重庆地区耐多药结核分枝杆菌对氟喹诺酮类药物耐药的相关基因特征分析

doi:10.3969/j.issn.1000-6621.2018.10.006

摘要/Abstract

摘要：

目的分析重庆地区耐多药结核分枝杆菌对氟喹诺酮类(FQs)药物耐药的相关基因特征,以及与结核分枝杆菌基因型的相关性。方法收集2015年1月至2017年6月重庆市39个区(县)967例耐多药结核病(MDR-TB)可疑患者的所有耐多药(MDR)结核分枝杆菌临床分离株229株,通过微孔板Alamar blue显色法检测4种FQs药物[氧氟沙星(Ofx)、左氧氟沙星(Lfx)、莫西沙星(Mfx)、加替沙星(Gfx)]的耐药性,用PCR测序方法对FQs药物耐药相关基因gyrA、gyrB进行分析,并采用实时荧光定量熔解曲线方法进行北京基因型鉴定。结果在229株MDR菌株中,94株(41.0%,94/229)对任一FQs药物耐药。其中,Ofx耐药率最高(41.0%,94/229);Lfx、Mfx耐药率居中,分别为31.4%(72/229)、30.6%(70/229);Gfx耐药率最低(20.1%,46/229)。94株对FQs耐药菌株中,81株(86.2%,81/94)发生gyrA基因突变,第94位密码子突变最常见(60.6%,57/94);10株(10.6%,10/94)发生gyrB基因突变。7株gyrA基因双位点突变均显示为高水平耐药;9株gyrA与gyrB联合突变中,2株为高水平耐药。重庆地区对FQs耐药菌株中北京基因型80株(85.1%,80/94),其中,现代北京基因型占60.0%(48/80)。结论重庆地区MDR结核分枝杆菌对FQs耐药的菌株以现代北京基因型为主,其耐药相关基因突变主要发生于gyrA基因。

关键词: 分枝杆菌, 结核, 结核, 抗多种药物性, 氟喹诺酮类, DNA突变分析, 基因型, 小地区分析

Abstract:

Objective To analyze the molecular characteristics of fluoroquinolones (FQs) resistance-associated gene mutations and the correlation with the different genotypes among the MDR-TB strains in Chongqing. Methods Two hundred and twenty-nine MDR-TB clinical strains were tested for susceptibility to ofloxacin, levofloxacin, moxifloxacin, and gatifloxacin using microplate alamar blue assay (MABA). PCR and sequencing were conducted for gyrA and gyrB genes conferring FQs resistance. The Beijing genotypes were identified with real-time polymerase chain reaction (PCR) melting curve assay. Results Among the 229 MDR-TB strains, 94 strains (41.0%, 94/229) were resistant to any FQ. The prevalence of FQs resistance in Chongqing were highest for ofloxacin (41.0%, 94/229), intermediate for levofloxacin (31.4%,72/229) and moxifloxacin (30.6%, 70/229), and lowest for gatifloxacin (20.1%, 46/229).The 94 strains resistant to FQs contained 81 gyrA mutation strains (86.2%, 81/94), and the highest mutation frequency was at codon 94 (60.6%,57/94). Mutations with gyrB were observed in 10 FQ-resistant strains (10.6%, 10/94). Seven strains with double mutations in gyrA, and 2 of the 9 strains with Asp94Gly and Ala504Thr/Ala504Val double mutations in gyrA and gyrB respectively, showed high-level FQ resistance. The proportion of Beijing genotype strains was 85.1% (80/94) among FQ-resistant strains in Chongqing. Of these, 60.0% (48/80) were modern-Beijing sublineage strains. Conclusion Modern-Beijing sublineage strains accounts for the majority of FQ-resistant strains and the resistant related mutations mainly occur in gyrA gene.

Key words: Mycobacterium tuberculosis, Tuberculosis, multidrug resistant fluoroquinolone, DNA mutational analysis, Genotype, Small-area analysis

胡彦,刘洁,沈静,朱大冕,冯鑫,陈林,詹建,欧喜超,周杨,赵雁林. 重庆地区耐多药结核分枝杆菌对氟喹诺酮类药物耐药的相关基因特征分析[J]. 中国防痨杂志, 2018, 40(10): 1060-1065. doi: 10.3969/j.issn.1000-6621.2018.10.006

Yan HU,Jie LIU,Jing SHEN,Da-mian ZHU,Xin FENG,Lin CHEN,Jian ZHAN,Xi-chao OU,Yang ZHOU,Yan-lin ZHAO. Prevalence and molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates resistant to fluoroquinolones in Chongqing[J]. Chinese Journal of Antituberculosis, 2018, 40(10): 1060-1065. doi: 10.3969/j.issn.1000-6621.2018.10.006

图/表 4

表1

表2

表3

表4

参考文献 22

[1]	World Health Organization . Golobal tuberculosis report 2017. Geneva: World Health Organization, 2017.
[2]	Pang Y, Zhu D, Zheng H , et al. Prevalence and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Southern China. BMC Infect Dis, 2017,17(1):711.
[3]	Hu Y, Xu L, He YL , et al. Prevalence and Molecular Characterization of Second-Line Drugs Resistance among Multidrug-Resistant Mycobacterium tuberculosis Isolates in Southwest of China. Biomed Res Int, 2017,2017:4563826.
[4]	Pang Y, Zhou Y, Zhao B , et al. Spoligotyping and drug resistance analysis of Mycobacterium tuberculosis strains from national survey in China. PLoS One, 2012,7(3):e32976.
[5]	Zhang Z, Pang Y, Wang Y , et al. Beijing genotype of Mycobacterium tuberculosis is significantly associated with linezolid resistance in multidmg-resistant and extensively drug-resistant tuberculosis in China. Int J Antimicrob Agents, 2014,43(3):231-235.
[6]	Parwati I, van Crevel R, van Soolingen D . Possible underlying mechanisms for successful emergence of the Mycobacterium tuberculosis Beijing genotype strains. Lancet Infect Dis, 2010,10(2):103-111.
[7]	Nosova EY, Bukatina AA, Isaeva YD , et al. Analysis of mutations in the gyrA and gyrB genes and their association with the resistance of Mycobacterium tuberculosis to levofloxacin, moxifloxacin and gatifloxacin. J Med Microbiol, 2013,62(Pt 1):108-113.
[8]	World Health Organization . Policy guidance on drug-susceptibility testing (DST) of second-line antituberculosis drugs. Geneva: World Health Organization, 2008. URL pmid: 26290924
[9]	Yang C, Luo T, Sun G , et al. Mycobacterium tuberculosis Beijing strains favor transmission but not drug resistance in China. Clin Infect Dis, 2012,55(9):1179-1187. doi: 10.1093/cid/cis670
[10]	张治国, 吴进凤, 王莉 , 等. 北京市昌平地区2011—2015年结核分枝杆菌的流行特征. 中华流行病学杂志, 2017,38(9):1236-1240. doi: 10.3760/cma.j.issn.0254-6450.2017.09.019 URL
[11]	Mestre O, Luo T, Dos Vultos T , et al. Phylogeny of Mycobacterium tuberculosis Beijing strains constructed from polymorphisms in genes involved in DNA replication, recombination and repair. PLoS One, 2011,6(1):e16020.
[12]	Luo T, Jiang L, Sun W , et al. Multiplex real-time PCR mel-ting curve assay to detect drug-resistant mutations of Mycobacterium tuberculosis. J Clin Microbiol, 2011,49(9):3132-3138. doi: 10.1128/JCM.02046-10
[13]	Javaid A, Hasan R, Zafar A , et al. Pattern of first- and second-line drug resistance among pulmonary tuberculosis retreatment cases in Pakistan. Int J Tuberc Lung Dis, 2017,21(3):303-308.
[14]	Kuo CY, Wang WH, Huang CH , et al. Resistance to first- and second-line antituberculosis drugs in Southern Taiwan: Implications for empirical treatment. J Microbiol Immunol Infect, 2018,51(1):88-93.
[15]	Zhang Z, Lu J, Wang Y , et al. Prevalence and molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis isolates in China. Antimicrob Agents Chemother, 2014,58(1):364-369.
[16]	Devasia R, Blackman A, Eden S , et al. High proportion of fluoroquinolone-resistant Mycobacterium tuberculosis isolates with novel gyrase polymorphisms and a gyrA region associated with fluoroquinolone susceptibility. J Clin Microbiol, 2012,50(4):1390-1396. doi: 10.1128/JCM.05286-11
[17]	Chien JY, Chiu WY, Chien ST , et al. Mutations in gyrA and gyrB among Fluoroquinolone- and Multidrug-Resistant Mycobacterium tuberculosis Isolates. Antimicrob Agents Chemother, 2016,60(4):2090-2096.
[18]	Yuan X, Zhang T, Kawakami K , et al. Molecular characte-rization of multidrug- and extensively drug-resistant Mycobacterium tuberculosis strains in Jiangxi, China. J Clin Microbiol, 2012,50(7):2404-2413. doi: 10.1128/JCM.06860-11
[19]	Cui Z, Wang J, Lu J , et al. Association of mutation patterns in gyrA/B genes and ofloxacin resistance levels in Mycobacterium tuberculosis isolates from East China in 2009. BMC Infect Dis, 2011,11:78.
[20]	王胜芬, 周杨, 逄宇等. 结核分枝杆菌北京基因型菌株的流行特征及其与耐药的关系. 中国防痨杂志, 2015,37(8):836-842.
[21]	李妍, 曾小红, 杨健 , 等. 陕西省87株耐药结核分枝杆菌寡核苷酸基因分型和耐药性分析. 现代预防医学, 2018,45(9):1679-1683.
[22]	罗东, 刘涛, 熊光初 . 等. 江西省MDR-TB的MIRU-VNTR分型研究. 中国病原生物学杂志, 2018,13(3):217-225.

基因	引物或探针	序列(5'-3')	浓度(μmol/L)
Rv2952	探针_W	ROX-TGGGAAGTACCCTCCTGGGC-BHQ2	0.20
	引物_F	CATGCGGCGATCTCATTGTT	0.02
	引物_R	GCACTGTTACCCGCACTTTC	0.40
mutT2	探针_W	FAM-AGAACTGGGACTCGAGGTCGC-BHQ1	0.20
	引物_F	GAACTTCCCGGCGGTAAGGTC	0.02
	引物_R	CCGTTCAACGCAATATCGTC	0.40

药物	不同MIC值(μg/ml)的菌株数											临界浓度 (μg/ml)	耐药菌株[株数(率,%)]
药物	≤0.31	0.063	0.125	0.25	0.5	1	2	4	8	16	32	临界浓度 (μg/ml)	初治 (84株)	复治 (145株)	合计 (229株)	χ²值	P值
Ofx	0	1	11	52	47	12	12	34	44	10	6	2.0	29(34.5)^abc	65(44.8)^ghi	94(41.0)	2.334	0.127
Lfx	0	4	48	57	10	10	28	44	17	8	3	2.0	22(26.2)^de	50(34.5)^jk	72(31.4)	1.697	0.193
Mfx	8	46	54	16	35	23	27	15	4	1	0	0.5	22(26.2)^f	48(33.1)^l	70(30.6)	1.198	0.274
Gfx	56	49	21	26	31	31	5	8	2	0	0	0.5	15(17.9)	31(21.4)	46(20.1)	0.411	0.521

耐药表型	gyrA基因QRDR 区突变	gyrB基因QRDR 区突变	突变菌株 [株数(率,%)]	MIC (μg/ml)
耐药表型	gyrA基因QRDR 区突变	gyrB基因QRDR 区突变	突变菌株 [株数(率,%)]	Ofx	Lfx	Mfx	Gfx
FQs耐药^a(94株)	Gly88Cys		1(1.1)	8.0	4.0	1.0	0.5
	Asp89Asn		1(1.1)	4.0	2.0	2.0	0.5
	Ala90Val		16(17.0)	4.0~8.0	2.0~4.0	0.25~1.0	0.125~0.5
	Ser91Pro		2(2.1)	4.0	2.0~4.0	0.5~1.0	0.5~1.0
	Asp94Asn		7(7.4)	4.0~8.0	2.0~8.0	0.25~4.0	0.125~1.0
	Asp94His		1(1.1)	8.0	4.0	2.0	1.0
	Asp94Ala		5(5.3)	4.0	2.0~4.0	0.5~1.0	0.125~0.5
	Asp94Gly		30(31.9)	4.0~16.0	4.0~8.0	1.0~4.0	0.25~2.0
	Asp94Tyr		2(2.1)	8.0~16.0	4.0~8.0	2.0~4.0	1.0~2.0
	Asp94Ala&Ala90Val		4(4.3)	16.0~32.0	16.0~32.0	4.0~8.0	4.0~8.0
	Asp94Ala&Ser91Pro		1(1.1)	16.0	16.0	8.0	4.0
	Asp94Gly&Ala74Ser		2(2.1)	16.0	16.0	4.0~8.0	4.0
		Gly512Arg	1(1.1)	4.0	4.0	2.0	1.0
	Ala90Val	Asp461Asn	3(3.2)	16.0~32.0	8.0~32.0	2.0~4.0	1.0~4.0
	Asp94Gly	Ala504Thr	1(1.1)	16.0	16.0	8.0	4.0
	Asp94Gly	Ala504Val	1(1.1)	32.0	16.0	16.0	8.0
	Asp94Ala	Gly512Arg	1(1.1)	8.0	4.0	1.0	0.5
	Asp94Gly	Gly512Arg	1(1.1)	8.0	4.0	1.0	1.0
	Ala90Val	Gly512Arg	1(1.1)	4.0	2.0	0.25	0.5
	Asp94Asn	Val496Val	1(1.1)	16.0	8.0	2.0	1.0
	合计		82(87.2)	4.0~32.0	2.0~32.0	0.25~16.0	0.125~8.0
FQs敏感^b(135株)	Asp94Ala		3(2.2)	0.5~2.0	0.25~1.0	0.125~0.5	0.063~0.125
	Ala90Val		6(4.4)	1.0~2.0	1.0~2.0	0.25~0.5	0.125~0.25
	Gly88Ala		1(0.7)	1.0	0.5	0.5	0.25
		Arg446Leu	1(0.7)	1.0	1.0	0.5	0.25
		Asp461Asn	4(3.0)	1.0~2.0	0.5~2.0	0.25~0.5	0.125~0.5
		Gly512Arg	1(0.7)	0.125	0.125	0.063	0.031
	合计		16(11.9)	0.125~2.0	0.125~2.0	0.063~0.5	0.031~0.5

	北京型(80株)			χ²值	P值	非北京型 (14株)	χ²值	P值
	古代北京型 (32株)	现代北京型 (48株)	合计	χ²值	P值	非北京型 (14株)	χ²值	P值
gyrA基因	26(81.3)	43(89.6)	69(86.3)	0.531	0.466	12(85.7)	0.000	1.000
Ala74Ser	0(0.0)	2(4.2)	2(2.5)	-	0.514	0(0.0)	-	1.000
Gly88Cys	0(0.0)	0(0.0)	0(0.0)	-	-	1(7.1)	-	0.149
Asp89Asn	1(3.1)	0(0.0)	1(1.3)	-	0.400	0(0.0)	-	1.000
Ala90Val	8(25.0)	13(27.1)	21(26.3)	0.043	0.836	3(21.4)	0.002	0.961
Ser91Pro	0(0.0)	2(4.2)	2(2.5)	-	0.514	1(7.1)	-	0.387
94位密码子	17(53.1)	33(68.8)	50(62.5)	2.000	0.157	7(50.0)	0.780	0.377
Asp94Asn	1(3.1)	4(8.3)	5(6.3)	0.222	0.637	3(21.4)	1.846	0.174
Asp94His	0(0.0)	1(2.1)	1(1.3)	-	1.000	0(0.0)	-	1.000
Asp94Ala	2(6.3)	9(18.8)	11(13.8)	1.585	0.208	0(0.0)	1.052	0.305
Asp94Gly	13(40.6)	19(39.6)	32(40.0)	0.009	0.926	3(21.4)	1.758	0.185
Asp94Tyr	1(3.1)	0(0.0)	1(1.3)	-	0.400	1(7.1)	-	0.277
gyrB基因	8(25.0)	0(0.0)	8(10.0)	10.700	0.001	2(14.3)	0.000	0.992
Asp461Asn	3(9.4)	0(0.0)	3(3.8)	2.439	0.118	0(0.0)	-	1.000
Gly512Arg	4(12.5)	0(0.0)	4(5.0)	3.958	0.047	0(0.0)	-	1.000
Ala504Thr	0(0.0)	0(0.0)	0(0.0)	-	-	1(7.1)	-	0.149
Ala504Val	1(3.1)	0(0.0)	1(1.3)	-	0.400	0(0.0)	-	1.000
Val496Val	0(0.0)	0(0.0)	0(0.0)	-	-	1(7.1)	-	0.149