MAB_2355c Confers Macrolide Resistance in Mycobacterium abscessus by Ribosome Protection.
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ABSTRACT:
SUBMITTER: Guo Q
PROVIDER: S-EPMC8373217 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mycobacterium abscessus has emerged as a successful pathogen owing to its intrinsic drug resistance. Macrolide and lincosamide antibiotics share overlapping binding sites within the ribosome and common resistance pathways. Nevertheless, while M. abscessus is initially susceptible to macrolides, they are completely resistant to the lincosamide antibiotics. Here, we have used RNA sequencing to determine the changes in gene expression in M. abscessus upon exposure to the lincosamide, clindamycin (CLY). We show that Mab_1846, encoding a putative ARE-ABCF protein, was upregulated upon exposure to macrolides and lincosamides but conferred resistance to CLY alone. A Mycobacterium smegmatis homologue of Mab_1846, Ms_5102, was similarly found to be required for CLY resistance in M. smegmatis. We demonstrate that Ms5102 mediates CLY resistance by directly interacting with the ribosomes and protecting it from CLY inhibition. Additional biochemical characterization showed that ribosome binding is not nucleotide dependent, but ATP hydrolysis is required for dissociation of Ms5102 from the ribosome as well as for its ability to confer CLY resistance. Finally, we show that in comparison to the macrolides, CLY is a potent inducer of Mab_1846 and the whiB7 regulon, such that exposure of M. abscessus to very low antibiotic concentrations induces a heightened expression of erm41, hflX, and Mab_1846, which likely function together to result in a particularly antibiotic-resistant state.
| S-EPMC8522731 | biostudies-literature
Project description:Mycobacterium abscessus infections tend to respond poorly to macrolide-based chemotherapy, even though the organisms appear to be susceptible to clarithromycin. Circumstantial evidence suggested that at least some M. abscessus isolates might be inducibly resistant to macrolides. Thus, the purpose of this study was to investigate the macrolide phenotype of M. abscessus clinical isolates. Inducible resistance to clarithromycin (MIC > 32 microg/ml) was found for 7 of 10 clinical isolates of M. abscessus previously considered susceptible; the remaining 3 isolates were deemed to be susceptible (MIC <or= 0.5 microg/ml). Inducible resistance was conferred by a novel erm gene, erm(41), which was present in all 10 isolates and in an isolate of Mycobacterium bolletii (M. abscessus type II). However, the erm(41) alleles were nonfunctional in the three susceptible M. abscessus isolates. No evidence of erm(41) was found in Mycobacterium chelonae, and an isolate of Mycobacterium massiliense appeared to be an erm(41) deletion mutant. Expression of erm(41) in M. abscessus conferred resistance to clarithromycin and erythromycin and the ketolide HMR3004. However, this species was found to be intrinsically resistant, independent of erm(41), to clindamycin, quinupristin (streptogramin B), and telithromycin. The ability to confer resistance to clindamycin and telithromycin, but not quinupristin, was demonstrated by expressing erm(41) in Maycobacterium smegmatis. Exposure of M. abscessus to the macrolide-lincosamide-streptogramin B-ketolide agents increased the levels of erm(41) mRNA 23- to 250-fold within 24 h. The inducible macrolide resistance phenotype of some M. abscessus isolates may explain the lack of efficacy of macrolide-based chemotherapy against this organism.
| S-EPMC2663066 | biostudies-literature
Project description:Mycobacterium abscessus is associated with antibiotic resistance and poor treatment outcomes. We described within-patient changes in M. abscessus resistance to clarithromycin and amikacin. Patients with amikacin exposure and a >50-month interval between M. abscessus isolates were identified. Antimicrobial susceptibility testing was performed on the first and last isolates by broth microdilution, and genetic markers of resistance were identified. 16 patients were identified with a median amikacin exposure of 2.3 years (range 0.6-8.6 years). 15 patients also received macrolides (median 7.2 years, range 1.3-10.7 years). All initial isolates were resistant to clarithromycin (minimum inhibitory concentration (MIC) ≥8 µg·mL-1). Two patients had later susceptible isolates, which were of a different subspecies (M. abscessus subsp. massiliense) than the initial isolates (M. abscessus subsp. abscessus). All initial isolates were susceptible or intermediately resistant to amikacin, and only one patient had a resistant final isolate (MIC >64 µg·mL-1), accompanied by an A→G mutation at position 1408 of the 16S ribosomal RNA. Forced expiratory volume in 1 s decreased significantly over the study period, while smear quantity and the proportions of patients with elevated C-reactive protein or cavitary lesions all increased significantly. Despite prolonged, mostly inhaled amikacin exposure, development of amikacin resistance was uncommon in this patient population; however, disease progression continued.
| S-EPMC6536947 | biostudies-literature
Project description:Mutations in the erm(41) gene of M.abscessus group organisms are associated with differences in inducible macrolide resistance, with current recommendations being to hold rapidly growing isolates for up to 14 days in order to ensure that resistance which develops more slowly can be detected. This study aimed to determine the ideal incubation time for accurate identification of inducible macrolide resistance as well as to determine if there was an association between the time taken to detect inducible resistance in M.abscessus group organisms and their erm(41) sequevar. We amplified and sequenced the erm(41) genes of a total of 104 M.abscessus group isolates and determined their sequevars. The isolates were tested for phenotypic clarithromycin resistance at days 7, 10, 14 and 21, using Trek Diagnostics Sensititre RAPMYCO microbroth dilution plates. Associations between erm(41) gene sequevars and time to detection of resistance were evaluated using Fisher's exact test in R. The samples included in this study fell into 14 sequevars, with the majority of samples falling into Sequevar02 (16), Sequevar06 (15), Sequevar08 (7) and Sequvar 15 (31), and several isolates that were in small clusters, or unique. The majority (82.7%) of samples exhibiting inducible macrolide resistance were interpreted as resistant by day 7. Two isolates in Sequevar02, which has a T28C mutation that is associated with sensitivity, showed intermediate resistance at day 14, though the majority (13) were sensitive at day 14. The majority of isolates with inducible macrolide resistance fell into Sequevars 06,08 and 15, none of which contain the T28C mutation. These sequevars were analyzed to determine if there was any correlation between sequevar and time to detection of resistance. None was found. Based on these findings, we recommend the addition of a day 7 read to the CLSI guidelines to improve turn-around-times for these isolates. It is also recommended that erm(41) gene sequencing be added to routine phenotypic testing for the resolution of cases with difficult-to-interpret phenotypic results.
| S-EPMC4973996 | biostudies-literature
Project description:BackgroundThe clinical impact of infection with Mycobacterium (M.) abscessus complex (MABC), a group of emerging non-tuberculosis mycobacteria (NTM), is increasing. M. abscessus subsp. abscessus/bolletii frequently shows natural resistance to macrolide antibiotics, whereas M. abscessus subsp. massiliense is generally susceptible. Therefore, rapid and accurate discrimination of macrolide-susceptible MABC subgroups is required for effective clinical decisions about macrolide treatments for MABC infection. We aimed to develop a simple and rapid diagnostic that can identify MABC isolates showing macrolide susceptibility.MethodsWhole genome sequencing (WGS) was performed for 148 clinical or environmental MABC isolates from Japan to identify genetic markers that can discriminate three MABC subspecies and the macrolide-susceptible erm(41) T28C sequevar. Using the identified genetic markers, we established PCR based- or DNA chromatography-based assays. Validation testing was performed using MABC isolates from Taiwan.FindingWe identified unique sequence regions that could be used to differentiate the three subspecies. Our WGS-based phylogenetic analysis indicated that M. abscessus carrying the macrolide-susceptible erm(41) T28C sequevar were tightly clustered, and identified 11 genes that were significantly associated with the lineage for use as genetic markers. To detect these genetic markers and the erm(41) locus, we developed a DNA chromatography method that identified three subspecies, the erm(41) T28C sequevar and intact erm(41) for MABC in a single assay within one hour. The agreement rate between the DNA chromatography-based and WGS-based identification was 99·7%.InterpretationWe developed a novel, rapid and simple DNA chromatography method for identification of MABC macrolide susceptibility with high accuracy.FundingAMED, JSPS KAKENHI.
| S-EPMC7910664 | biostudies-literature
Project description:Plasmid-mediated kanamycin resistance was detected in a strain of Mycobacterium abscessus subsp. bolletii responsible for a nationwide epidemic of surgical infections in Brazil. The plasmid did not influence susceptibility to tobramycin, streptomycin, trimethoprim-sulfamethoxazole, clarithromycin, or ciprofloxacin. Plasmid-mediated drug resistance has not been described so far in mycobacteria.
| S-EPMC3993635 | biostudies-literature
Project description:Mycobacterium abscessus is an emerging pathogen against which clarithromycin is the main drug used. Clinical failures are commonly observed and were first attributed to acquired mutations in rrl encoding 23S rRNA but were then attributed to the intrinsic production of the erm(41) 23S RNA methylase. Since strains of M. abscessus were recently distributed into subspecies and erm(41) sequevars, we investigated acquired clarithromycin resistance mechanisms in mutants selected in vitro from four representative strains. Mutants were sequenced for rrl, erm(41), whiB, rpIV, and rplD and studied for seven antibiotic MICs. For mutants obtained from strain M. abscessus subsp. abscessus erm(41) T28 sequevar and strain M. abscessus subsp. bolletii, which are both known to produce effective methylase, rrl was mutated in only 19% (4/21) and 32.5% (13/40) of mutants, respectively, at position 2058 (A2058C, A2058G) or position 2059 (A2059C, A2059G). No mutations were observed in any of the other genes studied, and resistance to other antibiotics (amikacin, cefoxitin, imipenem, tigecycline, linezolid, and ciprofloxacin) was mainly unchanged. For M. abscessus subsp. abscessus erm(41) C28 sequevar and M. abscessus subsp. massiliense, not producing effective methylase, 100% (26/26) and 97.5% (39/40) of mutants had rrl mutations at position 2058 (A2058C, A2058G, A2058T) or position 2059 (A2059C, A2059G). The remaining M. abscessus subsp. massiliense mutant showed an 18-bp repeat insertion in rpIV, encoding the L22 protein. Our results showed that acquisition of clarithromycin resistance is 100% mediated by structural 50S ribosomal subunit mutations for M. abscessus subsp. abscessus erm(41) C28 and M. abscessus subsp. massiliense, whereas it is less common for M. abscessus subsp. abscessus erm(41) T28 sequevar and M. abscessus subsp. bolletii, where other mechanisms may be responsible for failure.
| S-EPMC5192163 | biostudies-literature
Project description:Mycobacterium abscessus is a major nontuberculous mycobacterial (NTM) pathogen and is responsible for about 80% of all pulmonary infections caused by rapidly growing mycobacteria. Clofazimine is an effective drug active against M. abscessus, but the mechanism of resistance to clofazimine in M. abscessus is unknown. To investigate the molecular basis of clofazimine resistance in M. abscessus, we isolated 29 M. abscessus mutants resistant to clofazimine and subjected them to whole-genome sequencing to identify possible mutations associated with clofazimine resistance. We found that mutations in the MAB_2299c gene (which encodes a possible transcriptional regulatory protein), MAB_1483, and MAB_0540 are most commonly associated with clofazimine resistance. In addition, mutations in MAB_0416c, MAB_4099c, MAB_2613, MAB_0409, and MAB_1426 were also associated with clofazimine resistance but less frequently. Two identical mutations which are likely to be polymorphisms unrelated to clofazimine resistance were found in MAB_4605c and MAB_4323 in 13 mutants. We conclude that mutations in MAB_2299c, MAB_1483, and MAB_0540 are the major mechanisms of clofazimine resistance in M. abscessus Future studies are needed to address the role of the identified mutations in clofazimine resistance in M. abscessus Our findings have implications for understanding mechanisms of resistance to clofazimine and for rapid detection of clofazimine resistance in this organism.
| S-EPMC6021651 | biostudies-literature
Project description:Streptomycin, the first drug used for the treatment of tuberculosis, shows limited activity against the highly resistant pathogen Mycobacterium abscessus We recently identified two aminoglycoside-acetylating genes [aac(2') and eis2] which, however, do not affect susceptibility to streptomycin. This suggests the existence of a discrete mechanism of streptomycin resistance. M. abscessus BLASTP analysis identified MAB_2385 as a close homologue of the 3″-O-phosphotransferase [APH(3″)] from the opportunistic pathogen Mycobacterium fortuitum as a putative streptomycin resistance determinant. Heterologous expression of MAB_2385 in Mycobacterium smegmatis increased the streptomycin MIC, while the gene deletion mutant M. abscessus ΔMAB_2385 showed increased streptomycin susceptibility. The MICs of other aminoglycosides were not altered in M. abscessus ΔMAB_2385. This demonstrates that MAB_2385 encodes a specific and prime innate streptomycin resistance determinant in M. abscessus We further explored the feasibility of applying rpsL-based streptomycin counterselection to generate gene deletion mutants in M. abscessus Spontaneous streptomycin-resistant mutants of M. abscessus ΔMAB_2385 were selected, and we demonstrated that the wild-type rpsL is dominant over the mutated rpsLK43R in merodiploid strains. In a proof of concept study, we exploited this phenotype for construction of a targeted deletion mutant, thereby establishing an rpsL-based counterselection method in M. abscessus.
| S-EPMC5740355 | biostudies-literature
Project description:Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 Å resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.
| S-EPMC7990689 | biostudies-literature