| Tuberculosis (TB) is a chronic disease caused by Mycobacterium tuberculosis complex (MTBC) which primary infect the human respiratory system. According to WHO reports, there are nearly a third of the world population have been infected with Mycobacterium tuberculosis (MTB). Although the prevalence of TB is in the drop trend, the annual new cases have exceeded 9 million cases, and with the emergence of resistant strains, treatment and prevention of tuberculosis have been facing great challenge. Therefore, the identification and typing of MTB isolates and drug resistance monitoring have important meaning for disease diagnosis, treatment and prognosis. In this study, MTB isolates were collected in Yangzhou city, a set of techniques including the traditional bacteriological methods and multilocus PCR, MIRU-VNTR and Spoligotyping typing method were used in the preliminary identification of Mycobacterium species epidemiology research, respectively. Finally, the drug resistance of MTB was detected by the ratio method, MIC and the mutations of drug resistance gene had also been detected. 1 Isolation and identification of Mycobacterium tuberculosis in YangzhouAfter identified using Ziehl Neelsen staining, MTB isolates were further verified by PNB/ TCH test and multilocus PCR from those acid fast bacilli strains, and compared the results differences between two methods.289 of clinical isolates were collected as acid-fast bacilli in Yangzhou. Among them,270 strains of MTB,9 strains of nontuberculous mycobacteria (NTM), 2 strains of M. africanum,8 strains of nontypeable were identified using PNB/TCH test and multilocus PCR. It was shown that result of multilocus PCR has no significant differences compared with that of PNB/TCH test. The identification results showed that the majority of TB cases in Yangzhou are caused by MTB, but there are also few cases caused by NTM. 2 Subtyping of Mycobacterium tuberculosis isolates in YangzhouThe genome DNAs from a total of 270 MTB strains were extracted using CTAB method, and further genotyped using MIRU-VNTR and Spoligotyping techniques. Spoligotyping distinguished 8 groups, including 233 strains belonging to Beijing family,15 strains of T1 family, 11 strains of unknown family,4 strains of Beijing-Like family,3 strains of U family,2 strains of H3 family,1 strain of MANU 2 family,1 strain of T2 family. And the 270 isolates was divided into 154 genotypes by MIRU-VNTR method, which 28 clusters were consist of 144 isolates, the remaining 126 isolates were independent genotypes, and the resolution of the method was 0.960. In 15 VNTR loci, regardless of to all isolates or only to the Beijing family isolates, analysis showed that Mtub21 locus HGDI values were the highest, which were 0.545 and 0.451 respectively. Therefore, MIRU-VNTR method showed higher resolution (0.960) compared to Spoligotyping (0.253), and the combination of these two genotyping methods could make higher discrimination. We found that MTB isolates of Yangzhou had some kind of gene polymorphism by genotyping. Beijing family were the major epidemic genotype in this city, and the trend was highly concentrated. At the same time, the Spoligotyping method also found 11 new genotypes, which enriched the SpolDB4 database.3 Analysis of drug resistance phenotypes and resistance genes of Mycobacterium tuberculosis isolates in YangzhouDrug resistance testing were carried out by using the conventional ratio method and MIC method for 174 clinical resistant isolates, and then sequencing analyzed the resistant genes of identified resistant MTB isolates (mainly for the four first-line drugs:isoniazid INH, rifampicin RFP, streptomycin SM, ethambutol EMB). Ratio method results showed that 72 strains were drug resistance in 174 strains of MTB (41.4%),32 multidrug resistance strains accounted for 18.4%. There was no significant difference between the results of MIC method and the traditional ratio method in the drug-resistance detection of 72 resistance strains. Resistance gene sequence alignment results showed that in INH resistant strains, katG mutation rate for 72.0% (36/50), inhA mutation rate was 6.0%(3/50); in RFP resistant strains, rpoB mutation rate was 67.5%(27/40); in EMB resistant strains, embB mutation rate was 52.6%(10/19); in SM resistant strains, rrs mutation rate was 15.4%(6/39), rpsL mutation rate was 71.8%(28/39). Through the comparison analysis, resistance gene mutations of MTB isolates are closely related to the drug-resistant phenotype, appearance of resistance phenotype strains do not necessarily occur mutation of drug resistance gene, but resistance gene mutation strains are all drug resistant.In conclusion, MTB Yangzhou isolates between 2012 and 2015, were investigated through the isolation and identification, molecular typing, drug sensitivity tests and analysis of antibiotic resistance gene mutation, the distribution and propagation characteristics of MTB were demonstrated at the molecular level. This study provided scientific data for the control and prevention of TB in Yangzhou. It also have useful application on the investigation of MTB molecular epidemiology, and surveillance of MTB drug resistance. |
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