Mycobacterium Tuberculosis Resistant Molecular Characteristics Of The Beijing Area, And A Preliminary Study Of Mycobacterium Tuberculosis Resistant To Quinolones Pump Mechanism

Objective: To study the Beijing area isolates of Mycobacterium tuberculosis katG,inhA, oxyR, ahpC, ndh, dfrA, kasA, inhA promoter region, oxyR-ahpC intergene,rpoB, rpsL, rrs, gyrA, gyrB, tlyA and thyA genes mutation characteristics, and discussthe Mycobacterium tuberculosis to isoniazid, rifampicin, streptomycin, ethambutol,quinolones, amikacin, capreomycin and sodium para-aminosalicylic resistantmolecular mechanism. Methods:303cases of isoniazid-resistant strains and61casesof isoniazid-susceptible strains,195cases of rifampicin-resistant strains and169cases of rifampicin-susceptible strains,360cases of streptomycin-resistant strains and53cases of streptomycin-susceptible strains,266cases of ethambutol-resistant strainsand32cases of ethambutol-susceptible strains,118cases of quinolone-resistantstrains and128cases of quinolone-susceptible strains,10cases of amikacin resistantstrains,25cases of capreomycin-resistant strains,15cases of amikacin andcapreomycin cross-resistant strains and30cases of double-susceptible strains,31cases of sodium para-aminosalicylic resistant strains and65cases on the sodiumpara-aminosalicylic susceptible of clinical isolates related genes were amplified byPCR and DNA sequencing. Results:(1) The303cases of isoniazid-resistantMycobacterium tuberculosis, katG gene mutations rate was84.8%and97.4%,mutation was focus of katG85-463codon district, including the S315T sites mutationrate was up to55.4%, while80.3%of the R463L mutation sites rate in the susceptiblestrains was higher than62.7%in drug-resistant strains (P <0.05);18.8%pointmutations of inhA the promoter region in the-15(C→T) was higher than4.9%of thesusceptible strains (P <0.05);9.6%of oxyR-ahpC intergenic region mutant inresitance strains was higher than1.6%of susceptible strains (P <0.05).②In the195cases of rifampicin resistant strains,168cases (86.2%) had rpoB gene mutation, themutations were mainly in the531(41%),526(22.5%),516(9.7%) sites, while25cases (92.6%) were rifampicin resistant strains in27multisite mutations strains.③The360cases of streptomycin resistant strains, the rpsL gene mutation rate was71.7%and rrs gene mutation rate was11.6%, respectively; the rpsL gene of K43R and K88R point mutation proportion was60.6%and11.1%, respectively, while therrs gene A1401G site (2.2%) in the resistant strains was less than the susceptiblestrains (9.4%), statistically significant was different between the two groups (P <0.05).④The266cases of ethambutol resistant strains were detected, mutation rate of theembB gene was45.1%,98.3%of mutations was in the codon region of319-497,306codon is most common (26.1%).⑤In the118cases of quinolone resistant strainswere detected, mutation rate of gyrA gene was74.6%, the most common was94codon (49.9%) and90codon (19.5%).⑥The tlyA gene mutation results showed thatamikacin and susceptible strains had any mutant; capreomycin had six mutations(24%) in single drug-resistant strains, amikacin and capreomycin cross-resistantstrains had one case of synonymous mutations; mutation rate of A1401G in amikacin,capreomycin, amikacin and capreomycin cross resistant strains were10%,8%,13.3%,respectively; C1402T in amikacin, amikacin and capreomycin cross-resistance drugshad one case, respectively.⑦T he31cases of para-aminosalicylic resistant strains,mutation rate of thyA gene was71%,19deletion base C and168deletion bp Cmutation were most common, the mutation rate were54.8%,25.8%in the resistantstrains,35.5%and12.3%in susceptible strains; two sites mutation differences in theresistant and susceptible strains were not statistically significant (P>0.05).Conclusions: This study further clarifies the relationship between the eight kinds ofanti-TB drugs of Mycobacterium tuberculosis with gene in Beijing, and contribute tostudy the popular molecular characteristics of the Beijing and establish rapid, specific,and accurate drug susceptibility molecular diagnostic methods. Objective: Selecting of levofloxacin (LFX) resistant and sensitive clinicalisolates,to analyse the effect of efflux pump systems in the LFX resistance and screenof the LFX drug efflux pump genes. Methods: The minimum inhibitoryconcentration (MIC) changes was analysing before and after adding the three pumpinhibitor reserpine (Res),carbonyl cyanide-chlorophenyl hydrazone (CCCP) andverapamil (Ver) by the resazurin susceptibility testing methods,13kinds of effluxpump gene expression were detected by quantitative PCR after drug stimulating.Results:59cases were the LFX resistant strains, and four cases were sensitive strains.Before using the pump inhibitors,52drug-resistant strains of the MIC were greaterthan2mg/l (containing2mg/l),7strains of the MIC were less than2mg/l; afterapplication of pump inhibitors Res,43resistant strains of MIC were greater than2mg/l,16strains of the MIC were less than2mg/l, there was significant differencebefore and after application of pump inhibitors (X2=4.374, P<0.05), MIC values waslower2-16times; after application of pump inhibitor CCCP, all the59resistantstrains of MIC values were less than2mg/l, difference was statistically significantbefore and after the application of pump inhibitor (X2=970, P <0.05), MIC valueswas lower2-64times; after application of pump inhibitors Ver,31resistant strains ofMIC were greater than2mg/l,28strains of the MIC were less than2mg/l,resistance rate has dropped significantly, the difference was statistically significant(X2=17.913, P <0.05) before and after the application of pump inhibitors, the MICvalues was lower2-64times; pump inhibitors also had effected the four cases ofsusceptible strains.The effect of CCCP and Ver was most obvious: after applicationof pump inhibitors only a MIC value was2mg/l, while the application of the pumpinhibitors Res, MIC changes was not obvious. The gyrA and gyrB genes weredetected in59LFX resistant strains and4LFX-sensitive strains,45(95.8%,45/47)mutations strains of the MIC values were greater than2mg/l, only the two D94Gmutant strains MIC values were2mg/l. Furthermore,12resistant strains had no any mutant, all63strains of gyrB gene had no mutant.12resistant mutant strains MICvalues results show that the12resistant strains of the MIC values were lower2-16times after the application of pump inhibitors CCCP; the10resistant strains of MICvalues decreased2-4times after the application of pump inhibitor Ver, only451and575strains of the MIC values did not change;729,777,896,838,529,642and325strains of the MIC values reduced by2-4times after application of pump inhibitorsRes, the other five strains of the MIC values did not change after the application ofpump inhibitors. After the LFX drugs to stimulate, the results showed that the10kinds of pump gene level were decreased after drug treatment, including Rv1348andRv2686c, level of genes were0.52and0.42times comparing pre-drug treatment. Thelevel of three pump gene were increased, Rv2846c gene level was1.21timescomparing pre-drug treatment; Rv3239c gene level was1.17times comparingpre-drug treatment; Rv2936gene level was1.06times comparing pre-drug treatment.Conclusions:Target genes of gyrA mutation was the main mechanism of quinoloneresistance and associated with high level of quinolone resistance. Mycobacterialefflux pump systems involved in the occurrence of quinolone resistance, pumpinhibitor could inhibited efflux pump system, which CCCP of effect is the mostsignificant. Potential efflux pump genes level of Rv2846c, Rv3239c and Rv2936areincreased in the resistant strains, may be involved in the occurrence ofMycobacterium tuberculosis resistant to quinolone drugs.