Identification And Molecular Characterization Of Drug-resistant Mycobacteria Isolates Recovered From Wuhan,China

Tuberculosis (TB) is the greatest killer worldwide due to a single infectiousagent. Incidence and mortality caused by drug-resistant Mycobacterium tubeculosis(Mtb) is growing recently so fast that it is difficult to control TB. The number ofhuman diseases infected with NTM is increasing around the world, yet it is oftendifficult to distinguish NTM from Mtb, and NTMs are naturally resistant to mostanti-tuberculosis drugs.PCR and DNA sequencing technology were used for identifying the104strains isolated from pulmonary patients in Wuhan, China. According to sequencesof16S rDNA, ITS region, and INS gene fragment,85stains (81.7%) wereidentified as Mtb, the most common pathogens in patients with NTM pulmonaryinfection were M. abscessus (n=7,6.8%), followed by M. avium complex (MAC)(n=4,3.8%). Interestingly, it is found that7.7%of isolates belong to the genusGordonia, including three species: Gordonia bronchialis, Gordonia paraffinivorans,and Gordonia sptui. In addition, one strain was detected as Nocardia farcinica.Of the85Mtb isolates,20strains were pan-susceptible and65weredrug-resistances. The resistance-determining regions or hot spots of ten genomeregions associated with drug resistance, including rpoB for rifampin (RIF); katG,inhA, ahpC, mabA-inhA promoter, and intergenic region of oxyR-ahpC for isoniazid(INH); rpsL and rrs for streptomycin (SM); and embB for ethambutol (EMB), wereanalyzed by DNA sequencing-based method. Approximately91.7%(55/60) ofRIF-resistant isolates carried mutations in rpoB, the most frequently mutationcodons were531,526, and516, with mutation rate of51.7%(31/60),18.3%(11/60),and10.0%(6/60), respectively. Eight strains with seven different types of doublemutations were identified in rpoB gene. Three novel point mutations inRIF-resistant Mtb isolates were detected in rpoB: E458A, S509R, and P535S. Atleast one of the mutations in the katG, inhA, ahpC, mabA-inhA promoter, andintergenic region of oxyR-ahpC was found in84.4%(54/64) of the INH-resistant isolates, and mutations at katG codon315were detected at rate of68.8%(44/64).Five new mutations were detected in katG, including three point mutations: G273S,I266T, and P232S, one insertion mutation, and a10.8kb fragment deletion mutation.Of the45SM-resistant clinical isolates,34strains carried mutations in rpsL gene,with K43R and K88R as the most common mutation, and one novel mutation K88Ewas detected. Sequencing revealed four differernt mutations in rrs gene in13resistant isolates, and one mutation was also observed in one pan-susceptible strain.54.5%（15/22）of EMB-resistances were detected to carry mutations at embB306,but this mutation was detected in EMB-susceptible isolates, too. Our data confirmthat mutation at embB306does not confer resistance to EMB but is a rathercommon polymorphism in clinical strains of M. tuberculosis predisposing them tothe development of any type of drug resistance.These observations will contribute to developing a rapid and reliable detectionapproaches for species identification of mycobacteria and drug resistance screeningfor drug-resistant M. tuberculosis, and the drug resistance mechanisms of thesefirst-line antituberculosis drugs was not completely clear and still needs furtherresearch.