2017—2019年福建省结核分枝杆菌分离株基因型特征及其耐药性分析

doi:10.19982/j.issn.1000-6621.20220331

摘要/Abstract

摘要：

目的: 了解福建省结核分枝杆菌(Mycobacterium tuberculosis,MTB)基因型的分布特征和流行情况,同时分析MTB基因型与其耐药的关系。方法: 选取2017—2019年福建省结核病耐药监测点的477株MTB临床分离株作为研究对象。采用对硝基苯甲酸(PNB)/噻吩-2-羧酸肼(TCH)生长实验法进行菌种初步鉴定,并采用传统固体比例法对9种抗结核药物[异烟肼(isoniazid,INH)、利福平(rifampin,RFP)、链霉素(streptomycin,Sm)、乙胺丁醇(ethambutol,EMB)、卡那霉素(kanamycin,Km)、氧氟沙星(ofloxacin,Ofx)、卷曲霉素(capreomycin,Cm)、丙硫异烟胺(prothionamide,Pto)、对氨基水杨酸(para-aminosalicylic acid,PAS)]进行药物敏感性试验(简称“药敏试验”)。应用熔解曲线法间隔区寡核苷酸分型(McSpoligotyping)技术对菌株进行基因分型。结果: 477株MTB菌株中北京基因型有245株,占51.4%;T家族有44株(含2株T,28株T1,7株T2,T3和T4各3株,1株T5),占9.2%;H家族有35株(含13株H,1株H1,21株H3),占7.3%;EAI家族有11株(含10株EAI2-Manila,1株EAI5),占2.3%;LAM3、Manu2和X1家族各有1株,分别占0.2%;所属家族未定义或不明确的有139株,占29.1%。聚类分析显示,各基因型家族主要流行型为北京家族的SIT1(44.9%,214/477)、T家族的SIT53(2.5%,12/477)、H家族的SIT742(1.9%,9/477)、EAI家族的SIT19(1.5%,7/477)。各家族MTB菌株与INH的耐药率之间的差异有统计学意义(χ²=10.311,P=0.036),其中,EAI+LAM+Manu+X家族菌株对INH的耐药率最高[28.6%(4/14)];对RFP的耐药率之间的差异有统计学意义(χ²=14.366,P=0.006),其中,EAI+LAM+Manu+X家族菌株对RFP的耐药率最高[21.4%(3/14)];对Ofx的耐药率之间的差异有统计学意义(χ²=23.643,P=0.000),其中,H家族菌株对Ofx的耐药率最高[17.1%(6/35)];对PAS耐药率之间的差异有统计学意义(χ²=9.550,P=0.049),其中,未定义家族菌株对PAS的耐药率最高[4.3%(6/139)]。结论: 福建省MTB流行基因型以北京基因型为主,同时应重视未定义家族菌株的流行并加强对T家族、H家族和EAI等家族菌株的监测。菌株对INH、RFP、Ofx和PAS的耐药性与各基因家族相关。

关键词: 分枝杆菌,结核, 基因型, DNA, 核糖体间隔区, 聚合酶链反应, 抗药性,细菌

Abstract:

Objective: To investigate the genotype distribution and prevalence of Mycobacterium tuberculosis (MTB) isolates in Fujian Province, and to analyze the relationship between MTB genotypes and drug resistance. Methods: A total of 477 MTB clinical isolates from tuberculosis drug resistance surveillance sites in Fujian Province from 2017 to 2019 were selected as the research objects. The growth test method of p-nitrobenzoic acid (PNB)/thiophene-2-carboxylic acid hydrazide (TCH) was used for preliminary identification of bacteria. The drug sensitivity test of 9 kinds of anti-tuberculosis drugs (isoniazid (INH), rifampin (RFP), streptomycin (Sm), ethambutol (EMB), kanamycin (Km), ofloxacin (Ofx), capreomycin (Cm), propylamine (Pto), para aminosalicylic acid (PAS)) was carried out using the traditional solid proportion method. The strains were genotyped by the melting curve method of interval oligonucleotide typing (McSpoligotyping). Results: Among the 477 MTB strains, 245 strains (51.4%) were Beijing genotype, 44 strains (9.2%) were in T family (including 2 strains of T, 28 strains of T1, 7 strains of T2, 3 strains of T3 and T4, and 1 strain of T5), 35 strains (7.3%) were in H family (including 13 strains of H, 1 strain of H1, 21 strains of H3), 11 strains (2.3%) were in EAI family (including 10 strains of EAI2-Manila and 1 strain of EAI5), and 3 strains (0.2%) were in LAM3, Manu2 and X1 families, respectively, and the other 139 strains (29.1%) belonged to undefined or unknown families. Cluster analysis showed that the main epidemic types of each genotype family were SIT1 of Beijing family (44.9%, 214/477), SIT53 of T family (2.5%, 12/477), SIT742 of H family (1.9%, 9/477), and SIT19 of EAI family (1.5%, 7/477). The differences among the INH resistance rate of MTB strains of different family were statistically significant (χ²=10.311, P=0.036), the INH resistance rate of the EAI+LAM+Manu+X families was the highest (28.6%, 4/14). There was statistically significant difference among the RFP resistance rate MTB strains of different family (χ²=14.366, P=0.006), the RFP resistance rate of EAI+LAM+Manu+X families was the highest (21.4%, 3/14). There was statistically significant difference among Ofx resistance rate of MTB strains of different families (χ²=23.643, P=0.000), the Ofx resistance rate of H family strains was the highest (17.1%, 6/35). There was statistically significant difference among PAS resistance rate of MTB strains of different families (χ²=9.550, P=0.049), and the PAS resistance rate of strains of undefined family was the highest (4.3%, 6/139). Conclusion: The prevalent MTB genotype in Fujian Province is Beijing family, and the prevalence of the strains of T family, H family, EAI family and the undefined family should be pay attention and the monitoring should be strengthened. The resistance to INH, RFP, Ofx and PAS of MTB strains is related to different genotype families.

Key words: Mycobacterium tuberculosis, Genotype, DNA, ribosomal spacer, Polymerase chain reaction, Drug resistance, bacterial

中图分类号:

魏淑贞, 赵永, 林建, 林淑芳, 戴志松. 2017—2019年福建省结核分枝杆菌分离株基因型特征及其耐药性分析[J]. 中国防痨杂志, 2023, 45(1): 73-78. doi: 10.19982/j.issn.1000-6621.20220331

Wei Shuzhen, Zhao Yong, Lin Jian, Lin Shufang, Dai Zhisong. Analysis of genotype characteristics and drug resistance of Mycobacterium tuberculosis isolates in Fujian Province from 2017 to 2019[J]. Chinese Journal of Antituberculosis, 2023, 45(1): 73-78. doi: 10.19982/j.issn.1000-6621.20220331

图/表 3

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参考文献 16

[1]	Zeng X, Xu Y, Zhou Y, et al. McSpoligotyping, a One-Step Melting Curve Analysis-Based Protocol for Spoligotyping of Mycobacterium tuberculosis. J Clin Microbiol, 2018, 56(8): e00539-18. doi:10.1128/JCM.00539-18. doi: 10.1128/JCM.00539-18.
[2]	蓝如束, 叶婧, 罗丹, 等. 基于PCR熔解曲线技术的结核分枝杆菌Spoligotyping基因分型的临床应用研究. 中国人兽共患病学报, 2021, 37(4):285-291, 338. doi:10.3969/j.issn.1002-2694.2021.00.045. doi: 10.3969/j.issn.1002-2694.2021.00.045.
[3]	梁小烟, 林玫, 梁大斌, 等. 广西壮族自治区耐多药结核分枝杆菌耐药情况与基因型特征分析. 中国防痨杂志, 2020, 42(6):578-582. doi:10.3969/j.issn.1000-6621.2020.06.008. doi: 10.3969/j.issn.1000-6621.2020.06.008.
[4]	李辉, 王少华, 石洁, 等. 结核分枝杆菌新型间隔区寡核甘酸分型技术临床应用价值研究. 河南预防医学杂志, 2019, 30(12):881-885, 899. doi:10.13515/j.cnki.hnjpm.1006-8414.2019.12.001. doi: 10.13515/j.cnki.hnjpm.1006-8414.2019.12.001.
[5]	Luo T, Comas I, Luo D, et al. Southern East Asian origin and coexpansion of Mycobacterium tuberculosis Beijing family with Han Chinese. Proc Natl Acad Sci U S A, 2015, 112(26):8136-8141. doi:10.1073/pnas.1424063112. doi: 10.1073/pnas.1424063112. URL
[6]	王兆芬, 李斌, 蒋明霞, 等. 青海省251株结核分枝杆菌Spoligo-typing基因型与4种一线药物耐药表型的研究. 中国人兽共患病学报, 2017, 33(4):332-336. doi:10.3969/j.issn.1002-2694.2017.04.008. doi: 10.3969/j.issn.1002-2694.2017.04.008.
[7]	梁晨, 张旭霞, 邢青, 等. 北京基因型结核分枝杆菌的流行及与二线抗结核药物耐药性的相关性. 中华结核和呼吸杂志, 2020, 43(4):356-361. doi:10.3760/cma.j.cn112147-20191215-00827. doi: 10.3760/cma.j.cn112147-20191215-00827.
[8]	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.
[9]	庞慧, 刘巧, 刘海灿, 等. 江苏农村地区结核杆菌基因分型及耐药性分析. 现代预防医学, 2016, 43(19):3566-3570,3575.
[10]	袁薇, 郑雯琳, 何昱颖, 等. 我国西部贵州地区结核分枝杆菌分子流行病学初步研究. 中华疾病控制杂志, 2017, 21(1):19-21,34. doi:10.16462/j.cnki.zhjbkz.2017.01.004. doi: 10.16462/j.cnki.zhjbkz.2017.01.004.
[11]	林明冠, 吴元东, 林翀, 等. 海南地区484株结核分枝杆菌菌株基因型特征分析. 中国热带医学, 2018, 18(8):770-774. doi:10.13604/j.cnki.46-1064/r.2018.08.06. doi: 10.13604/j.cnki.46-1064/r.2018.08.06.
[12]	罗丹, 蓝兰, 赵锦明, 等. 结核分枝杆菌可变数目串联重复序列基因型成簇特征分析. 中华传染病杂志, 2016, 34(8):490-493. doi:10.3760/cma.j.issn.1000-6680.2016.08.008. doi: 10.3760/cma.j.issn.1000-6680.2016.08.008.
[13]	陈连勇, 杨星, 茹浩浩, 等. 云南省271株结核分枝杆菌分离株基因分型研究. 中华预防医学杂志, 2018, 52(1):62-67. doi:10.3760/cma.j.issn.0253-9624.2018.01.012. doi: 10.3760/cma.j.issn.0253-9624.2018.01.012.
[14]	Pan J, Ye H, Wu Z, et al. One-step melting curve analysis-based McSpoligotyping reveals genotypes of Mycobacterium tuberculosis in a coastal city, China. Arch Microbiol, 2021, 203(7):4579-4585. doi:10.1007/s00203-021-02441-0. doi: 10.1007/s00203-021-02441-0. URL
[15]	张建梅, 郑蓉蓉, 曾小红, 等. 厦门市临床分离的466株结核分枝杆菌基因分型研究. 现代预防医学, 2016, 43(22):4159-4162, 4187.
[16]	蔡杏珊, 许蕴怡, 张院良, 等. 广东省地区结核分枝杆菌寡核苷酸基因分型及耐药相关性. 中国人兽共患病学报, 2018, 34(7):643-647, 652. doi:10.3969/j.issn.1002-2694.2018.00.110. doi: 10.3969/j.issn.1002-2694.2018.00.110.

基因型	福州市	龙岩市	南平市	宁德市	莆田市	泉州市	三明市	厦门市	合计
北京家族	25(46.3)	40(51.3)	10(55.5)	12(44.5)	50(50.0)	80(55.9)	6(25.0)	22(66.7)	245(51.4)
EAI家族	0(0.0)	1(1.3)	0(0.0)	0(0.0)	1(1.0)	9(6.3)	0(0.0)	0(0.0)	11(2.3)
EAI2-Manila	0(0.0)	1(1.3)	0(0.0)	0(0.0)	1(1.0)	8(5.6)	0(0.0)	0(0.0)	10(2.1)
EAI5	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	1(0.7)	0(0.0)	0(0.0)	1(0.2)
H家族	3(5.6)	7(9.0)	3(16.7)	9(33.3)	4(4.0)	6(4.2)	3(12.5)	0(0.0)	35(7.4)
H	1(1.9)	0(0.0)	0(0.0)	3(11.1)	3(3.0)	3(2.1)	3(12.5)	0(0.0)	13(2.7)
H1	0(0.0)	1(1.3)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	1(0.2)
H3	2(3.7)	6(7.7)	3(16.7)	6(22.2)	1(1.0)	3(2.1)	0(0.0)	0(0.0)	21(4.4)
LAM3	0(0.0)	0(0.0)	0(0.0)	0(0.0)	1(1.0)	0(0.0)	0(0.0)	0(0.0)	1(0.2)
Manu2	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	1(4.2)	0(0.0)	1(0.2)
X1	0(0.0)	0(0.0)	0(0.0)	1(3.7)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	1(0.2)
T家族	3(5.6)	5(6.4)	2(11.1)	4(14.8)	12(12.0)	15(10.5)	3(12.5)	0(0.0)	44(9.2)
T	0(0.0)	0(0.0)	0(0.0)	1(3.7)	0(0.0)	1(0.7)	0(0.0)	0(0.0)	2(0.4)
T1	3(5.6)	2(2.6)	2(11.1)	3(11.1)	6(6.0)	9(6.3)	3(12.5)	0(0.0)	28(5.9)
T2	0(0.0)	0(0.0)	0(0.0)	0(0.0)	4(4.0)	3(2.1)	0(0.0)	0(0.0)	7(1.5)
T3	0(0.0)	0(0.0)	0(0.0)	0(0.0)	1(1.0)	2(1.4)	0(0.0)	0(0.0)	3(0.6)
T4	0(0.0)	3(3.8)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	3(0.6)
T5	0(0.0)	0(0.0)	0(0.0)	0(0.0)	1(1.0)	0(0.0)	0(0.0)	0(0.0)	1(0.2)
未定义	23(42.5)	25(32.0)	3(16.7)	1(3.7)	31(31.0)	32(22.4)	11(45.8)	9(27.3)	135(28.3)
不明确	0(0.0)	0(0.0)	0(0.0)	0(0.0)	1(1.0)	1(0.7)	0(0.0)	2(6.0)	4(0.8)
合计	54(11.3)	78(16.3)	18(3.8)	27(5.7)	100(21.0)	143(30.0)	24(5.0)	33(6.9)	477(100.0)

药品	北京家族	H家族	T家族	未定义家族	EAI+LAM+ Manu+X1	χ²值	P值
异烟肼						10.311	0.036
敏感	225(91.8)	31(88.6)	43(97.7)	122(87.8)	10(71.6)
耐药	20(8.2)	4(11.4)	1(2.3)	17(12.2)	4(28.6)
利福平						14.366	0.006
敏感	235(95.9)	34(97.1)	44(100.0)	126(90.6)	11(78.6)
耐药	10(4.1)	1(2.9)	0(0.0)	13(9.4)	3(21.4)
乙胺丁醇						8.080	0.089
敏感	239(97.6)	34(97.1)	44(100.0)	134(96.4)	12(85.7)
耐药	6(2.4)	1(2.9)	0(0.0)	5(3.6)	2(14.3)
链霉素						4.981	0.289
敏感	227(92.7)	33(94.3)	42(95.5)	130(93.5)	11(78.6)
耐药	18(7.3)	2(5.7)	2(4.5)	9(6.5)	3(21.4)
卡那霉素						4.686	0.321
敏感	244(99.6)	34(97.1)	43(97.7)	135(97.1)	14(100.0)
耐药	1(0.4)	1(2.9)	1(2.3)	4(2.9)	0(0.0)
氧氟沙星						23.643	0.000
敏感	242(98.8)	29(82.9)	43(97.7)	133(95.7)	13(92.9)
耐药	3(1.2)	6(17.1)	1(2.3)	6(4.3)	1(7.1)
卷曲霉素						9.119	0.058
敏感	245(100.0)	34(97.1)	42(95.5)	135(97.1)	14(100.0)
耐药	0(0.0)	1(2.9)	2(4.5)	4(2.9)	0(0.0)
丙硫异烟胺						8.864	0.065
敏感	243(99.2)	35(100.0)	43(97.7)	132(95.0)	14(100.0)
耐药	2(0.8)	0(0.0)	1(2.3)	7(5.0)	0(0.0)
对氨基水杨酸						9.550	0.049
敏感	244(99.6)	34(97.1)	44(100.0)	133(95.7)	14(100.0)
耐药	1(0.4)	1(2.9)	0(0.0)	6(4.3)	0(0.0)