| The incidence of Mycobacterium abscessus(M.abscessus)infections has increased significantly in recent years.M.abscessus accounts for a large proportion of lung disease cases caused by Non-Tuberculous Mycobacteria(NTM).As a highly drugresistant opportunistic pathogen,infections caused by M.abscessus are difficult to treat and options are limited.Macrolide,represented by clarithromycin(CLA),are currently recognized as the core drugs for the treatment of M.abscessus infections,and are the only oral antibiotics proven to be effective for long-term use.However,the emergence of CLA resistance is a challenge in treating M.abscessus infections.In addition,the currently known CLA resistance mechanisms cannot fully explain the resistance of clinical strains,and new genes and mechanisms related to CLA resistance need to be further supplemented.This three-part study assesses the impact of CLA resistance development on clinical outcomes in patients with M.abscessus lung disease,explored the role of efflux pump in CLA resistance isolates,and provides insight into the mechanisms by which the ABC-F protein MAB＿2355c confers resistance to macrolides.Part I Objective: To compare the clinical features and therapeutic efficacy in M.abscessus lung disease patients with different CLA-susceptibility genotypes and to evaluate the predictive value of genotyping on the treatment outcomes.Methods:Patients with M.abscessus lung disease were divided into a CLA-resistant genotype group [including rrl 2058/2059 mut type and erm(41)T28 type] and a sensitive genotype group [including erm(41)C28 type and erm(41)M type] based upon the CLAsusceptibility genotype of the organism isolated.A retrospective analysis was conducted to compare the clinical features and treatment outcomes of the two genotype groups.Potential predictors of the response to treatment were also assessed.Results:Sixty-nine patients were included in the CLA-resistant genotype group,includeing 5patients with rrl 2058/2059 mutants and 64 patients with erm(41)T28 type;31 patients were included in the CLA-sensitive group,including 6 patients with erm(41)C28 type and 25 patients with erm(41)M type,respectively.Patients in CLA-sensitive group exhibited higher sputum negative conversion,radiological improvement rates,and better therapeutic outcomes than those in the CLA-resistant group(P=0.006;P=0.013;P<0.001).In multivariate analysis,genotype(OR=0.185,95%CI [0.059,0.579],P=0.004)was a predictor of treatment effect.Conclusion: Significant differences in the outcome of CLA-based combination therapy between patients infected with CLAsensitive and-resistant M.abscessus genotypes,with a therapeutic advantage in the sensitive genotype group.Accurate and rapid genotyping is a reliable predictor of the efficacy of CLA-based combination antibiotics therapy.Part II Objective: To screen efflux pumps related to CLA resistance in M.abscessus and explore the role of efflux pumps in the development of resistance in clinical strains.Methods: 194 clinical M.abscessus isolates were collected from patients with M.abscessus lung infections and the whole-genome of each isolate was sequenced.A comprehensive examination of the molecular mechanisms underlying intrinsic CLA resistance was performed,combining CLA susceptibility assays and genome sequence analysis.The efflux pump genes related to CLA resistance were further screened by bioinformatics analysis and verified in clinical strains by real-time quantitative PCR(q RT-PCR).Results: Of the 194 isolates,13(6.7%)were CLA resistant;No specific resistance-associated mutation were observed in the ribosomal proteins,rpl C,rpl D,rpl V,or target-site modified gene erm(41)in all resistant strains.Only 7 of these harbored a rrl 2270/2271 mutation,and the remaining 6 acquired resistant strains could not be explained by known resistance mechanisms,with MICs were mostly distributed in the range of 8-32 mg/L.In addition,the presence of efflux pump inhibitors significantly decreased the MIC of CLA for non-susceptible isolates,especially the intrinsic resistant isolates that exhibited no rrl 2270/2271 mutation.Bioinformatics predicted 7 efflux pump genes associated with CLA resistance.Further q RT-PCR analysis showed that the increased expression of the efflux pump genes,MAB＿2355c,MAB＿1409c and MAB＿1846,consistently correlated with increased CLA resistance.Conclusion: These findings indicate that efflux pump-mediated resistance mechanism is complementary to currently known CLA resistance mechanism in M.abscessus and is primarily involved in mediating low-level resistance in clinical isolates.MAB＿2355c,MAB＿1409c,MAB＿1846 are candidate M.abscessus CLA resistanceassociated efflux pump genes.Part III Objective: To elucidate the function of MAB＿2355c and the mechanism mediating macrolide resistance in M.abscessus.Methods: The potential mechanism by which MAB＿2355c exerts macrolide resistance was explored by bioinformatics analysis,MAB＿2355c protein expression and purification,ATP hydrolysis assay,gene knockout and complementation,antibiotic sensitivity,and transcription-translation assays.Results: MAB＿2355c is a putative ATP-binding cassette F(ABC-F)family protein.Purified MAB＿2355c protein exhibits ATP hydrolysis ability that can be inhibited by ribosomal targeting antibiotics.The transcript level of MAB＿2355c could be significantly upregulated after exposure to macrolides.MAB＿2355 deleted M.abscessus was more sensitive to macrolides than it wild control.Colony morphology of M.abscessus changed from smooth to rough after MAB＿2355c knockout.Finally,MAB＿2355c rescued the transcription and translation activities affected by macrolides in vitro.Conclusion: MAB＿2355c is the first reported antibiotic resistance-related ABC-F subfamily protein in M.abscessus,which has ATPase activity and confers M.abscessus resistance to macrolide through a ribosome protection mechanism. |
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