Study On The Cause Of Tuberculosis Recurrence And The Molecular Mechanism Of Drug Resistance

Tuberculosis (TB) is an ancient infectious disease, which killed millions of people in history. Recently, this disease has reemerged worldwide due to the migration of population, the prevalence of HIV infection, the transmission of drug resistant strains and the effects of political and economic policies. China is a country with a high burden of tuberculosis. Although China has made significant progress controlling this disease, there are still many challenges for tuberculosis prevention and control. One of major obstacles in tuberculosis control is the high rate of tuberculosis recurrence. In this study, molecular epidemiology methods were used to describe tuberculosis recurrence and to determine whether each case of tuberculosis was due to relapse or reinfection in Shanghai, China. The rate of drug resistant is high among the recurrent patients and the molecular mechanism of ethambutol resistance is still debated, so the potential mechanism of ethambutol resistance in mycobacteria was explored by epidemiological method and by screening a transposon mutagenesis library. Therefore, the study is divided into two parts.Part I: Study on the cause of tuberculosis recurrenceIn order to determine whether tuberculosis recurrence among TB patients was due to relapse or reinfection, the recently developed mycobacterial interspersed repetitive units (MIRU) typing, a genotyping method never before used in China, was first evaluated for its suitability to differentiate the isolates in the local region. Ninety-one M. tuberculosis clinical isolates were randomly selected from the collection of isolates at the Shanghai Municipal Center for Disease Control and Prevention (SCDC), and were genotyped using the MIRU method. The 91 isolates were divided into 46 genotypes. In this sample, 12 MIRU showed different discriminative power according to their allelic diversity. Locus 26 showed the highest discrimination, while locus 16, 31, 40 were moderately discriminative. Compared with another PCR-based genotyping method, specifically spoligotyping, the MIRU typing method had higher discriminative power. The MIRU method then was used to study the cause of tuberculosis recurrence. M. tuberculosis isolates from patients who experienced 2 successive tuberculosis episodes during the years from 1999 to 2004 were genotyped. In 32 of the 52 tuberculosis recurrent cases, isolates from the two tuberculosis episodes showed different MIRU patterns, indicating that 62% of therecurrences were due to exogenous reinfection. The frequency of exogenous reinfection decreased with the patients' age from 100% (4/4, ≤30 years of age) to 67% (12/18, 31 to 60 years) and 53 % (16/30, ≥60 years). However, the frequency of exogenous reinfection increased with the time interval of recurrent tuberculosis. In the recurrent cases that occurred within 6 months, 47% were caused by exogenous reinfection. If the time interval to the recurrent episode was over one year, 74% of the recurrent cases were due to reinfection. The high proportion of exogenous reinfection in recurrent TB patients suggests that there are high levels of transmission of M. tuberculosis and that ongoing transmission is an important cause of new cases of tuberculosis. Therefore, more attention should be paid to interrupting the route of transmission. Our findings indicate that the immunity evoked by the primary infection with M. tuberculosis couldn't effectively protect the host against a second infection with M. tuberculosis. Determining the mechanism behind this phenomenon is a challenge for the development of a more efficacious vaccine.Part II: Study of the molecular mechanism of ethambutolEthambutol (EMB) is used extensively as a first-line antituberculosis drug. Previous studies suggested that ethambutol resistance is associated with resistance to other first-line antituberculosis drugs. In order to study whether this phenomenon is widespread, drug susceptibility test data of the isolates collected by SCDC during the past ten years were analyzed. The frequency of EMB mono-resistance was significantly lower than that of other first-line drugs (isoniazid, rifampin and streptomycin). Also, the number of EMB drug resistant strains increased in proportion with the number of other drugs that the strain was resistant to. These observations indicate that the EMB drug resistance was associated with resistance to other drugs. In order to investigate the recent controversy surrounding the association between the mutation at condon 306 of embB (embB306) and EMB resistance, 116 drug resistant strains and 54 pan-susceptible strains of M. tuberculosis were analyzed for mutations in embB306. Among the 27 EMB drug resistant strains, 17(17/27, 63%) strains had the embB306 mutation, while among 89 EMB drug susceptible strains and strains with resistant to other drugs, 17(17/89, 19%) strains had the embB306 mutation. Statistical analysis showed that embB306 was associated with EMB drug resistance and with resistance to other antituberculosis drugs. In addition, the frequency of the embB306 mutation increased with the number of other drugs to which the strain was resistant. It may not be useful to identify EMB mono-resistant strain by screening forthe embB306 mutation, as the prevalence of this drug resistance type in clinical isolates is extremely low. However, since the embB306 mutation is associated with multiple drug resistance (MDR-TB), this locus could be a potential marker to rapidly screen for the multiple drug resistant strains in the clinical setting.The mechanism of drug resistance for mycobacteria differs from that of other bacteria. Genetically horizontal transfer by plasmids, transposons and integrons occurs frequently in other bacteria, but rarely occurs in mycobacteria. The mutation and deletion of genomic DNA in the chromosome is likely the main way that contributes to drug resistance. In order to fully understand the molecular mechanism of ethambutol resistance in tuberculosis, M. smegmatis was used as a model to search for new genes related to EMB drug resistance. Using the himar-based transposon system, a mutagenesis library was constructed. Southern blot showed that himar1 transposon randomly inserted into the chromosome as a single copy. This library was screened for EMB drug resistant mutants. Four drug resistant mutants, designated as ER2A, ER4A, ER5A and ER7A, respectively, were isolated. The minimal inhibitor concentration (MIC) test showed that all 4 mutants were resistant to EMB at least 20 fold more than the wild type strain. After identifying the locus of transposon insertion of the mutants by adaptor PCR and marker rescue methods, bioinformatics methods was used to analyze the gene function of the insertion locus. A gene coding for monoxide enzyme in ER2A, a gene probably coding for imported lipoprotein in ER4A, and a gene named arsA coding for the transport protein in ER5A were disrupted by transposon. In ER7A, the transposon inserted a gene of unknown function. The detailed mechanisms about the association of these genes and EMB drug resistance in mycobacteria need to be further explored.