| Objectives1.To assess the isolation rates of Mycobacterium avium complex(MAC)in patients with nontuberculous Mycobacteria(NTM)lung disease and evaluate the composition of MAC subspecies;2.To assess the drug resistance profiles in MAC and its subspecies and try to find potentially effective anti-MAC drugs;3.To evaluate the genetic polymorphism and clusters of Mycobacterium avium and Mycobacterium intracellular,respectively,and explore the correlation between antimicrobial resistance phenotype and variable-number tandem-repea(VNTR)genotype in Mycobacterium avium and Mycobacterium intracellular.Methods1.Collected the NTM clinical isolates initially identified by Pnitro benzoic acid(PNB)selected medium in Shenzhen Third People’s Hospital from January 2018 to December2018,all isolates come from respiratory specimens of suspected TB and NTM lung disease patients.16 S r RNA gene sequence was used to identify the composition of NTM and Multi target gene PCR-sequencing of 16 S r RNA,hsp65,rpo B and ITS was performed to identify MAC subspecies.2.Broth microdilution method was used to evaluate the minimum inhibitory concentration of MAC to commonly used medications of MAC lung disease and potentially effective anti-MAC drugs.Commonly used medications of MAC lung disease contains clarithromycin,ethambutol,rifampin,rifabutin,amikacin,streptomycin,linezolid and moxifloxacin recommended by guideline for diagnosis and treatment of NTM disease issued by American Thoracic Society,Infectious Disease Society of America,European Respiratory Society and European Society of Clinical Microbiology and Infectious Diseases and by guideline for in vitro drug susceptibility testing in NTM issued by American Clinical and Laboratory Standards Institute.Referring to the treatment of tuberculosis,potential anti-MAC drugs included 2 kind of three-generation fluoroquinolone drugs(levofloxacin and ciprofloxacin)and 3 kinds of new anti-TB drugs(bedaquiline,clofazimine and delamanid),there were 13 drugs in all.3.15-loci and 16-loci VNTR genotyping were performed for Mycobacterium avium and Mycobacterium intracellular,respectively.Genetic polymorphism,clusters and the correlation between the antimicrobial resistance phenotype and VNTR genotype were counted and analyzed.Results1.Identification of MAC: Through the identified of 16 S r RNA gene sequence,there were 371 NTM isolates and 2 TB in 373 NTM initially identified by PNB selected medium,the false-positive rate of PNB selected medium was 0.54%(2/373).There were 176 MAC isolated in slow-grow NTM(47.44%)and 98 Mycobacterium abscessus in fast-grow NTM.2.Identification of MAC subspecies: There were 7 subspecies in MAC identified by multi target gene PCR-sequencing: Mycobacterium intracellular(27.27%,48/176),Mycobacterium avium(27.27%,48/176),Mycobacterium chimaera(10.80%,19/176),Mycobacterium paraintracellular(10.23%,18/176),Mycobacterium marseillense(4.55%,8/176),Mycobacterium colombiense(7.39%,13/176)and Mycobacterium timonense(1.14%,2/176).3.Drug susceptibility profiles of MAC: The resistance rates of MAC to commonly used medications of MAC lung disease: clarithromycin,ethambutol,rifampicin,rifabutin,amikacin,streptomycin,linezolid and moxifloxacin were 8.38%,22.75%,40.12%,14.97%,11.98%,32.93%,42.51% and 44.31%,respectively;to potentially effective anti-MAC drugs: levofloxacin,ciprofloxacin,bedaquiline,clofazimine and delamanid were 83.83%,90.42%,2.99%,8.98% and 86.83%,respectively.4.Differnence of drug susceptibility profiles of MAC subspecies: Among subspecies of MAC,the resistance rates of Mycobacterium chimaera to clarithromycin were significantly higher than that in Mycobacterium intracellular(26.32% vs.4.69%,P=0.014)and Mycobacterium avium(26.32% vs.6.25%,P=0.014);the resistance rates of Mycobacterium colombiense to moxifloxacin were significantly lower than that in Mycobacterium avium(81.82% vs.37.50%,P=0.0.016)and Mycobacterium chimaera(81.82% vs.26.32%,P=0.007;whereas the resistance rates of Mycobacterium paraintracellular to moxifloxacin were significantly higher than that in Mycobacterium chimaera(64.71% vs.26.32%,P=0.024);moreover,the resistance rates of Mycobacterium intracellular to levofloxacin were significantly higher than that in Mycobacterium avium(92.19% vs.64.58%,P=0.007).5.VNTR genotype: Every VNTR loci of Mycobacterium avium had obvious polymorphism and the HGI was 0.989.There were 3 genotypes in Mycobacterium avium and the clustering rates were 31.25%.Every VNTR loci of Mycobacterium intracellular also had obvious polymorphism and the HGI was 0.994.There were 2genotypes in Mycobacterium intracellular and the clustering rates were 22.34%.6.The correlation between the drug-resistant phenotype and genotype: the resistance rates of clustered isolates to 13 drugs differed from that in non-clustered isolates in Mycobacterium avium and Mycobacterium intracellular,but those differences were not statistically significant.Conclusions1.The predominant species in fast-grow and slow-grow NTM were Mycobacterium abscessus and MAC,respectively,in Shenzhen,and MAC was the most common species in NTM.There were 7 different subspecies in MAC and the most frequently subspecies was Mycobacterium intracellular,followed by Mycobacterium avium.2.The drug resistant profiles of MAC greatly varied,commonly used medications of MAC lung disease showed excellent antimicrobial activities to MAC in vitro,In potentially effective anti-MAC drugs,fluoroquinolones in all showed mediocre antimicrobial activities,and four-generation fluoroquinolone were better than threegeneration fluoroquinolone;among the 3 new anti-TB drugs,bedaquiline and clofazimine showed excellent antimicrobial activities to MAC in vitro while delamanid were resistant to most of MAC.The drug resistant profiles of subspecies varied and the resistant rates of MAC subspecies to clarithromycin,streptomycin,moxifloxacin and levofloxacin were significantly different.3.VNTR genotyping Had high resolution to Mycobacterium avium and Mycobacterium intracellular.The drug resistant profiles of clustered and non-clustered isolates varied but not that significantly,no evidence supported that there was association between antimicrobial resistance phenotype and VNTR genotype in Mycobacterium avium and Mycobacterium intracellular. |
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