| Objectives To understand the drug resistance and drug target gene mutation characteristics of Mycobacterium tuberculosis(MTB)in Tangshan area;to understand the expression levels of MTB DNA repair system genes and proteins in different clinical strains;to analyze the relationship between drug resistance of clinical isolates strains and drug target gene mutations and DNA repair system expression level.To explore the correlation between Nth gene,an important gene in the DNA repair system,and drug target gene mutations and MTB resistance,and to further clarify the drug resistance mechanism of Mycobacterium tuberculosis.Methods 193 cases of pulmonary tuberculosis patients diagnosed and hospitalized by Tangshan Fourth Hospital from October 2015 to November 2016 were selected as the research subjects,and their general conditions were investigated.The bacteria in the patient's sputum were isolated and cultured,and acid-fast staining was used to identify Mycobacterium tuberculosis.Drug sensitivities of Mycobacterium tuberculosis,isolated from the sputum of the research subjects,were identified by the drug sensitivity kit.According to reports in the literature,15 target gene loci of four first-line antituberculosis drugs were selected for sequencing.The above two results were descriptively analyzed to clarify the drug resistance characteristics and target gene site mutation characteristics of MTB in Tangshan.The changes of gene and protein expression levels in the DNA repair system may be one of the causes of gene mutations and drug resistance.RT-PCR and Western Blot methods were used to detect the expression of mRNA and protein respectively,which are in MTB DNA repair system.Expression levels of the repair gene and proteins in different susceptibility-type strains were analyzed.And more in-depth research was implemented among their expressions with mutations in drug target gene sites.Phage-mediated homologous recombination gene knockout method and seamless cloning and ligation technology were used to perform knockout and supplementary over-expression experiments on the specific gene,Nth gene,which was detected from clinical strains.The results of experiments were identified by PCR technology and bacterial culture.Four kinds of first-line anti-tuberculosis drugs,isoniazid,rifampicin,ethambutol and streptomycin were used to induce the drug resistance to the standard strains H37 Rv,Nth knockout strain and Nth over-expression strain.The choices of drug concentrations were referred to the clinical drug dosage and drug sensitivity test concentration.The results of drug resistance were detected by 960 automatic Mycobacterium tuberculosis culture systems.The growth curves of the standard strains H37 Rv,Nth knockout and Nth over-expression strains were measured and recorded.The minimum inhibitory concentration of the three strains was identified by the micro-broth dilution method to further clarify the effect of Nth gene on MTB resistance.Perform drug target gene loci sequencing on the strains with successful resistance induction and compare the gene mutations.Results Among the 193 clinically isolated strains,96.37% are human-type strains;the rates of resistance to first-line and second-line anti-tuberculosis drugs are 37.37% and 75.65% respectively,of which the proportion of multi-drug resistant strains is 3.63% and the proportion of polyresistance strains is 15.54%;the resistance rates of isoniazid,rifampicin,streptomycin and ethambutol are 12.44%,12.95%,21.76%,and 17.62% respectively;the host's body mass index,occupation,and history of tuberculosis treatment are the influencing factors for drug-resistant tuberculosis;the mutation rates of EmbB 306 locus,RpoB 531 and 526 loci are the highest among 15 drug target gene loci,which are 80.66%,72.62% and 55.36% respectively,the mutation rates of the remaining loci are less than 50%;the mRNA and protein expression levels of some genes in the DNA repair system are significantly different among different drug-susceptible strains;the mRNA and protein expression trends of End,Nth,Nei,and Mpg are consistent,and the expressions in sensitive strains are higher than that in drug-resistant strains(P<0.05);SigA,DnaQ and DnaE1,they show the opposite expression trends;the mRNA and protein expression levels of Nth gene are significantly different between the mutated and non-mutated groups on RpoB 531 locus,which is target gene of rifampin(P<0.05).This result was analyzed by the research on the relationship between the mutation of the target gene loci and the expression levels of the repair genes;the induction of drug resistance was performed on the standard strain H37 Rv,Nth knockout strain and Nth over-expressing strain.The results of comparison show that the strains without Nth gene entered the logarithmic growth phase and the plateau phase in the drug environment earlier than the H37 Rv and Nth over-expression strains;during single-drug induction,only Nth over-expressed strains were not detected bacterial growth in high concentrations of rifampicin and ethambutol,and other strains were resistant to all drugs;when the two drugs were combined,Nth knockout strains and the standard strain H37 Rv are resistant to low concentration drugs;when the three drugs are used together,Nth over-expressed strains have not detected growth in all drug environments,and only Nth knockout strains are resistant to low concentration drugs.In the environment of three-drug combined application,the survival rate of the strain is significantly lower than that of the two-drug combined and single-drug environments;Nth over-expressed strains are more difficult to develop drug resistance in the drug environment,especially in which rifampin participates;the minimum inhibitory concentration of Nth over-expressed strains in rifampicin is 5.29 times as that of standard strain H37 Rv and 9.25 times as that of Nth knock-out strains;among the strains that successfully induced drug resistance,only 4 strains were detected with gene mutations,and all are Nth knockout strains.Conclusions 1 The resistance rates of clinically isolated Mycobacterium tuberculosis in Tangshan area to first-line and second-line drugs are high,and the target gene mutation level is close to the national average;body mass index,occupation,and history of tuberculosis treatment are influencing factors for drug-resistant tuberculosis;2 The differences in mRNA and protein expression of some genes in the DNA repair system are related to the development of resistance to MTB.The mRNA and protein expression differences of some genes in the DNA repair system are related to the occurrence of MTB resistance.The changes in the Nth gene level of this system are related to bacterial resistance and mutation in the RpoB 531 locus,which is the target gene of rifampin;3 Deletion and over-expression of Nth gene are related to MTB growth,drug target mutations and drug resistance;4 Combined application of multiple drugs can reduce the growth of MTB in the pharmaceutical environment.Figure 33;Table 32;Reference 292... |
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