn5, as previously published.11 Amplification products were predicted to be ~6.5 kb in size; so Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA) was used for ‘long-range’ PCR. For each purified rRNA gene fragment, the domain V region spanning base pairs 2280–2699 (Escherichia coli numbering) was amplified. The primers used were 5'-GCGGTCGCCTCCTAAAAG-3' (upper primer) and 5'-ATCCCGGTCCTCTCGTACTA-3' (lower primer).

After separation by agarose gel electrophoresis and gel extraction/purification, the resulting products were sequenced using the standard dideoxynucleotide method (Molecular Biology Core Facility, Dana-Farber Cancer Institute, Boston, MA, USA). Sequence data were analysed using MEGALIGN (DNASTAR Inc., Madison, WI, USA) and CHROMAS version 1.45 (Conor McCarthy, School of Health Sciences, Griffith University, Gold Coast Campus, Southport, Queensland, Australia).

To study the stability of this resistant phenotype in the absence of antimicrobial selective pressure, a single colony of the linezolid-resistant S. aureus isolate, A8761, was serially passaged 60 times (over a 75 day period) on antibiotic-free tryptic soy agar with 5% sheep blood (Northeast Laboratory, Waterville, ME, USA) and incubated overnight at 35°C. The linezolid MIC was determined by broth macrodilution testing for the original isolate (designated S. aureus A8761 isolate), the organism recovered after 30 passages (designated S. aureus A8761A) and the organism recovered after 60 passages (designated S. aureus A8761B). Individual copies of the 23S rRNA genes in the serially passaged strains were amplified and sequenced, as described above. Clonal relationships were determined by pulsed-field gel electrophoresis (PFGE) with SmaI-macrorestricted genomic DNA.

The number of all rRNA genes and the number of those with the G2576T mutation were determined by Southern-blot hybridization, as previously described.11


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This clinical isolate, S. aureus A8761, was initially found to be linezolid resistant (MIC > 4 mg/L using a broth microdilution technique; Dade Microscan Inc., West Sacramento, CA, USA) and the resistance was subsequently confirmed by Etest (MIC of linezolid of 12 mg/L) in the clinical microbiology laboratory. Susceptibility testing in our reference laboratory by broth macrodilution (using two-fold dilutions) determined the MIC of linezolid to be 16 mg/L (Table 1).


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Table 1. Characteristics of original linezolid-resistant clinical Staphylococcus aureus and its two laboratory derivatives analysed in this study

 
Sequencing of S. aureus A8761 identified a G->T mutation at position 2576 (E. coli numbering) in four of the five copies of the 23S rRNA gene in this organism. Sequencing and MIC testing by broth macrodilution was repeated on the isolates after 30 and 60 passages. These results are summarized in Table 1. The original isolate and the two more susceptible derivatives were indistinguishable by PFGE (data not shown).

Following digestion of genomic DNA with EcoRI and then hybridization with the domain V probe, all three organisms showed five discrete bands on Southern blotting. In 23S rRNA genes that contain the G2576T mutation, digestion with NheI results in two fragments to which the domain V probe can bind. This method (data not shown) confirmed the results of the DNA sequencing.

All three organisms were fully resistant to erythromycin by disc diffusion testing (Table 1).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Linezolid binds to ribosomal RNA (rRNA), specifically to domain V of the 23S rRNA, on the 50S ribosomal subunit near its interface with the 30S subunit. These two subunits join to form the 70S ribosome, used by prokaryotes in protein synthesis.2 The genes encoding this rRNA are present in multiple copies in clinically relevant bacterial species. Therefore, it was thought that the multiple copies of rRNA genes would make it more difficult to select for mutational resistance to linezolid.3 However, reports of linezolid resistance in clinical S. aureus isolates have been published,610 and all involved mutations in more than one copy of the 23S rRNA gene.

For comparison, the isolate reported by Tsiodras et al.6 had all five copies of the 23S rRNA gene mutated with the G2576T mutation, and the MIC of linezolid was >32 mg/L. In the series of isolates published by Wilson et al.,7 one isolate with an MIC of linezolid of 8 mg/L had the G2576T mutation in two of six copies. Another isolate with an MIC of linezolid of 32 mg/L had five of six copies mutated.7 Finally, Paterson et al.9 reported an isolate with an MIC of linezolid of 8 mg/L, which had two of five copies with the G2576T mutation.

The results of our study document a relationship between the proportion of mutant copies of the 23S rRNA gene and the level of linezolid resistance. Wilson et al.7 also demonstrated this relationship in their series of linezolid-resistant clinical isolates of S. aureus recovered in the UK. Such mutant gene dosage effects have also been seen in laboratory-derived S. aureus isolates4 and reported in clinical Enterococcus faecium and Enterococcus faecalis isolates.12,13

The emergence of isolates with G2576T mutations in multiple copies of 23S rRNA is believed to occur via a process called gene conversion. Under antimicrobial selective pressure, it is believed that wild-type genes are replaced with mutant copies by homologous recombination. This mechanism was implicated in resistance to aminoglycosides in laboratory strains of Mycobacterium smegmatis.14 Prammananan et al.14 found that in organisms with a heterozygous rRNA genotype, RecA-mediated gene conversion was required to select for aminoglycoside resistance. Evidence supporting this mechanism of resistance in E. faecalis has also been reported. In that study, Lobritz et al.15 found that a recombination-capable isolate was able to mutate to linezolid resistance by the selection of the G2576T mutation in multiple copies of 23S rDNA, but a recombination-deficient mutant was unable to do so. A different 23S rDNA mutation conferring resistance to linezolid was selected in the recombination-deficient mutant, but in only a single copy. Presumably, whereas it may have been able to mutate a single copy to G2576T at the same rate as the wild-type strain, the recombination-deficient strain could not perform the subsequent recombination events to acquire a sufficient percentage of mutated copies to show detectable resistance.15

In the present study, we have shown that in the absence of antibiotic pressure, restoration of susceptibility to linezolid can occur, associated with the loss of the G2576T mutation in most copies of the 23S rRNA gene. In contrast, Pillai et al.11 had found that the MIC of linezolid remained unchanged when a linezolid-resistant clinical S. aureus isolate, designated A7817, was passaged 15 times on antibiotic-free medium. Although we performed additional passages in the current study, the difference between the two isolates may be explained by the fact that A7817 had only mutant 23S rDNA, whereas our isolate, A8761, had a wild-type copy preserved. For homologous recombination to occur, the isolate requires a wild-type copy of the 23S rRNA gene to be present, which can act as a template.

Another interesting aspect of our findings was that despite 60 passages in the absence of selective pressure over a 75 day period, the isolate maintained a single mutant rRNA gene, suggesting that a single mutated copy of the gene may result in only a small cost to the fitness of the organism. It is important to note that such a single copy mutant would not be detected by standard susceptibility testing in a clinical microbiology laboratory. How quickly such an isolate may become more resistant if re-exposed to linezolid is not known. Clinicians should be cautious, however, in restarting linezolid treatment for patients in whom linezolid-resistant isolates have been previously recovered, even if subsequent isolates of the same species test susceptible.

The results of this study may be useful in designing clinical strategies for minimizing the persistence of resistance to oxazolidinones among S. aureus and possibly other Gram-positive cocci.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to acknowledge that this study was supported in part through an Independent Medical Grant from Pfizer, Inc.


    Footnotes
 
* Correspondence address. Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine Bldg, Room 219, 4 Blackfan Circle, Boston, MA 02115, USA. Tel: +1-617-667-0039; Fax: +1-617-975-5235; Email: vmeka{at}bidmc.harvard.edu


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Shinabarger, D. L. (1999). Mechanism of action of the oxazolidinone antibacterial agents. Expert Opinion on Investigational Drugs 8, 1195–202.

2 . Matassova, N. B., Rodnina, M. V., Endermann, R. et al. (1999). Ribosomal RNA is the target for oxazolidinones, a novel class of translational inhibitors. RNA 5, 939–46.[Abstract/Free Full Text]

3 . Xiong, L., Kloss, P., Douthwaite, S. et al. (2000). Oxazolidinone resistance mutations in 23S rRNA of Escherichia coli reveal the central region of domain V as the primary site of drug action. Journal of Bacteriology 182, 5325–31.[Abstract/Free Full Text]

4 . Swaney, S. M., Shinabarger, D. L., Schaadt, R. D., et al. (1998). Oxazolidinone resistance is associated with a mutation in the peptidyl transferase region of 23S rRNA. In Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract C-104, p. 98. American Society for Microbiology, Washington, DC, USA.

5 . Klappenbach, J. A., Saxman, P. R., Cole, J. R. et al. (2001). rrndb: the Ribosomal RNA Operon Copy Number Database. Nucleic Acids Research 29, 181–4.[Abstract/Free Full Text]

6 . Tsiodras, S., Gold, H. S., Sakoulas, G. et al. (2001). Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet 358, 207–8.[CrossRef][ISI][Medline]

7 . Wilson, P., Andrews, J. A., Charlesworth, R. et al. (2003). Linezolid resistance in clinical isolates of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 51, 186–8.[Free Full Text]

8 . Machado, A. R. L., Andrade, S. S., Barth, A. L., et al. (2003). The emergence of linezolid-resistance among Staphylococcus aureus from cystic fibrosis patients. In Abstracts of the Forty-third Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2003. Abstract C2-1825, p. 144. American Society for Microbiology, Washington, DC, USA.

9 . Paterson, D. L., Potoski, B. A., Kolano, J., et al. (2003). Fatal infection due to Staphylococcus aureus with decreased linezolid susceptibility. In Abstracts of the Forty-third Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2003. Abstract K-1405, p. 385. American Society for Microbiology, Washington, DC, USA.

10 . Meka, V. G., Pillai, S. K., Sakoulas, G. et al. (2004). Linezolid resistance in sequential Staphylococcus aureus isolates associated with a T2500A mutation in the 23S rRNA gene and loss of a single copy of rRNA. Journal of Infectious Diseases 190, 311–7.[CrossRef][ISI][Medline]

11 . Pillai, S. K., Sakoulas, G., Wennersten, C. et al. (2002). Linezolid resistance in Staphylococcus aureus: characterization and stability of resistant phenotype. Journal of Infectious Diseases 186, 1603–7.[CrossRef][ISI][Medline]

12 . Marshall, S. H., Donskey, C. J., Hutton-Thomas, R. et al. (2002). Gene dosage and linezolid resistance in Enterococcus faecium and Enterococcus faecalis. Antimicrobial Agents and Chemotherapy 46, 3334–6.[Abstract/Free Full Text]

13 . Zurenko, G. E., Todd, W. M., Hafkin, B., et al. (1999). Development of linezolid-resistant Enterococcus faecium in two compassionate use program patients treated with linezolid. In Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 848, p. 118. American Society for Microbiology, Washington, DC, USA.

14 . Prammananan, T., Sander, P., Springer, B. et al. (1999). RecA-mediated gene conversion and aminoglycoside resistance in strains heterozygous for rRNA. Antimicrobial Agents and Chemotherapy 43, 447–53.[Abstract/Free Full Text]

15 . Lobritz, M., Hutton-Thomas, R., Marshall, S. et al. (2003). Recombination proficiency influences frequency and locus of mutational resistance to linezolid in Enterococcus faecalis. Antimicrobial Agents and Chemotherapy 47, 3318–20.[Abstract/Free Full Text]