| Tuberculosis(TB)is a respiratory infectious disease caused by Mycobacterium tuberculosis {Mtb),which seriously affects human health.According to the 2018 global report on tuberculosis,the estimated number of new TB cases was 10 million in 2017.About 1.6 million people died of TB,and 550,000 people developed MDR-TB,which is also resistant to isoniazid and rifampicin.China is the second largest country with high burden of tuberculosis and also one of the countries with the largest number of drug-resistant tuberculosis cases.The biggest challenge facing WHO’s goal of ending TB by 2035 comes from the emergence and spread of drug-resistant TB.Rapid and accurate identification of drug-resistant tuberculosis bacteria can provide a basis for formulating effective treatment programs,thereby reducing the production of drug-resistant bacteria.The identification of drug-resistant tuberculosis bacteria mainly includes drug susceptibility test and molecular biology diagnosis.Compared with drug susceptibility test based on culture,molecular biology diagnosis is based on the detection of drug-resistant gene mutation sites.It has advantages of easy operation,high sensitivity and rapidity.It is more and more used in the clinical diagnosis of drug-resistant tuberculosis.However,there are still several challenges for identification of drug resistant isolates with molecular biology diagnosis techniques,such as the relationship between specific gene mutations and phenotypic drug resistance,and impaction of the combined gene mutations to the level of drug resistance.Isoniazid(INH)is an important kind of first-line anti-tuberculosis drugs.It is recommended by WHO for DOTS treatment.It can also be used for preventive treatment of latent tuberculosis infection.INH resistance involves many genes of Mtb.At present,it is known that at least 20 genes are related to INH resistance,including genes related to metabolic enzyme activity,cell wall synthesis and efflux pump.Due to the limited information obtained by conventional PCR amplification,previous studies mainly focused on some genes and high-frequency mutation sites,such as tefG 315 and inhA promoter region-15 mutations.Because of limit samples and representativeness,there is little information about those fewer frequent INH resistance-related genes.At present,there is no systematic and complete report on all known INH resistance-related gene mutations in clinical strains.In this study,high-throughput genome sequencing was used to analyze the mutations of INH resistance-related genes of clinical isolates in China.The focus was the impact of combined mutations of multiple genes on the evolution of INH resistance.The genome-wide sequencing analysis of 20 INH drug resistance related genes were performed among 201 tuberculosis isolates,which included 168 MDR-TB,20 INH single-drug-resistant and 13 susceptible strains randomly from the collection of the recent national survelence of drug resistance.At the same time,the corresponding result of INH resistance level for each isolate was determined by minimum inhibitory concentration(MIC).Our study showed that:1)There were 96.8%(182/188)of INH-resistant clinical isolates detected mutations in INH-resistant genes,including 18 structural genes and 2 promoter regions.There was no mutation detected in accD6 and fabG1.Among these mutation types,katG,inhA/inhA-P and ahpC/ahpC-oxyR regions had the highest mutation rate,accounting for 85.7%(162/188),respectively.21.8%(41/188)and 18.6%(35/188),the mutation frequencies of other genes were between 0.53%-4.26%.2)For katG gene,56 non-315 site mutations(45.21%(85/188)were detected in addition to three katG 315 mutations(51.6%(97/188).39 of them were reported for the first time.3)INH resistance gene mutations were highly differentiated in China.A total of 96 mutation combinations were detected,including 28 single gene mutations and 68 multi-gene combined mutations,accounting for 51.60%and 45.21%of resistant strains respectively.3)The combined mutations were mainly katG combined mutations,including katG 315 combined mutations and katG non 315 combined mutations.The co-mutations of high drug resistance were katG 315 and inhA-P,katG non315 and ahpC-oxyR.Our conclusion is that combined gene mutations are common in clinical isolates of INH-resistant strains in China.The combined katG 315 with inhA-P.katG non315 with ahpC-oxyR genes are related to high-level INH resistance.In addition,the frequency of katG 315 mutations was 54.76%of MDR-TB strains,it means that the current commercial kit could not meet the clinical needs in China,which used katG 315 mutations as the main target for the identification of INH resistance.This study provides valuable information for understanding the mechanism of INH resistance in M.tuberculosis,and gives data support for the establishment of more sensitive INH detection method. |
