Analysis Of The Correlation Between The Phenotype And Genotype Of Common Pathogenic Non-Tuberculosis Mycobacterium

ObjectivesTo explore the prevalence of nontuberculous mycobacterium(NTM)in South China,and to make clear the drug susceptibility profiles and analyze the genetic polymorphism of nontuberculous mycobacterium,further to analyze the association between the genotype and drug susceptibility.Methods1.A total of 1571 clinical isolates identified as nontuberculous mycobacterium between 2012 and 2014 from two TB specialized hospitals were recruited in this study.Sequencing of partial genes,including 16S rRNA,hsp65,rpoB,and the 16S-23S rRNA internal transcribed spacer(ITS)was performed to identify the strains identified as NTM with paranitrobenzoic acid(PNB)in solid media at species level.2.Minimal inhibitory concentrations(MIC)were tested by microplate alamar blue assays to assess the drug susceptibility of 20 drugs to nontuberculous mycobacterium.3.Genotyping was performed by using variable number of tandem repeats(VNTR)for Mycobacterium avium,Mycobacterium intracellulare and Mycobacterium abscessus complex.PCR-restricted fragment length polymorphisms(RRA)was performed to genotype Mycobacterium kansasii.4.Comparing the association between the genotype and drug susceptibility.Results1.1450 isolates were identified as NTM.The most frequently isolate was Mycobacterium abscessus(23.7%),followed by Mycobacterium massiliense(17.9%)and Mycobacterium intracellulare(17.3%).2.In vitro drug susceptibility testing,amikacin was the most effective agents against Mycobacterium avium(87.8%),and tigecycline had the highest resistance rate(100%);clarithromycin was the most effective agents against Mycobacterium intracellulare(88.6%),and tigecycline,imipenem and meropenem had the highest resistance rate(100%);amikacin was the most effective agents against Mycobacterium kansasii(94.9%),and tigecycline had the highest resistance rate(97.4%);amikacin was the most effective agents against Mycobacterium abscessus(93.1%),and rifampicin had the highest resistance rate(100%);amikacin was the most effective agents against Mycobacterium massiliense(89.0%),and stremptomycin and imipenem had the highest resistance rate(98.8%).Compared with Mycobacterium intracellulare isolates,the significant higher susceptibility rates of linezolid,meropenem,imipenem,cefoxitin,gatifloxacin and sulfamethoxazole were observed in Mycobacterium avium isolates(P<0.05).And the higher susceptibility rates of moxifloxacin,gatifloxacin,sulfamethoxazole and tigecycline were observed in Mycobacterium abscessus isolates than that in Mycobacterium massiliense isolates,but minocycline-susceptibility isolates were significantly higher among Mycobacterium massiliense than that among Mycobacterium abscessus(P<0.05).3.Each VNTR loci of Mycobacterium avium,Mycobacterium intracellulare,Mycobacterium abscessus and Mycobacterium massiliense has obvious polymorphism and HGI was between 0.933 and 0.999,and the cluster rate was 29.3%,36.4%,13.8%and 27.0%,respectively.By using PCR-restriction fragment analysis of the hsp65 gene(PRA),all the Mycobacterium kansasii isolates were classified as four different subtypes with subtype I(71.8%)was the most frequently genotype in China.4.The proportion of clustered isolates resistant to capreomycin,azithromycin,levofloxacin,streptomycin,meropenem and cefoxitin was higher than that of unclustered isolates in Mycobacterium avium,but there was no significant difference(P<0.05).The proportion of unclustered isolates resistant to levofloxacin,cefoxitin and minocycline was significantly higher than that of clustered isolates in Mycobacterium intracellulare,Mycobacterium abscessus and Mycobacterium massiliense respectively(96.4%vs.81.3%,28.2%vs.10.0%,54.6%vs.40.9%)(P<0.05).26.8%of the predominant subtype I of Mycobacterium kansasii was associated with clarithromycin resistance.ConclusionsThe species of NTM varies in South China with 12 different species,and the five most frequently isolates were Mycobacterium abscessus complex,Mycobacterium intracellulare cmoplex,Mycobacterium gordonae,Mycobacterium kansasii and Mycobacterium fortuitum.The drug susceptibility profiles of different species,and the clustered and unclustered isolates vary greatly.So accurate identification,genotype and drug susceptibility testing is essential to guide clinical treatment of NTM lung disease.