Molecular Analysis of Linezolid-Resistant Clinical Isolates of Mycobacterium abscessus

A total of 194 Mycobacterium abscessus isolates were collected from patients, and the whole genomes were sequenced. Eighty-five (43.8%) isolates showed linezolid (LZD) resistance.

Alternations in the LZD target sites. Whole genomes of the 194 strains were sequenced (BioProject PRJNA488058 from this study and PRJNA448987 and PRJNA448987 from our previous studies), including 96 isolated in 2017 and 98 isolated during 2012 to 2016 (13,17). The sequences of the entire 23S rRNA, L3, L4, and L22 proteins were extracted from the whole-genome sequence data of each strain and compared with those from reference strain ATCC 19977. A total of 26 mutation types were observed in 23S rRNA. Detailed information about the mutations is listed in Table S1 in the supplemental material. Nine mutations were found in 7 (8.2%) LZD-resistant strains, indicating that these mutations contributed to LZD resistance (Fig. 1B, red). Other 17 mutations in 23S rRNA were present in either susceptible strains or in both susceptible and resistant strains, suggesting that they do not contribute to LZD resistance. No meaningful mutations were found in L3, L4, and L22 in LZD-resistant strains. These results suggest that a mutation in ribosomal proteins is not responsible for LZD resistance in most of the strains isolated in this study.
The methyltransferase genes cfr, rlmN, and spr033 and the pseudouridine synthase gene rulC that modify the 23S rRNA at the LZD binding sites are known to affect LZD susceptibility (18)(19)(20)(21). However, none of them were found in our 194 isolates.
Sequence alignment showed that the homologs of drrABC, rv0987, lmrS, acrAB, mmpL9, and acrF were present in all of the 194 M. abscessus isolates, except for optrA.
Therefore, LZD-resistant isolates with a significant MIC fold change (4-fold) upon efflux pump inhibition (n ϭ 6), along with 6 randomly selected LZD-susceptible isolates (MICs, 0.5 to 4 mg/liter), were selected and subjected to quantitative real-time PCR (qRT-PCR) analysis, as previously described (17). Primer pairs for amplification of each gene were as follows: mmpL9, ACGTCATTTCAGCTCTGCCA/AAGGGGCGGGTGATACTTTG; drrC, GTCGAGTACAGCACGCGATA/TAATCCGACCAGCAACCCAC; drrA, GTCCCGGATTGGC GAAATTG/GCTGCTTTTCCATCTCGCTG; lmrS, TGGTCAATGCTCGCATTCCT/ATCGGGTATCC CCTTGGTCA; acrF, ACTTCGTTGCGTTCCTCGAT/AGCGTTGTCACTCAACACCA; and acrB, GATTCGGTATCGGTGGCTGT/CCGGATTCTCCTCGACGAAC. As shown in Fig. 2, the LZDresistant strains had Ͼ50-fold (P ϭ 0.004) and Ͼ5-fold (P ϭ 0.04) increased transcriptional levels of lmrS and mmpL9, respectively, compared to the LZD-susceptible strains.  These results indicated that efflux pumps lmrS and mmpL9 play an important role in LZD resistance in M. abscessus. No difference in the transcription levels of drrABC, rv0987, acrAB, or acrF was observed between the LZD-susceptible and resistant groups (data not shown). Whole-genome comparative analysis. For 25% of the LZD-resistant M. abscessus isolates in this study, resistance could not be explained by known mechanisms, suggesting the presence of novel mechanisms for LZD resistance. Accordingly, genome comparative analysis was conducted and identified 24 genes that were highly associated with LZD resistance (P Ͻ 0.01), such as genes encoding MmpL10, which is known to mediate drug resistance in M. tuberculosis (25), and FabG, which is required for antibiotic resistance in P. aeruginosa (26). Detailed information for these genes is listed in Table S2.
In conclusion, this study suggests that rather than mutations or modifications of LZD target sites, efflux pumps played a predominant role in LZD resistance of M. abscessus. Whole-genome sequencing and comparative analyses also identified new LZD resistance-associated genes, which set the foundation for elucidation of the mechanism of LZD resistance in M. abscessus.
Accession number(s). Whole-genome sequences have been deposited under Bio-Project no. PRJNA488058.

SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/AAC .01842-18.