| Background: Tuberculosis is a chronic infectious disease which seriously threats public health in recently years. The drug-resistant TB is still a striking problem. Therefore, it is worthy to making research on drug-resistant mechanisms of M.tuberculosis. In our previous study, the expression of transcription regulatory gene dev R was up-regulated in the clinical isolated multi-drug resistant strains. It could be induced by IN H, SM and EMB in drug-resistant strains obviously. It was suggested that dev R was significiently associated with drug-resistant of MTB. In this study, we plan to create a multiple-stress dormancy model in vitro for M. tuberculosis,Then further to explore the relationship between dormant and drug resistance and expression of hbh A.Methods: 1. Construction of In Vitro Dormancy Model for Mycobacterium Tuberculosis Created M.bovis BCG dormancy model by using combined stresses of low oxygen(5%), high CO2(10%), low nutrient(10% Dubos medium) and acidic p H(5.0) to incubate and harvest at diffenert time point in vitro multiple-stress dormancy model. Then carried out the growth curve by detect the OD600 at different time point. Dual staining with the combination of Auramine-O and Nile Red had been used to examine acid- fastness of Mtb cells and lipid body accumulation. quantitative real time PC R was used to check the expression level of dormancy related genes. 2. To explore the relation of the dormancy and resistant of MTB Using Alamar Blue stain to identify the MIC of the BCG dormancy strains. PCR was employed to amplify the resistant related gene of the dormancy strains, Then products were sequenced and performed sequence alignment. 3. The effect of hbh A on M.bovis BCG and dev R-knockout strains BCG and dev R-knockout strains on dormancy culture was detected by the OD600 and stained by Auramine-O and N ile Red at different time point. The expression of hbh A of the BCG and dev R knockout strains was checked it by RT-PCR during dormancy culture.Results: 1. Construction of In Vitro Dormancy Model for Mycobacterium Tuberculosis Comparing to conventional strains, the growth of dormancy strains was significantly decelerated at all phase(P<0.01). It was showed that the only acid- fast stain(green) positive cells gradually decreased and the other type steadily increased during multiple-stress treatment. The expression of dormancy related genes hsp X and dev R was up-regulated at 0-18 days, especially the expression of 18 day was significantly higher than the start day(P<0.05). 2. To explore the relation of the dormancy and resistant of MTB It was showed that the increase of MIC by the days of dormancy culture. By day 18 under multiple-stress, phenotypic resistance of BCG cells against INH increased by about 93.33-fold and resistance against RIF increased by about 22.50- fold. All of the strains did not show gene mutation. 3. The effect of hbh A on M.bovis BCG and dev R-knockout strains Comparing to conventional strains, BCG strains was significantly decelerated at all phase.So was the dev R-knockout strains. The stain of Auramine-O and N ile Red showed that the decrease of acid- fast stain(green) positive cells by the days of dormancy culture on BCG strains. But the dev R-knockout strains still remain the green positive cells on18 th day, The strains keep alive.The expression of hbh A was up-regulated at 1-18 days on the BCG strains, especially the expression of 18 day was significantly higher than the start day(P<0.05). But the number of dev R-knockout strains was reduced at 1-18 days, especially the expression of 18 day was significantly lower than the start day(P <0.05). The BCG strains induced hbh A gene expression than dev R-knockout strains on 18 th day.Conclusions: A multiple-stress dormancy model had been create by using combined stresses of low oxygen(5%), high CO2(10%), low nutrient(10% Dubos medium) and acidic p H(5.0). Then we demonstrated the relationship of dormant and the drug resistance and hbh A. |
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