Drug Resistance In Mycobacterium Tuberculosis: Molecular Mechanism And The Development Of Rapid Detection Method

Tuberculosis (TB) is the most common cause of death due to a single infectious agent; it is responsible for approximately 2 million deaths annually around the world. The problem is becoming more critical with the emergence and spread of drug resistant strains of Mycobacterium tuberculosis, together with infection of HIV. China is not only one of the 22 high-burden countries that collectively account for approximately 80% of the world's tuberculosis cases, but also the hotspot area of very high prevalence of drug-resistant tuberculosis identified by WHO. To study on the molecular mechanism of drug-resistance in M. tuberculosis and establish a rapid molecular approach for detection of drug-resistant M. tuberculosis is urgent for control of tuberculosis.This study focus on the drug resistance of rifampin (RFP) and isoniazid (INH), which are the most important first-line anti-tuberculosis drugs, and ethambutol(EMB) which drug-resistance-remains unknown. DNA sequencing analysis was used to investigate genetic alterations in the rpoB, katG, inhA regulatory region and embB in 87 Mycobacterium tuberculosis isolates recovered from Henan, China. Of the 47 RFP-resistant isolates, 43 (91.5%) had mutations in the amplified region of rpoB. The most frequent mutation (58.1%, 25/43) occurs at codon 531. Fourteen types of missense mutation involving 15 kinds of amino acid were detected. A new kind of double mutation was discovered. Among 49 INH-resistant isolates, 36 isolates (73.5%) were detected to harbor mutation at codon 315 of katG. Four INH-resistant isolates were detected to carry C→T mutation at-15 positon of inhA. At least one mutation was found in the katG and inhA regulatory region in 79.6% (39/49) of the INH-resistant isolates. Among EMB-resistant isolates, 16 isolates (53.4%) had mutations in embB306. However, among 24 EMB-susceptible but RFP and NIH-resistant isolates, 4 isolates (16.7%) were also found to carry embB306 mutation. At the same time, a new type of mutation M306I (ATG→ATC) was reported first in China. Alterations at embB306 may not confer resistance to EMB. EmbB306 mutants were more frequently accompanied by rpoB mutations (100%, 19/19) than by katG 315 mutations 73.7% (14/19). Our results show that geographic variation in the molecular genetic mechanism is responsible for drug resistance in drug-resistant M. tuberculosis. This observation will facilitate the development of a rapid and reliable molecular drug resistance approach for screening drug-resistant M. tuberculosis.It is essential to develop a rapid approach for detection of rifampicin and isoniazid resistance of in multidrug-resistant M. tuberculosis isolates. Base on PCR-reverse dot blot, a multiplex probe array was designed for simultaneously screening the mutations in rpoB, katG, inhA, and ahpC genes that are associated with multidrug-resistant M. tuberculosis. Among the 52 multidrug-resistant isolates, the genotypic test identified 46 isolates (88.5%) with mutations in rpoB. Compared with PCR-DNA sequencing, there was 98.1% agreement between the wild-type probes assay and DNA sequencing, and there is 100% agreement between the mutant-type probes assay and DNA sequencing. By using the novel probe array, 45 isolates (86.5%, 45/52) were detected to harvor at least one mutaion at codon 315 of katG, or inhA regulatory region, or ahpC intergenic region. There was complete agreement between wild-and mutant-probes assay, and DNA sequencing by testing 10 INH-resistant and 10 INH-susceptible isolates. All 35 susceptible isolates showed a wild type hybridization pattern. Compared with conventional drug susceptibility testing, the sensitivity and specificity of the probe array were 88.5% and 100% for rifampicin resistance and 86.5% and 100% for isoniazid resistance, respectively. The novel genotypic test developed was a rapid, easy-to-perform, and reliable assay for the detection of the most frequent mutations leading to rifampicin and isoniazid resistance in multidrug-resistant strains of M. tuberculosis. This genotypic test has the potential to be developed into a clinical application for the rapid detection of resistant strains before chemotherapy is initiated, thus prevent the transmission of multidrug-resistant strains of tuberculosis in community.At present, mycobacteriosis is on the increase. Non-tuberculous mycobacteria (NTM) are resistant to most anti-tuberculosis drugs naturally. Furthermore, the transmission approach is different between mycobacteriosis and tuberculosis. Accordingly, differential identification of NTM and M. tuberculosis is very important for clinical therapy and management. Conventional identification of mycobacteria is based on the culture of the isolates which is time consuming, laborious, and is not always conclusive. Base on PCR-reverse dot blot, an efficient and affordable novel multiplex probe array which allows simultaneous identification of 15 medically important mycobacterial species was developed. Sixteen genus-, complex-, and species-specific probes were designed according to the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer region, and 23S rRNA gene of mycobacteria. This novel multiplex probe array was applied for the identification of 92 mycobacterial clinical isolates recovered from Henan, China. The results showed that the specificity and sensitivity of the probe array were 100% for both genus-specific probe and M. tuberculosis complex-specific probe. Among 62 isolates of non-tuberculous mycobacteria, 50 isolates (80.4%) can be rapidly identified to the species level. Genetic and geographical variability of 16S-23S rRNA gene internal transcribed spacer were analyzed in some isolates of M. avium, M. intracellulare, M. chelonae, M. abscessus and M. fortuitum. With the accumulation of the sequences of internal transcribed spacer, and further optimization of probes, the multiplex probe array may be developed into a useful tool for rapid and accurate. identification of mycobacterial species in clinical laboratory.