重庆地区耐多药结核分枝杆菌吡嗪酰胺耐药基因突变的特征分析

doi:10.3969/j.issn.1000-6621.2018.02.013

摘要/Abstract

摘要： 目的

分析重庆地区分离的耐多药结核分枝杆菌(MDR-MTB)菌株pncA和rpsA基因突变特征及其与吡嗪酰胺(PZA)耐药的相关性。

方法

收集2014年11月至2016年2月重庆市结核病防治所“MTB耐多药项目”中所属39个区(县)的133株MDR-MTB临床分离菌株。应用BACTEC MGIT 960系统作PZA药物敏感性试验(简称“药敏试验”),再通过PCR-DNA测序技术,分析菌株pncA和rpsA基因突变类型及其与PZA耐药的相关性。

结果

133株MDR-MTB中PZA 耐药83株,耐药率为62.4%。83株PZA耐药株中73株pncA基因突变,突变率为87.9%;50株PZA敏感株中,4株pncA基因突变,突变率为8.0%;PZA耐药株pncA基因突变率明显高于PZA敏感株(χ ²=81.82,P=0.000)。PZA耐药株pncA基因改变以碱基置换为主,碱基位点G395A的改变最多见(9株)。突变位点随机散布在整个pncA基因,位于核苷酸-11~515位。碱基突变的区域集中在位点20~40、136~146、185~226、392~408。pncA基因序列分析发现18种新的突变位点:T2C、T37G、T94G、C206T、G319A、C437T、T488C、52插入GCC、130插入C、139插入CA、232插入C、243插入T、288插入A、393插入GGT、408插入CA、341缺失ACGCC、342缺失GCCAC、376缺失 GATGAGGTC。PZA敏感株pncA基因改变均为碱基置换,且突变位点与耐药株突变位点均不相同。133株MDR-TB均无rpsA基因突变。

结论

重庆地区MDR-MTB中PZA耐药率较高。pncA基因突变是重庆地区MDR-MTB中PZA耐药的主要机制之一。

关键词: 分枝杆菌, 结核, 抗药性, 多种, 细菌, 吡嗪酰胺, 基因, DNA突变分析

Abstract: Objective

To investigate the mutation characteristics in pncA and rpsA genes and its correlation with pyrazinamide (PZA) resistance in multidrug-resistant Mycobacterium tuberculosis (MDR-MTB) strains isolated from Chongqing.

Methods

A total of 133 MDR-MTB clinical isolates were collected from Chongqing from November 2014 to February 2016. PZA susceptibility was determined by BACTEC MGIT 960 TB system. The pncA and rpsA genes were amplified by PCR and the products were analyzed by DNA sequencing in order to identify the mutations in pncA and rpsA genes and the correlations with PZA resistance.

Results

Among the 133 MDR-TB clinical strains, 83 isolates were resistant to PZA, yielding a resistance rate of 62.4%. Sequence analysis revealed that 73 out of 83 (87.9%) MDR-MTB strains harbored a mutation located in the pncA gene. Four out of 50 (8.0%) PZA-sensitive isolates occurred pncA gene mutation. The PZA-resistant strains showed higher mutation rate than the PZA-sensitive strains (χ ²=81.82, P=0.000). Most of the pncA gene mutations in the genomes of PZA-resistant strains were base substitution mutation, especially the G395A substitution (9 isolates). The identified mutations were dispersed along the pncA gene, distributing in the region from nucleotide -11 to nucleotide 515. The pncA gene mutations centralized in the regions of 20-40, 136-146, 185-226, and 392-408. Eighteen novel mutation sites of pncA gene were observed including T2C, T37G, T94G, C206T, G319A, C437T, T488C, GCC insertion at 52, C insertion at 130, CA insertion at 139, C insertion at 232, T insertion at 243, A insertion at 288, GGT insertion at 393, CA insertion at 408, ACGCC deletion at 341, GCCAC deletion at 342, and GATGAGGTC deletion at 376. The pncA gene mutations in the genomes of PZA-sensitive strains were only the substitution mutations, and the sites were different from those of PZA-resistant strains. While no genetic mutation associated with PZA resistance was found in the rpsA gene.

Conclusion

The MDR-MTB strains showed higher resistance rate to PZA in Chongqing. Mutation in pncA gene might be one of the major mechanisms of resistance to PZA in MDR-MTB isolates in Chongqing.

Key words: Mycobacterium tuberculosis, Drug resistance, multiple, bacterial, Pyrazinamide, Genes, DNA mutational analysis

朱大冕,胡代玉,刘洁,逄宇,罗明,沈静,陈林. 重庆地区耐多药结核分枝杆菌吡嗪酰胺耐药基因突变的特征分析[J]. 中国防痨杂志, 2018, 40(2): 177-182. doi: 10.3969/j.issn.1000-6621.2018.02.013

Da-mian ZHU,Dai-yu HU,Jie LIU,Yu PANG,Ming LUO,Jing SHEN,Lin. CHEN. Analysis of mutation in pyrazinamide-resistance gene among the multidrug-resistant Mycobacterium tuberculosis strains isolated from Chongqing municipality[J]. Chinese Journal of Antituberculosis, 2018, 40(2): 177-182. doi: 10.3969/j.issn.1000-6621.2018.02.013

图/表 4

表1

表2

表3

对PZA耐药的MDR-MTB菌株pncA基因突变情况

核苷酸位点	核苷酸置换	氨基酸位点	氨基酸改变类型	菌株数
-11	TAT→TGT	-4	酪氨酸→半胱氨酸	4
2	ATG→ACG	1	甲硫氨酸→苏氨酸	1
20	GTC→GGC	7	缬氨酸→甘氨酸	2
24	GAC→GAG	8	天冬氨酸→谷氨酸	1
35	GAC→GCC	12	天冬氨酸→丙氨酸	2
37	TTC→GTC	13	苯丙氨酸→缬氨酸	1
40	TGC→CGC	14	半胱氨酸→精氨酸	3
94	TTC→GTC	32	苯丙氨酸→缬氨酸	1
146	GAC→GCC	49	天冬氨酸→甘氨酸	1
146	GAC→GGC	49	天冬氨酸→丙氨酸	2
151	CAC→TAC	51	组氨酸→酪氨酸	1
151	CAC→CGC	51	组氨酸→精氨酸	1
152	CAC→CCC	51	组氨酸→脯氨酸	1
170	CAC→CGC	57	组氨酸→精氨酸	1
185	CCG→CTG	62	脯氨酸→亮氨酸	5
206	CCG→CTG	69	脯氨酸→亮氨酸	1
213	CAT→CAG	71	组氨酸→谷氨酸	1
226	ACT→CCT	76	苏氨酸→脯氨酸	3
232	GGC→AGC	78	甘氨酸→天冬氨酸	1
245	CAT→CGT	82	组氨酸→精氨酸	1
286	AAG→CAG	96	赖氨酸→谷氨酸	1
307	TAC→CAC	103	酪氨酸→组氨酸	1
319	GAA→AAA	107	谷氨酸→赖氨酸	1
395	GGT→GAT	132	甘氨酸→天冬氨酸	9
425	ACG→ATG	142	苏氨酸→甲硫氨酸	1
437	GCG→GTG	146	丙氨酸→缬氨酸	1
464	GTG→GGG	155	缬氨酸→甘氨酸	1
488	GTG→GCG	163	缬氨酸→丙氨酸	1
515	CTG→CCG	172	亮氨酸→脯氨酸	2
28	CAG→TAG	10	终止	1
123	TAC→TAG	41	终止	1
309	TAC→TAG	103	终止	1
52	插入GC		移码突变	1
130	插入 C		移码突变	1
136	缺失 G		移码突变	2
139	插入CA		移码突变	1
232	插入C		移码突变	1
243	插入T		移码突变	1
288	插入A		移码突变	1
341	缺失ACGCC		移码突变	1
342	缺失GCCAC		移码突变	2
392	插入 G		移码突变	1
392	插入GG		移码突变	1
408	插入 CA		移码突变	1
408	插入 A		移码突变	2
376	缺失 GATGAGGTC		整码突变	1
393	插入GGT		整码突变	1

表3

表4

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基因名称	引物序列	产物长度(bp)	退火温度(℃)
pncA	上游:5'-GTAACCGGTCGTAGGTCATA-3'	1288	56
	下游:5'-CCCGAGAGATGTACGAAACC-3'
rpsA	上游:5'-CGGAGCAACCCAACAATA-3'	1544	54
	下游:5'-GTGGACAGCAACGACTTC-3'

影响因素	检测菌株 [株(构成比,%)]	PZA耐药 [株(构成比,%)]	PZA敏感 [株(构成比,%)]	χ²值	P值	OR(95%CI)值
性别				1.14	0.285
男	80(60.2)	47(56.6)	33(66.0)			0.67(0.33~1.39)
女	53(39.8)	36(43.4)	17(34.0)			1.00
年龄(岁)				4.32	0.116
<30	31(23.3)	18(21.7)	13(26.0)			1.00
30~59	80(60.2)	55(66.3)	25(50.0)			1.59(0.68~3.74)
≥60	22(16.5)	10(12.0)	12(24.0)			0.60(0.20~1.81)
治疗史				10.14	0.001
初治	49(36.8)	22(26.5)	27(54.0)			1.00
复治	84(63.2)	61(73.5)	23(46.0)			3.26(1.55~6.82)

核苷酸位点	核苷酸置换	氨基酸位点	氨基酸改变类型	菌株数
184	CCG→ACG	62	脯氨酸→苏氨酸	1
185	CCG→CAG	62	脯氨酸→谷氨酸	1
200	TCG→TGG	67	丝氨酸→色氨酸	1
461	AGG→AAG	154	精氨酸→赖氨酸	1