Linezolid, the first oxazolidinone in clinical use, is effective in the treatment of infections caused by various gram-positive pathogens, including ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2008, p. 1570–1572 0066-4804/08/$08.00⫹0 doi:10.1128/AAC.01098-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 52, No. 4
Linezolid Resistance in Staphylococcus aureus: Gene Dosage Effect, Stability, Fitness Costs, and Cross-Resistances䌤 Silke Besier, Albrecht Ludwig, Johannes Zander, Volker Brade, and Thomas A. Wichelhaus* Institut fu ¨r Medizinische Mikrobiologie und Krankenhaushygiene, Klinikum der Johann Wolfgang Goethe-Universita ¨t, Frankfurt am Main, Germany Received 21 August 2007/Returned for modification 24 September 2007/Accepted 15 January 2008
Linezolid resistance in Staphylococcus aureus is typically associated with mutations in the 23S rRNA gene. Here we show that the accumulation of a single point mutation, G2576T, in the different copies of this gene causes stepwise increases in resistance, impairment of the biological fitness, and cross-resistance to quinupristin-dalfopristin and chloramphenicol. T991 with a 883-bp DNA probe containing domain V of the S. aureus 23S rRNA gene revealed that T991 carries five copies of this gene (data not shown). To characterize the domain V regions of each of the five 23S rRNA gene copies from strain T991 and its descendants, these genes were individually amplified as previously described (15). DNA sequence analysis demonstrated that S. aureus T991 does not contain any mutations in domain V of its five 23S rRNA gene copies. The five T991 derivatives selected under linezolid selective pressure, however, showed a G2576T substitution in domain V within one or more of their 23S rRNA gene copies (Table 1). Other mutations were not found. The level of linezolid resistance observed for the different T991 derivatives directly correlated with the number of 23S rRNA gene copies carrying this nucleotide exchange; i.e., the higher the resistance the more gene copies showed the G2576T exchange (Table 1). Interestingly, the first G2576T mutation (observed in isolate T1887) required by far the longest time to occur. The faster acquisition of the same nucleotide exchange in the other 23S rRNA gene copies suggested that mechanisms other than independent mutations, such as homologous recombination, might have been involved in the spreading of this mutation. Stability of the G2576T mutation. To determine the stability of linezolid resistance associated with the G2576T mutation, a single colony of S. aureus T2019, the T991 descendant containing this mutation in all five 23S rRNA gene copies, was passaged 50 times in antibiotic-free medium, resulting in the isolation of S. aureus T2019/50. MIC testing demonstrated an approximately twofold decrease in the MIC for S. aureus T2019/50 compared to T2019 results. Furthermore, sequencing of the 23S rRNA genes of T2019/50 revealed a reversion of the G2576T mutation, i.e., T2576G, in one of the five 23S rRNA gene copies (Table 1). Fitness costs of linezolid resistance associated with the G2576T mutation. To investigate whether the G2576T mutation in the 23S rRNA genes influences the biological fitness of S. aureus, we analyzed the growth kinetics for S. aureus T991 and the different T991 descendants. For this purpose, S. aureus strains were grown in Mueller-Hinton broth to the logarithmic growth phase (optical density at 600 nm [OD600] ⫽ 1). A total of 100 l of each culture with an OD of 1 was then inoculated into 10 ml of fresh medium and incubated with shaking (200
Linezolid, the first oxazolidinone in clinical use, is effective in the treatment of infections caused by various gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (2, 11). Linezolid binds to the 50S subunit of the bacterial ribosome via interaction with the 23S rRNA, thereby blocking protein synthesis (2, 9, 16). Linezolid resistance in S. aureus has been encountered in the clinical setting and has also been selected in vitro, but it is still a rare phenomenon (2, 6, 8, 19, 21). The resistance to this antibiotic has been associated with distinct nucleotide substitutions in domain V of the 23S rRNA gene, particularly G2447T, T2500A, and G2576T (3, 14, 18, 19, 21, 22). S. aureus possesses five to six rRNA (rrn) operons (14), which suggests that the presence of a resistance-mediating mutation in one, several, or all of the 23S rRNA gene copies might be associated with gradually increased levels of resistance. This study was aimed at (i) correlating the level of linezolid resistance in S. aureus with the presence of mutations in the different copies of the 23S rRNA gene, (ii) analyzing the stability of linezolid resistance, (iii) determining the fitness costs associated with linezolid resistance, and (iv) assessing the issue of cross-resistance to other protein synthesis inhibitors. Isolation of linezolid-resistant mutants of S. aureus T991. S. aureus T991, a clinical linezolid-susceptible strain, was grown at 37°C in Mueller-Hinton broth and then serially passaged in medium containing successively increasing concentrations (1 to 128 mg/liter) of linezolid. In the course of these passages, five S. aureus T991 descendants, T1887, T1888, T1900, T1910, and T2019, were isolated which showed gradually increasing linezolid MICs between 4 and 128 mg/liter (Table 1). Clonal identity of the parental strain T991 and the most resistant derivative, T2019, was proven by pulsed-field gel electrophoresis (PFGE) as previously described (20) (data not shown). Association of linezolid resistance with the presence of the G2576T mutation in the 23S rRNA genes. Southern blot hybridization of EcoRI-digested genomic DNA from S. aureus
* Corresponding author. Mailing address: Institut fu ¨r Medizinische Mikrobiologie und Krankenhaushygiene, Klinikum der J. W. GoetheUniversita¨t, Paul-Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany. Phone: 49 69 6301 6438. Fax: 49 69 6301 5767. E-mail: Wichelhaus @em.uni-frankfurt.de. 䌤 Published ahead of print on 22 January 2008. 1570
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NOTES
TABLE 1. Correlation of the presence of point mutation G2576T in the 23S rRNA genes with the level of linezolid resistance in S. aureus Strain
T991 T1887 T1888 T1900 T1910 T2019 T2019/50
No. of passages
21c 22c 34c 44c 54c 50d
Nucleotide at position 2576a in the 23S rRNA gene copies
Linezolid MICb (mg/liter)
rrn 1
rrn 2
rrn 3
rrn 4
rrn 5
1 4 8 32 64 128 64
G G G G G T T
G G G G T T G
G T T T T T T
G G G T T T T
G G T T T T T
a
Escherichia coli numbering. MICs were determined by the Etest method (AB Biodisk, Solna, Sweden), using Columbia sheep blood agar (Heipha) for growth of the bacteria. MICs are given as the medians of the results of three experiments. c Result represents passages of strain T991 under linezolid selective pressure. d Result represents passages of T2019 in the absence of antimicrobial selective pressure. b
rpm) at 37°C. Bacterial growth was recorded for a total period of 12 h by measuring the OD600 of the cultures at intervals of 1 h. As shown in Fig. 1, the growth rates decreased gradually with increasing numbers of mutant 23S rRNA gene copies. Several CFU determinations performed during the experiment also confirmed these observations (data not shown). The differences in the growth yields (OD600 values) observed in the midlogarithmic phase (after 7 h of growth) between T991, T1887, T1888, T1900, T1910, and T2019 were statistically significant (P ⬍ 0.05 as determined by analysis of variance). In three experiments, the ratios of the OD600 values (linezolidresistant mutants versus linezolid-susceptible wild type) measured after 7 h of growth were 0.816 ⫾ 0.007 (means ⫾ standard deviations) for T1887 versus T991, 0.613 ⫾ 0.003 for T1888 versus T991, 0.552 ⫾ 0.004 for T1900 versus T991, 0.432 ⫾ 0.004 for T1910 versus T991, and 0.302 ⫾ 0.006 for T2019
FIG. 1. Growth kinetics of S. aureus strain T991 and its descendants T1887, T1888, T1900, T1910, and T2019. Data are expressed as the means ⫾ standard deviations of the results of three experiments.
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TABLE 2. Cross-resistances associated with mutation G2576T in the 23S rRNA genes of S. aureus MIC (mg/liter) or strain sensitivitya Antibiotic
Linezolid Erythromycin Clindamycin Quinupristin-dalfopristin Doxycycline Tigecycline Gentamicin Fusidic acid Chloramphenicol
T991
T2019
1 ⬎256 ⬎256 0.5 6 1 256 0.19 S (22 mm)
128 ⬎256 ⬎256 6 6 0.75 256 0.5 R (0 mm)
a Data were determined by the Etest method. MICs are given as the medians of the results of three experiments. S, susceptible; R, resistant. Zones of inhibition were determined by the disc diffusion method according to CLSI guidelines (4). Inhibition zone diameters are given in parentheses.
versus T991. Although the possibility cannot totally be excluded that other mutations adversely affecting the fitness may have been selected during passaging of S. aureus T991, these data suggested that the accumulation of the G2576T mutation in the 23S rRNA gene copies causes a successive decline in the biological fitness of S. aureus. Interestingly, isolate T2019/50, showing a reversion of the G2576T mutation in one of the five 23S rRNA gene copies, exhibited a marked fitness gain compared to T2019; i.e., the ratio of OD600 values measured after 7 h of growth was 0.707 ⫾ 0.007 for T2019/50 versus T991. Given that the fitness of T2019/50 is considerably higher than that of T1910 (both strains carry four G2576T mutations), it seems, however, unlikely that the fitness increase observed for T2019/50 is exclusively due to the reversion of one of the G2576T exchanges. In fact, previous studies indicated that the fitness loss caused in S. aureus by certain mutations conferring resistance to antibiotics such as fusidic acid can be compensated for by secondary mutations in the genome (1). It is hence tempting to speculate that a compensatory mutation(s) outside the domain V region of the 23S rRNA genes may be involved in the partial restoration of the biological fitness observed for S. aureus T2019/50. Cross-resistances associated with the G2576T mutation. Linezolid possesses a distinctive mechanism of action, suggesting that there is no cross-resistance with other antibiotics (5, 14). Nonetheless, while S. aureus T991 proved to be susceptible to chloramphenicol and quinupristin-dalfopristin, the linezolid-resistant mutant T2019 showed cross-resistances to these antibiotics (Table 2). Chloramphenicol acts on the 50S subunit by binding to the central loop of domain V of the 23S rRNA, thereby inhibiting the peptidyl transferase reaction (10). Footprint studies have indicated that chloramphenicol interacts strongly with the A2451 and G2505 sites in the peptidyl transferase region (10). Similarly, quinupristin-dalfopristin interacts closely with G2505 and C2586 (7). Thus, the close vicinity of G2505 and C2586 to G2576 might explain the observed phenomenon of cross-resistance, i.e., the G2576T mutation possibly decreases the binding affinity not only for linezolid but also for chloramphenicol and the streptogramins quinupristin-dalfopristin. In this regard, the recent identification of the cfr gene as part of a mobile genetic element from a human isolate of S. aureus that encodes a rRNA methyltrans-
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ferase is of interest (17). This methyltransferase modifies adenosine at position 2503 in the 23S rRNA and has been shown to confer resistance to linezolid and chloramphenicol. Again, the close vicinity of A2503 to G2576 argues for overlapping binding sites of these antibiotics and the observed phenomenon of cross-resistance. Conclusions. The data presented here show that the successive accumulation of a single point mutation, G2576T, in the 23S rRNA gene copies of S. aureus is associated with, and apparently causative of, a stepwise increase in the level of linezolid resistance. The current report thus ultimately verifies the similar gene dosage effects that have been described for clinical isolates as well as laboratory-derived linezolid-resistant mutants of S. aureus and enterococci (11–13, 15). Furthermore, we found that the increase in linezolid resistance mediated by the spreading of the G2576T mutation is paralleled by a decline in the biological fitness of the bacteria, which suggests a negative effect of the G2576T mutation on protein synthesis. In the S. aureus T991 descendant carrying the G2576T mutation in all five 23S rRNA gene copies, this mutation proved to be rather stable in the absence of linezolid-selective pressure, which is consistent with findings of Pillai et al. (15). Nevertheless, the reversion of this nucleotide exchange in one of these gene copies observed after multiple serial passages in antibiotic-free medium indicates that the fitness burden associated with the G2576T mutation is a driving force for the restoration of the wild-type sequence and hence for reversion to a more linezolid-susceptible phenotype. We thank Denia Frank for excellent technical assistance.
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REFERENCES 1. Besier, S., A. Ludwig, V. Brade, and T. A. Wichelhaus. 2005. Compensatory adaptation to the loss of biological fitness associated with acquisition of fusidic acid resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 49:1426–1431. 2. Bozdogan, B., and P. C. Appelbaum. 2004. Oxazolidinones: activity, mode of action, and mechanism of resistance. Int. J. Antimicrob. Agents 23:113–119. 3. Bozdogan, B., M. V. Patel, S. V. Gupte, N. De Souza, H. Khorakiwala, K. Sreenivas, S. Nair, M. R. Jacobs, and P. C. Appelbaum. 2002. Characterization of linezolid-resistant mutants of Staphylococcus aureus and Streptococcus pneumoniae isolated from mouse in terms of 23S rRNA mutations. Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-1609. 4. Clinical and Laboratory Standards Institute (CLSI). 2005. Performance standards for antimicrobial susceptibility testing; fifteenth informational supplement—M100-S15, vol. 25, no. 1. Clinical and Laboratory Standards Institute, Wayne, PA. 5. Fines, M., and R. Leclercq. 2000. Activity of linezolid against Gram-positive cocci possessing genes conferring resistance to protein synthesis inhibitors. J. Antimicrob. Chemother. 45:797–802. 6. Gales, A. C., H. S. Sader, S. S. Andrade, L. Lutz, A. Machado, and A. L. Barth. 2006. Emergence of linezolid-resistant Staphylococcus aureus during
18.
19.
20.
21.
22.
treatment of pulmonary infection in a patient with cystic fibrosis. Int. J. Antimicrob. Agents 27:300–302. Harms, J. M., F. Schlunzen, P. Fucini, H. Bartels, and A. Yonath. 2004. Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin. BMC Biol. 2:4–14. Kola, A., P. Kirschner, B. Gohrbandt, I. F. Chaberny, F. Mattner, M. Struber, P. Gastmeier, and S. Suerbaum. 2007. An infection with linezolid-resistant S. aureus in a patient with left ventricular assist system. Scand. J. Infect. Dis. 39:463–465. Leach, K. L., S. M. Swaney, J. R. Colca, W. G. McDonald, J. R. Blinn, L. M. Thomasco, R. C. Gadwood, D. Shinabarger, L. Xiong, and A. S. Mankin. 2007. The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria. Mol. Cell 26:393–402. Lin, A. H., R. W. Murray, T. J. Vidmar, and K. R. Marotti. 1997. The oxazolidinone eperezolid binds to the 50S ribosomal subunit and competes with binding of chloramphenicol and lincomycin. Antimicrob. Agents Chemother. 41:2127–2131. Livermore, D. M. 2003. Linezolid in vitro: mechanism and antibacterial spectrum. J. Antimicrob. Chemother. 51:ii9–ii16. Marshall, S. H., C. J. Donskey, R. Hutton-Thomas, R. A. Salata, and L. B. Rice. 2002. Gene dosage and linezolid resistance in Enterococcus faecium and Enterococcus faecalis. Antimicrob. Agents Chemother. 46:3334–3336. Meka, V. G., H. S. Gold, A. Cooke, L. Venkataraman, G. M. Eliopoulos, R. C. Moellering, Jr., and S. G. Jenkins. 2004. Reversion to susceptibility in a linezolid-resistant clinical isolate of Staphylococcus aureus. J. Antimicrob. Chemother. 54:818–820. Meka, V. G., S. K. Pillai, G. Sakoulas, C. Wennersten, L. Venkataraman, P. C. DeGirolami, G. M. Eliopoulos, R. C. Moellering, Jr., and H. S. Gold. 2004. Linezolid resistance in sequential Staphylococcus aureus isolates associated with a T2500A mutation in the 23SrRNA gene and loss of a single copy of rRNA. J. Infect. Dis. 190:311–317. Pillai, S. K., G. Sakoulas, C. Wennersten, G. M. Eliopoulos, R. C. Moellering, Jr., M. J. Ferraro, and H. S. Gold. 2002. Linezolid resistance in Staphylococcus aureus: characterization and stability of resistant phenotype. J. Infect. Dis. 186: 1603–1607. Swaney, S. M., H. Aoki, M. C. Ganoza, and D. L. Shinabarger. 1998. The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrob. Agents Chemother. 42:3251–3255. Toh, S. M., L. Xiong, C. A. Arias, M. V. Villegas, K. Lolans, J. Quinn, and A. S. Mankin. 2007. Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid. Mol. Microbiol. 64:1506–1514. Tsakris, A., S. K. Pillai, H. S. Gold, C. Thauvin-Eliopoulos, L. Venkataraman, C. Wennersten, R. C. Moellering, Jr., and G. M. Eliopoulos. 2007. Persistence of rRNA operon mutated copies and rapid re-emergence of linezolid resistance in Staphylococcus aureus. J. Antimicrob. Chemother. 60:649–651. Tsiodras, S., H. S. Gold, G. Sakoulas, G. M. Eliopoulos, C. Wennersten, L. Venkataraman, R. C. Moellering, and M. J. Ferraro. 2001. Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet 358:207–208. Wichelhaus, T. A., J. Schulze, K. P. Hunfeld, V. Scha ¨fer, and V. Brade. 1997. Clonal heterogeneity, distribution, and pathogenicity of methicillin-resistant Staphylococcus aureus. Eur. J. Clin. Microbiol. Infect. Dis. 16:893–897. Wilson, P., J. A. Andrews, R. Charlesworth, R. Walesby, M. Singer, D. J. Farrell, and M. Robbins. 2003. Linezolid resistance in clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 51:186–188. Zhu, W., F. C. Tenover, J. Limor, D. Lonsway, D. Prince, W. M. Dunne, Jr., and J. B. Patel. 2007. Use of pyrosequencing to identify point mutations in domain V of 23S rRNA genes of linezolid-resistant Staphylococcus aureus and Staphylococcus epidermidis. Eur. J. Clin. Microbiol. Infect. Dis. 26:161– 165.
