JAC Advance Access originally published online on July 10, 2007
Journal of Antimicrobial Chemotherapy 2007 60(3):649-651; doi:10.1093/jac/dkm246
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Persistence of rRNA operon mutated copies and rapid re-emergence of linezolid resistance in Staphylococcus aureus
Division of Infectious Diseases, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02115, USA
* Corresponding author. E-mail: atsakris{at}bidmc.harvard.edu
Received 12 February 2007; returned 10 April 2007; revised 18 May 2007; accepted 12 June 2007
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Abstract |
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Objectives: The G2576T mutation in domain V of 23S rRNA has been most often associated with the rare cases of linezolid resistance in Staphylococcus aureus. In a linezolid-susceptible S. aureus (A8761B) possessing a single mutated (G2576T) copy, originally derived from a resistant clinical isolate, we assessed the persistence of the mutation on further passage on antibiotic-free medium and the selection of resistance upon re-exposure of the susceptible strain to linezolid.
Methods: The stability of the mutant rRNA copy was tested through 40 serial passages on antibiotic-free medium. The re-emergence of linezolid-resistant mutants was examined after serial passage on successively increasing linezolid concentrations. The efficacy of novobiocin, at subinhibitory concentrations, to prevent or delay the emergence of resistant mutants was examined. Strain relatedness was confirmed by PFGE and domain V of individual rRNA copies was sequenced.
Results: After 40 passages in antibiotic-free medium, the linezolid MIC of derived strain A9584 remained stable at 2 mg/L and the G2576T mutation persisted in one 23S rRNA gene copy (copy number 2). Upon re-exposure of the strain to increasing concentrations of linezolid, linezolid resistance (MIC of 64 mg/L) emerged rapidly. In a representative derivative (A9753), the G2576T mutation was found in four of the five rRNA copies. All laboratory derivates were closely related by PFGE. When A9584 was applied to plates containing linezolid at 4 x MIC, resistant colonies emerged at a frequency of 8 x 10–6. Novobiocin at 1/4 x MIC prevented the emergence of resistant colonies.
Conclusions: The persistence of the G2576T mutation in one rRNA operon copy in the absence of selective pressure suggests that the mutation has a minimal impact on the organism's fitness in vitro. Resistance to linezolid, associated with acquisition of multiple mutant copies, emerges rapidly upon re-exposure to linezolid. Novobiocin, predicted to interfere with gene conversion, may reduce the likelihood of rapid development of linezolid resistance.
Keywords: oxazolidinone , passage , resistant mutants , 23S rRNA , domain V , novobiocin
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Introduction |
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Linezolid, a member of the oxazolidinone class of antibiotics, has become an important therapeutic option for multidrug-resistant staphylococcal infections. Very low rates (<10–9) of spontaneous resistance to oxazolidinones are reported for Staphylococcus aureus,1 which is in accordance with the rarity with which linezolid-resistant clinical isolates of S. aureus are detected.2 In the sporadic reports of linezolid-resistant staphylococci (MIC >4 mg/L), linezolid resistance has been associated with mutations in the central loop of the domain V region and G2576T mutation is the one most commonly reported.3,4 We have previously shown that in the absence of antibiotic pressure, linezolid resistance was unstable in a clinical isolate with the G2576T mutation in four of the five copies of 23S rRNA.3 However, despite reversion to susceptibility after passage in antibiotic-free agar, a single mutant rRNA copy remained. In the present report, we document the persistence of the mutant rRNA operon copy despite further passage on antibiotic-free medium and the rapid re-emergence of resistance under antimicrobial selective pressure with linezolid. In addition, because novobiocin is able to inhibit DNA repair, preventing homologous recombination,5 we examined whether subinhibitory concentrations of novobiocin could prevent the re-emergence of linezolid resistance in our laboratory-derived strain.
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Materials and methods |
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S. aureus A8761 is a linezolid-resistant (MIC 16 mg/L) clinical isolate and S. aureus A8761B is its linezolid-susceptible (MIC 2 mg/L) derivative (obtained after 60 serial passages in an antibiotic-free medium) that contains one mutant copy among its five 23S rRNA genes (Table 1).3 The persistence of the mutant copy in the absence of antibiotic selective pressure was studied. A single colony of A8761B was streaked onto antibiotic-free tryptic soy agar with 5% sheep blood (Northeast Laboratory, Waterville, ME, USA) and the plate incubated overnight at 35°C. This process was repeated for a total of 40 additional passages and the resulting strain was designated A9584.
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The emergence of spontaneous mutants of S. aureus A9584 that were resistant to linezolid was examined by plating 0.1 mL of an
108 cfu/mL cell suspension onto brain heart infusion agar plates containing 2 x linezolid MIC (4 mg/L). The plates were incubated at 35°C for 48 h and colonies recovered were tested for spontaneous mutants to progressively higher linezolid concentrations (8, 16, 32 and 64 mg/L). To examine the stability of resistance, the mutants recovered after passage on the higher linezolid concentration (32 mg/L) were subcultured twice in an antibiotic-free medium and then tested by the broth macrodilution method to determine the final linezolid MIC.
To test whether novobiocin could prevent or delay the generation of linezolid-resistant mutants, spontaneous single-step mutants were sought by plating 0.1 mL of a cell suspension (108 cfu/mL) of A9584 onto brain heart infusion agar plates containing linezolid at concentrations of 4, 8, 16 or 32 mg/L (2–16 times the MIC) alone or together with novobiocin at the subinhibitory concentration of 0.015 mg/L (broth macrodilution MIC of novobiocin 0.06 mg/L). The stability of mutants derived from mutation frequency experiments was tested as above.
Individual copies of the five 23S rRNA genes in S. aureus A9584 as well as of the linezolid-resistant mutant that exhibited the higher MIC (A9753) were amplified using primer pairs that give amplified products of 6.5 kb in size.6 For each rRNA operon gene fragment, the domain V region spanning base pairs 2280–2699 (Escherichia coli numbering) was then amplified. The primers used were 5'-GCGGTCGCCTCCTAAAAG-3' (upper primer) and 5'-ATCCCGGTCCTCTCGTACTA-3' (lower primer). Amplicons were purified and sequenced (Molecular Biology Core Facility, Boston Children's Hospital, Boston, MA, USA). Clonal relationships were determined by PFGE of SmaI-digested genomic DNA.
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Results |
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Passage of S. aureus A8761B in antibiotic-free medium 40 times yielded strain A9584. Both A8761B and A9584 exhibited the same MIC of linezolid (2 mg/L) by broth macrodilution and Etest. All strains exhibited resistance to erythromycin as well as to trimethoprim/sulfamethoxazole, ciprofloxacin and aminoglycosides (gentamicin, tobramycin and amikacin). Resistance testing to erythromycin was carried out because of observations that macrolide resistance may be lost coincidentally with development of linezolid resistance.7 DNA sequencing of the 23S rRNA genes revealed that S. aureus A9584 still contained the G to T transversion at position 2576 (E. coli numbering) in one of the five rRNA operon copies (Table 1).
Upon re-exposure to increasing concentrations of linezolid, strain A9584 rapidly developed resistance to linezolid. Resistant mutants were always selected at mutation frequencies >5 x 10–6 after serial passages on agar plates containing 4, 8, 16 and 32 mg/L of linezolid. Mutants were not selected on plates containing 64 mg/L of linezolid. The linezolid MIC for mutants able to grow on agar containing 32 mg/L linezolid ranged from 32 to 64 mg/L. For one mutant with an MIC of 64 mg/L (S. aureus A9753 strain), the individual domain V regions were sequenced and revealed the G to T transversion at position 2576 in four of the five rRNA operon copies (rrn1, rrn2, rrn3 and rrn5), identical to the progenitor strain A8761 (Table 1). The isolate A8761 as well as the mutants A9584 and A9753 belonged to the same clonal strain.
The addition of novobiocin at 1/4 x MIC (0.015 mg/L) did not affect the number of colonies arising when A9584 was plated on linezolid at a concentration of 2 x MIC (3 x 10–4 on linezolid alone versus 2 x 10–4 on plates containing the combination). In contrast, there was a substantial impact of novobiocin on mutation frequency in the presence of higher concentrations of linezolid. The mutation frequency for A9584 in the presence of 4 x linezolid MIC (8 mg/L) was 8 x 10–6, but there was no growth on plates containing 4 x linezolid MIC combined with novobiocin. Consistent with these differences in the mutation frequency to linezolid, representative mutants selected on plates containing linezolid alone exhibited MICs of linezolid up to 32 mg/L, whereas 8 mg/L linezolid was the highest MIC reached for mutants recovered from linezolid plus novobiocin plates (Table 2).
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Discussion |
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Linezolid resistance is rare in S. aureus. Our previous study of linezolid-resistant clinical isolates, which were associated with a T2500A mutation in two of five or three of six copies of the 23S rRNA gene, suggested that there was a fitness cost associated with such mutations.8 In that case, an isolate recovered after discontinuation of linezolid demonstrated susceptibility to the drug and was wild-type in all six rrn genes. The present study gives indications that a single copy of the 23S rRNA gene with the G2576 mutation exerts very little cost of fitness in vitro and remains stable despite 100 serial passages on antibiotic-free medium. However, because the laboratory strain that reverted to linezolid susceptibility retained a copy of the mutant gene (G2576T), we wondered whether the presence of this gene posed a risk for rapid re-emergence of resistance upon exposure to linezolid. Our study suggested that this was indeed the case, with resistance re-emerging at rates at least 1000-fold greater than those reported spontaneous linezolid resistance rates.1 We believe that the presence of the single G2576T gene serves as a template for homologous recombination that has been proposed as integral to the development of progressively higher levels of resistance associated with increased copy numbers of mutated genes.3
Miller et al.9 have shown that a number of antimicrobials that might potentially interfere with gene conversion may delay emergence of linezolid resistance. Because our isolate was resistant to ciprofloxacin and gentamicin, we examined the ability of novobiocin to delay or prevent the emergence of resistance. We showed that this combination can delay the emergence of resistance. The concentrations of both linezolid (8 mg/L) and novobiocin (0.015 mg/L) that in combination prevented the appearance of any linezolid-resistant colonies in our study are both clinically attainable, even taking into account the high degree of protein binding of novobiocin.10 Although the latter is no longer used clinically, these findings suggest that other inhibitors of bacterial recombination may well be effective in forestalling linezolid resistance.
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This study was supported in part through an Independent Medical Grant from Pfizer, Inc. (to H. S. G., R. C. M. and G. M. E.).
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R. C. M. and G. M. E. have served as consultants for Pfizer, Inc. All the other authors: none to declare.
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References |
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1 Kaatz GW, Seo SM. In vitro activities of oxazolidinone compounds U100592 and U100766 against Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother (1996) 40:799–801.[Abstract]
2
Jones RN, Ross JE, Fritsche TR, et al. Oxazolidinone susceptibility patterns in 2004: report from the Zyvox Annual Appraisal of Potency and Spectrum (ZAAPS) Program assessing isolates from 16 nations. J Antimicrob Chemother (2006) 57:279–87.
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Meka VG, Gold HS, Cooke A, et al. Reversion to susceptibility in a linezolid-resistant clinical isolate of Staphylococcus aureus. J Antimicrob Chemother (2004) 54:818–20.
4 Tsiodras S, Gold HS, Sakoulas G, et al. Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet (2001) 358:207–8.[CrossRef][Web of Science][Medline]
5
Purdy MA, Yielding KL. Enhancement of post-ultraviolet killing in Escherichia coli K-12 through the action of gyrase inhibitors: evidence for associated gyrase-recBC deoxyribonuclease function. Antimicrob Agents Chemother (1979) 16:247–51.
6 Pillai SK, Sakoulas G, Wennersten C, et al. Linezolid resistance in Staphylococcus aureus: characterization and stability of resistance phenotype. J Infect Dis (2002) 186:1603–7.[CrossRef][Web of Science][Medline]
7 Howe RA, Noel A, Bowker KE, et al. Emergence of linezolid resistance in Staphylococcus aureus can be associated with loss of erythromycin resistance. Abstracts of the Forty-second Interscience Conference on Antimicrobial Agents and Chemotherapy, 2002: San Diego, CA. Washington, DC, USA: American Society for Microbiology. Abstract C1-1608, p. 75.
8 Meka VG, Pillai SK, Sakoulas G, et al. 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. J Infect Dis (2004) 190:311–7.[CrossRef][Web of Science][Medline]
9 Miller K, O'Neill AJ, Wilcox H, et al. Suppression of linezolid resistance in Staphylococcus aureus using drug combinations. Abstracts of the Forty-fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, 2004: Washington, DC. Washington, DC, USA: American Society for Microbiology. Abstract C1-1498, p. 85.
10 Kucers A, Crowe SM, Grayson ML, et al. The Use of Antibiotics (1997) 5th Edition. Oxford: Butterworth-Heinemann. 663–6.
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