| ObjectiveResults of the ethambutol resistance in clinic and its molecular mechanism are very complicated and need further investigation, of which the gene mutations associated with EMB resistance especially need the extensively study. For those purpose we investigated the clinical data and the mutations patterns and frequency in those genes related to EMB resistance. We also analyzed the effects of the base alteration on the protein structure and functions and predicted the secondary structure changes of the noncoding RNA in intergenetic regions. Method1. Total 2129 clinical Mycobacterium tuberculosis strains and the clinical data were included for the clinical retrospective study. Demographic data of the clinical cases and the EMB resistance of the clinical isolates were analyzed by comparison of EMB resistant group with EMB sensitive groups. EMB-resistant risk factors focused on the irregular medication, adverse drug reaction and the primary drug resistance. In contrast with INH and RFP, the irregular medication of the drug medication times, irregular treatment duration and the treatment strategy were investigated.2. Total 23 genes or intergenetic regions(IGs) related to EMB resistance were included to find their mutations. They were Rv0179 c, Rv0026, Rv1058, Rv1699, Rv2807, Rv2947 c, Rv3756 c, Rv3877, Rv1080c-Rv1081 c, Rv1482c-Rv1483, Rv2416c-Rv2417 c, Rv3796-Rv3797, Rv2427c-Rv2428, Rv2733-Rv273, Rv2754c-Rv2755 c, Rv3428c-Rv3429, Rv3793-Rv3794, Rv3901c-Rv3902 c, Rv3806, emb B, emb A, emb C and emb R. All 183 EMB-resistant isolates were included in this study. The DNA extracted from the 183 isolates was used as the templates for amplifying hot spot regions or full length of the 23 genes or IG by PCR method. Then the PCR products were sequenced twice. Mutations were found by Aligning with the Mycobacterium tuberculosis H37Rv(ATCC 27294). The affection of the aino acid changes for those genes was analyzed by the software of Predict SNP. And the synonymous mutations were analyzed by compared with the codon preference data base for Mycobacterium tuberculosis H37Rv(ATCC 27294). The secondary structure changes of the non-coding RNA with mutations were analyzed by the software of Gene Quest. Results1. In total, 228(10.71%) ethambutol(EMB) resistant clinical isolates were found in 2129 clinical isolates. There were 1130 were pan-sensitive strains(53.08%) in 2129 clinical isolates. Of the 228 EMB-resistant strains 181 cases(79.4%) were from the primary EMB-resistant patients and 47 strains(20.6%) were from the retreatment patients. Of the 228 EMB-resistant isolates 164(71.9%) were resistant to INH, 171(75%) were resistant to RFP and 134(58.8%) were MDR-TB. Comparing with the EMB sensitive group, the patient ratio of primary and retreatment was significantly high(P=0.01), and there were no difference in the EMB rsistant patients for their age, gender, history of smoking and the concurrent other diseases(P>0.05) in comparison with the EMB sensitive patients.In concern to the resistant spectrum of the EMB resistant clinical isoates, the average resistant drug numbers of the EMB resistant isolates were significantly more than the EMB sensitive ones(P<0.001). The average resistant drug number of the EMB resistance group was 2.19± 0.10(mean±SD), and 0.79 ±0.04 for the EMB sensitive group. The proportion of MDR-TB(58.8%) was significantly higher in EMB resistant group than that of EMB sensitive group(17.0%),which indicated that the high proportion of MDR-TB in EMB-resistant isolates were the main cause of the broad resistant spectrum of the EMB-resistant isolates.Risk factors resulting in the EMB resistance were analyzed and the results showed that there were 47.25% of the EMB resistant patients were resulted from the irregular treatment, 4.40% from the adverse drug reaction and 48.35% from primary drug resistance. Further analysis on the irregular treatment including the treatment times and medication duration showed that the EMB resistance aroused after medication for 2.86±0.69 times, which was significant higher than that of INH(1.67±0.88) and that of RFP(1.39±0.61) and that the medication duration(less than 6 months) before generating the resistance was 5±0.94 months for EMB, 3.78±1.28 months for INH and 3.89±0.60 months for RFP. While the medication durations(6 months or more) were not significant fifferent before generating the resistance among the EMB, INH and RFP.2. Of all the 23 tested genes mutations in the following genes of Rv0026, Rv1058, Rv1699, Rv2807, Rv3756, and intergetic regions of Rv2416c-Rv2417 c, Rv3796-Rv3797, Rv2733-Rv2734, Rv2754c-Rv2755 c, Rv3428c-Rv3429 were seldom found, with the mutantion frequency of 2.5%, 1.25%, 1.25%, 2.5%, 0, 0, 0, 0, 2.5% and 1.25%, respectively. Each mutation was found in no more than two isoaltes. Some mutations were found in Rv0179 c, Rv2947 c and Rv3877.One hundred and sixty isolates were tested for the mutations in Rv0179 c, of which 18 mutation patterns were found in 46 isoaltes(28.75%). They were S102*, A198 G, S214 L, L215 P, D236 E, F254 L, V62 A, G164 A, S166 I, W167 C, S169 T, T170 T, N182 T, H183 R, Q186 R, K202 E, G204 A and Y227 F. One hundred and sixty seven isolates were tested for the mutations in Rv2947 c, of which two mutations patterns, D213 G and P448 S, were found in 8 isoaltes(4.79%). One hundred and sixty eight isolates were tested for the mutations in Rv3877, of which 11 mutations patterns were found in 16 isoaltes(9.52%). Their mutations patterns were P39 S, E45 D, G370 G, I439 I, L454 L, L88 L, C203*, Q281 H, I289 T, A354 A and S466 S.The base alteration frequency of the emb R, emb A, emb C, emb B and Rv3806 was 3.3%, 3.8%, 4.1%, 74.17% and 8.46%, respectively. Mutations in emb R, emb A, emb C and Rv3806 mostly co-existed with the mutations in emb B. In emb B mutations were found at six mutation sites. They were emb B306, emb B328, emb B354, emb B406, emb B439, emb B497 and emb B531, with the frequency of 54.6%, 1.8%, 3.7%, 31.7%, 8.3% and 5%, respectively. Six types of mutant showed three amino acid changes(M306V> M306I> M306L) at the site of emb B306. Totally 54.6 percent clinical isolates showed amino acid changes at emb B306 and31.7 percent at emb B406. But emb B406 always did not happen in combination with emb B306.The prediction of the drug-resistance related mutations showed that some mutations were deleterious. They were P243 S in P243 S, L105 V and R380 P in emb A, M306 L, D328 H, G406 D, Q497 K and Q497 R in emb B, S166 I, W167 C, K202 E, L215 P, D236 E and F254 L in Rv0179 c, P39 S and I289 T in Rv3877 and D213G in Rv2947 c. Others mutation patterns were have neutral effects on the protein functions.Five of ten tested intergenetic regions Rv1080c-Rv1081 c, Rv1482c-Rv1483, IG1872(Rv2427c-Rv2428), IG2934(Rv3793-Rv3794) and IG3021(Rv3901c-Rv3902c) were found with more than one mutations and each mutation pattern was always found in more than one isolates. The non-coding RNA changed their secondary structure when those mutations happened. Conclusion1. EMB resistance was common in TB clinic, which happened mainly in primary EMB resistance patients. The EMB resistance occurrence was not related with the patient’s age, gender, history of smoking and tuberculosis complicated with other diseases.2. The EMB resistant isoaltes had broader drug resistant spectrum, of which MDR-TB was the major.3. In clinic the EMB resistance was not easier to produce than INH and RFP, which was supported by the longer continued medication duration and more medication times before the arouse of their resistance.4. Of all 23 tested genes emb gene showed the most mutation frequency. The codons of emb B306 and emb B406 in emb B had the highest mutation frequency. But emb B406 always did not happen in combination with emb B306(P=0.037).5. Mutations on three genes were frequently found. They were Rv0179 c, Rv2947 c and Rv3877.6. Five of ten intergenetic regions Rv1080c-Rv1081, Rv1482c-Rv1483, IG1872(Rv2427c-Rv2428), IG2934(Rv3793-Rv3794), IG2991(Rv3862c-Rv3863)and IG3021(Rv3901c-Rv3902c) showed higher mutation frequency in EMB-resistant clinical isolates. Those mutations make the non-coding RNA change their secondary structure.7. Some synonymous mutations resulted in the deleterious effect of the protein functions by reducing the the codon preference, which might influence protein synthesis, then affected the resistance phenotype. |
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