In Vitro Isolation and Characterization of Oxazolidinone-Resistant Mycobacterium tuberculosis

ABSTRACT Oxazolidinones are promising candidates for the treatment of Mycobacterium tuberculosis infections. We isolated linezolid-resistant strains from H37Rv (Euro-American) and HN878 (East-Asian) strains; resistance frequencies were similar in the two strains. Mutations were identified in ribosomal protein L3 (RplC) and the 23S rRNA (rrl). All mutant strains were cross resistant to sutezolid; a subset was cross resistant to chloramphenicol. Mutations in rrl led to growth impairment and decreased fitness that may limit spread in clinical settings.

T he oxazolidinone class of antibiotics inhibits the formation of protein synthesis initiation complexes by binding to domain V of the 23S rRNA (1). Linezolid (LZD), the first member of the oxazolidinones approved for clinical use, has recently been investigated as a potential treatment for drug-resistant strains of Mycobacterium tuberculosis (2,3). LZD demonstrates time-dependent kill kinetics against replicating M. tuberculosis (4), bactericidal activity against nonreplicating bacilli (5), and good efficacy in mouse models (5). However, the long-term administration of LZD is limited due to side effects that include neuropathy and anemia (2,6). Sutezolid (SZD; previously PNU-100480) is a next-generation oxazolidinone that has improved tolerance over long-term administration and improved efficacy against M. tuberculosis in a mouse model (7). Resistance to oxazolidinones has been studied in other bacteria and is mediated via mutations in domain V of the 23S rRNA (rrl), in the ribosomal protein L3 (rplC), or by the transporter OptrA (8). We sought to further characterize the mechanisms of oxazolidinone resistance in M. tuberculosis.
We isolated resistant mutant strains (RMs) against LZD by plating late-log-phase cultures of M. tuberculosis H37Rv (ATCC 25618) (Euro-American lineage) and HN878 (East-Asian lineage) on Middlebrook 7H10 agar with 10% (vol/vol) OADC (oleic acidalbumin-dextrose-catalase) supplement (Becton Dickinson) and 8 M (2.7 g/ml) of LZD (5ϫ MIC) (9). We confirmed resistance by determining MICs on solid medium or in liquid medium. Solid MICs were determined in 24-well plates on 7H10-OADC agar and were defined as the lowest concentrations that prevented growth. MIC 90 was determined in Middlebrook 7H9 liquid medium with 10% (vol/vol) OADC and 0.05% (wt/vol) Tween 80 (Tw); bacterial growth was measured by the optical density at 590 nm (OD 590 ) after 5 days, and the MIC 90 was defined as the concentration at which 90% of growth was inhibited (10). Sixteen resistant strains were isolated in H37Rv at a frequency of 2.3 ϫ 10 Ϫ9 (Table 1); 12 resistant strains were isolated in HN878 at a similar frequency of 3.3 ϫ 10 Ϫ9 (Table 2). All strains were confirmed as resistant to LZD and were also cross resistant to sutezolid (SZD) ( Tables 1 and 2).
Mutations in 23S rRNA are commonly associated with growth defects in a diverse range of bacterial species, including M. smegmatis and M. tuberculosis (17,19,23). We conducted growth curves for representative strains in liquid medium; cultures were grown in 16-mm borosilicate tubes containing 5 ml of 7H9-OADC-Tw and incubated at 37°C with stirring at 250 rpm using an 8-mm stirrer bar. All strains with rrl mutations demonstrated impaired growth compared to WT strains ( Fig. 1D and E), while strains with mutations in L3 were unimpaired (Fig. 1D).
The fitness cost of resistance mutations is an important contributor to the emergence and expansion of drug-resistant strains (24,25). To investigate the fitness cost of LZD resistance, we conducted in vitro competition experiments as described previously (25). Briefly, 100 ml of 7H9-OADC was inoculated with ϳ10 6 CFU of the WT and mutant strains in a 450-cm 2 roller bottle. Coculture experiments were grown at 37°until stationary phase (OD 590 , ϳ1). Serial dilutions were plated onto 7H10-OADC with and without 5 M (1.7 g/ml) LZD at day 0 and at stationary phase. CFU were counted after 4 to 5 weeks of incubation at 37°C. The relative fitness (W) of resistant (R) compared to that of susceptible (S) strains was calculated by W ϭ ln(R F /R I )/ln(S F /S I ) (25), where R I and S I are the number of resistant and susceptible cells at day 0, and R F and S F are the number of resistant and susceptible cells at stationary phase. Experiments were performed in biological triplicate. The H37Rv rrl G2814T strain had a fitness cost compared to that of the susceptible parental strain (Fig. 1F). In contrast, the L3 C154R mutant strain had no fitness cost relative to that of the parent (Fig. 1F). Relative fitness costs have been previously shown to influence the spread of resistance, with low-cost resistance phenotypes being the most prevalent within clinical populations (25). From a limited number of clinical studies, the L3 C154R single nucleotide polymorphism (SNP) is more prevalent than rrl SNPs within LZD-resistant strains (6,11,13). Whether or not this is because of the associated fitness cost requires further investigation. Resistance defects can be overcome by compensatory mechanisms as, for example, in S. aureus where changes in the copy number of 23S rRNA can achieve a balance between fitness and resistance (19). M. tuberculosis is unique in that it contains only a single copy of 23S rRNA, so it may not have access to the same compensatory mutations. Identifying compensatory mechanisms that overcome the fitness defects associated with rrl SNPs would provide further insights.
In conclusion, we demonstrate that mutations in the 23S rRNA (rrl) and the ribosomal protein L3 (RplC) are associated with resistance to the oxazolidinones LZD and SZD. Resistance led to decreased bactericidal activity from LZD. Mutations in rrl, but not L3, had a competitive fitness cost in vitro, suggesting that their appearance may be limited in clinical settings.
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