A Novel Oxazolidinone, Contezolid (MRX-I), Expresses Anti-Mycobacterium abscessus Activity In Vitro

ABSTRACT An evaluation of the anti-Mycobacterium abscessus activity expressed by a novel oxazolidinone, contezolid (MRX-I), toward 12 reference strains and 194 clinical isolates was conducted. Contezolid was active against M. abscessus in vitro, with effects comparable to the anti-M. abscessus effects of linezolid both extracellularly and intracellularly. Contezolid did not antagonize the most frequently used anti-M. abscessus drugs, and preexposure to contezolid did not induce drug resistance. These results provide a novel approach to treating M. abscessus infections.

isolates were also tested and were found to be contezolid insensitive (see Table S1 in the supplemental material). Previous studies conducted by other investigators similarly reported that the majority of M. abscessus isolates were sensitive to linezolid, while .80% of M. avium and M. intracellulare isolates were insensitive (10)(11)(12)(13)(14)(15).
Contezolid exhibited anti-M. abscessus activity toward extracellular M. abscessus in culture that was comparable to that of linezolid. The MICs ranged from 0.25 to 64 mg/liter; the MIC 50 was 16 mg/liter and the MIC 90 was 32 mg/liter for M. abscessus subsp. abscessus, and the MIC 50 was 8 mg/liter and the MIC 90 was 32 mg/liter for M. abscessus subsp. massiliense ( Table 2). The detailed MIC distribution for all clinical isolates is shown in Table S2. Notably, while linezolid and tedizolid exhibited normal MIC distributions, the distribution for contezolid appeared biphasic. A lack of diversity could potentially contribute to this finding, since all the isolates were obtained at a single center. Genotypic and phylogenetic analyses were performed to exclude this possibility, and no duplicate clones were found (see Fig. S1). Therefore, the isolates were genetically diverse, and the biphasic response to contezolid remains to be clarified.
Contezolid inhibits the intracellular replication of M. abscessus. Killing assays were performed according to methods described previously to assess and compare the effects of contezolid and linezolid on the intracellular survival of two reference strains, i.e., ATCC 19977 (M. abscessus subsp. abscessus) and CIP108297 (M. abscessus subsp. massiliense), and two clinical isolates, i.e., A243 (M. abscessus subsp. abscessus) and G71 (M. abscessus subsp. abscessus), in primary mouse peritoneal macrophages (16). The cells of both the experimental and control groups were washed three times with warm phosphate-buffered saline to remove the extracellular organisms. Serial dilutions of the supernatants collected after the final wash were cultured on agar plates, and the CFU were counted to ensure that the number of residual extracellular bacteria was negligible.
Both contezolid and linezolid inhibited the intracellular growth of M. abscessus, relative to the untreated control, for all tested strains; inhibition was dose dependent (Fig. 1). There was no difference in the effects of contezolid and linezolid, indicating comparable intracellular anti-M. abscessus activity. Notably, the structural change in contezolid that results in lower toxicity did not weaken its ability to penetrate cells in our study. Contezolid was equivalent to linezolid and effective in inhibiting both the intracellular and extracellular growth of M. abscessus in vitro at the same concentration.
Contezolid is compatible with drugs most frequently used to treat M. abscessus infections. M. abscessus infections generally require treatment with multidrug combinations (2, 4). The compatibility between contezolid and eight antimycobacterial drugs that are frequently used therapeutically (i.e., clarithromycin, azithromycin, amikacin, imipenem, cefoxitin, tigecycline, bedaquinoline, and moxifloxacin) was assessed in vitro using the broth microdilution chequerboard titration technique and five randomly selected clinical M. abscessus isolates. No antagonism between contezolid and the aforementioned antimycobacterial drugs was evident (see Table S3).  Preexposure to contezolid does not induce antibiotic resistance in M. abscessus. The risk of resistance induced by contezolid exposure was determined by preexposing M. abscessus strains (ATCC 19977 and two randomly selected clinical M. abscessus isolates) to contezolid at one-fourth and one-half the MIC and then subsequently quantifying the MICs of contezolid and eight other antibiotics postexposure. The MIC values of contezolid, as well as those of the other eight drugs listed above, did not increase following contezolid preexposure (see Table S4). Huang and coworkers reported similar results, i.e., contezolid exhibited a lower potential than linezolid to induce mutations and resistance in S. aureus (17).
In conclusion, contezolid is active against M. abscessus in vitro and is compatible with antibiotics that are most frequently used to treat M. abscessus infections. Therefore, contezolid is a potential candidate to include in novel therapeutic anti-M. abscessus regimens.

ACKNOWLEDGMENTS
We sincerely thank Stephen H. Gregory (Providence, RI, USA) for his help writing and editing this manuscript. We have no conflicts of interest to declare.