【作者】 陈曦；

【导师】 马玙； 金奇；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 2003， 硕士

【摘要】 目的 阐明结核分枝杆菌耐异烟肼临床分离株katG基因、inhA基因、ahpC基因、kasA基因及oxyR基因突变特点，探讨是否可能通过DNA序列分析来判断结核分枝杆菌对异烟肼的敏感性。方法 对144株结核分枝杆菌临床分离株(101株异烟肼耐药株及43株异烟肼敏感株)katG，inhA，ahpC，kasA及oxyR基因进行PCR扩增及DNA序列分析。结果 (1)101株耐异烟肼结核分枝杆菌临床分离株中未发现katG基因完全缺失。共检测到80.20％(81/101)的菌株存在点突变、小的缺失或插入。其中16个突变位点未见过报道。异烟肼耐药株中共有38.61％(39/101)的菌株发生Ser315Thr或Ser315Ash突变(Ser315Thr[n=31]，Ser315Ash[n=8])，突变形式为AGC→ACC(Ser→Thr)或AGC→AAC(Ser→Ash)。结合菌株耐药程度，低耐药组(1ug/ml)315位氨基酸突变率显著高于高耐药组(10ug/ml)315位氨基酸突变率(P＜0.05)。57.43％(58/101)的耐异烟肼菌株存在Arg4631eu突变，此位点氨基酸突变与细菌耐异烟肼无关；其中44株联合其它类型的突变，占75.86％(44/58)。本项研究中还发现12株耐异烟肼株发生碱基缺失；还在1株耐药株中检测到单碱基插入，此株细菌还联合同义突变及Arg4631eu突变。12株发生缺失的菌株中，7株对异烟肼低度耐药，其中6株是1716位碱基缺失的菌株；发生碱基插入的唯一1株细菌对异烟肼高度耐药。另外，其它氨基酸位点发生突变的菌株为15.84％(16/101)株。在43株异烟肼敏感株中发现有53.49％(23/43)株存在463位点氨基酸突变，突变形式与异烟肼耐药株相同；另外在43株异烟肼敏感株中检测到发生同义突变的1株细菌。(2)101株耐异烟肼结核分枝杆菌临床分离株中共有4.95％(5/101)株细菌inhA基因发生突变(Ser94Ala[n=2]，Ile21Thr[=2]，缺失[n=1])，其中4株为错义突变，均与katG基因463位点联合突变；1株为单碱基缺失，且联合katG315突变。94位氨基酸突变的2株细菌低度耐药，21位氨基酸突变的2株细菌高度耐药，碱基缺失的细菌高度耐药。43株异烟肼敏感株中没有检测到inhA基因突变。(3)101株耐异烟肼结核分枝杆菌临床分离株中共有3株(2.97％)细菌有错义突变(Ala195Asp[n=2]，Gln169Arg[n=1])，均为高度耐药，其中1株细菌合并katG基因463位点突变。摘要Glnl 69Aig为新发现的突变位点。43株异烟脐敏感株中没有检测到ahpc基因突变。 (4)101株耐异烟阱结核分枝杆菌临床分离株中共有16.83%(17/101)株发生KasA基因突变(GlylZILys[n=21，Gly312Ser【n=7]，Gly387Aspln=3]，phe413Leu[n=l]，AsP66Asn汇n=4J)，均为低度耐药。在7株312位点发生突变的耐异烟脱株中，5株与katG基因463位点联合突变;其它位点突变占9.90%(10/101)，此10株细菌katG，1 nhA，ahpC，oxyR基因中未检测到突变。43株异烟脐敏感株中检测到3株细菌存在oly3 12Ser突变。(5)在144株临床分离株中未发现oxyR基因突变。(6)综合本项研究中katG基因、inllA基因、曲pC基因及kasA基因的突变情况，共有85株耐异烟脱菌株发生与异烟脱耐药相关的突变(katG67株、inhAS株、ahpC3株、kasA10株)，占84%(85/101)。结论由于与异烟脱耐药相关的基因数量多，因此给分析耐异烟脐结核分枝杆菌临床分离株的基因型带来不便。本项研究进一步证实了结核分枝杆菌耐异烟脐与katG，illhA，曲pC，kasA基因突变之间的关系，并且提示还有其它尚未阐明的机制参与异烟脱耐药。本项研究结果还说明在不同的地理区域有不同的突变发生。更多还原

【Abstract】 Objective: To elucidate the characterization of katG, inhA, ahpC, kasA, and oxyR gene mutations in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis, and discuss the value of judging the susceptibility of Mycobacterium tuberculosis strains to isoniazid by DNA sequencing. Method: A total of 101 isoniazid-resistant and 43 susceptible strains of Mycobacterium tuberculosis were analyzed by PCR and sequence analysis of their katG, inhA, ahpC, kasA, oxyR genes. Results: (1) Sequencing of katG from 101 INH-resistant strains showed point mutations, small deletions or insertions in 81 isolates(80.20%), but no complete deletions were identified. The mutations at 16 positions were found for the first time. Point mutations at position 315 including AGC→ACC(Ser-to-Thr) and AAC(Ser-to-Asn) substitutions were found in the genomes of 38.61%(39/101) of isoniazid-resistant strains. Among the 39 strains the substitutions of Ser-to-Thr were identified in 31 isolates and the substitutions of Ser-to-Asn were detected in 8 isolates. Low level isoniazid resistant strains (lug/ml) had higher mutation frequency at 315-Ser than high level isoniazid resistant strains (10ug/ml) (P<0.05). Mutations at position 463 causing an Arg-to-leu substitution were detected in 58 (57.43%) isoniazid-resistant strains, combinative mutation rate is 75.86%(44/58). mutations at 463 were independent of isoniazid-resistant. Partial gene deletions were found in 12 of 101 isoniazid-resistant strains, Frameshift insertion was detected only in 1 of 101 isoniazid-resistant isolates combining with synonymous mutation and Arg4631eu. 7 of 12 strains having small deletions showed low level isoniazid resistant including 6 strains at 1716 bp position deletion, and 1 isolates having insertion showed high level isoniazid resistant. In addition, Other types missense mutations were identified in 16 of 101 (15.84%) isolates. Arg4631eu was also present in 23 of 43 susceptible strains, In addition, 1 susceptible strains had synonymous mutation. (2) Among the 101 isoniazid-resistant clinical isolates, mutations in inhA genes were identified in 5 isolates ( Ser94Ala[n=2], Ile21Thr[n=2], small deletion[n=1]) (4.95%). Among the 5 isolates, 4 strains showing missense mutations all combined with Arg4631eu , and 1 strain having deletion combined with Ser315Thr. An Ser-to-Ala substitution at position 94 was detected in 2 isolates showing low level isoniazid resistant, An Ile-to-Thr substitution at posotion 21 was found in 2 isolates showing high level isoniazid resistant, and small deletion was identified in 1 isolates showing high level isoniazid resistant. None of the susceptible strains contained any mutation in inhA genes. (3) Only 3 isolates in the 101 (2.97%) isoniazid-resistant clinical isolates had mutations in ahpC genes (Ala195Asp[n=2], Glnl69Arg[n=l]); All the 3 isolates showed high level isoniazid resistant. 1 strain showing Ala 195Asp combined with Arg4631eu substitution. Glnl69Arg was first identified. No mutations were identified inthe ahpC genes in 43 isoniazid-susceptible isolates. (4) Mutations in kasA genes were present in 17 of 101 (16.83%) isoniazid-resistant isolates (Glyl21Lys[n=2], Gly312Ser[n=7], Gly387Asp[n=3], Phe413Leu[n=1], Asp66Asn[n=4] ). These strains all showed low level isoniazid resistant. 5 strains concluding Gly312Ser combined with Arg4631eu mutation. No mutations in katG, inhA, ahpC, oxyR genes were identified in 10 strains showing other types mutations. However, G312S was also present in 3 of 43 susceptible strains. (5) None of the isoniazid-resistant strains and susceptible isolates contained oxyR gene mutation. (6) Summarize: 85 of 101(84%) isoniazid-resistant strains had mutations in katG, inhA, ahpC, kasA genes associated with drug resistance. Conclusion: Genotypic analysis of resistant to isoniazid in Mycobacterium tuberculosis is complex due to the various genes potencially invovled. These studies further support the association between katG, inhA, ahpC, kasA gene mutations and INH resistant in Mycobacterium tu更多还原