北京市昌平区耐多药结核分枝杆菌对新抗结核药品的耐药情况分析

doi:10.3969/j.issn.2096-8493.2021.02.014

摘要/Abstract

摘要：

目的探讨北京市昌平区耐多药结核分枝杆菌对左氧氟沙星(levofloxacin,Lfx)、莫西沙星(moxifloxacin,Mfx)、贝达喹啉(bedaquiline,Bdq)、利奈唑胺(linezolid,Lzd)、氯法齐明(clofazimine,Cfz)和德拉马尼(delamanid,Dlm)的耐药特点和耐药突变类型,为临床用药提供依据。方法收集2011—2015年北京市昌平区结核病防治所收治的经分离培养及菌种鉴定后获得的83株耐多药结核分枝杆菌。采用最低药物浓度法对耐多药结核分枝杆菌进行药物敏感性检测,并扩增耐药相关基因序列,将耐药基因测序结果与标准株进行比对分析。结果 83株耐多药结核分枝杆菌菌株对Lfx和Mfx的耐药率最高,均为41.0%(34/83);对Bdq的耐药率最低,为2.4%(2/83),对Lzd、Cfz和Dlm的耐药率分别为4.8%(4/83)、3.6%(3/83)和4.8%(4/83)。39株对Lfx和Mfx耐药的菌株中,gyrA基因第94位密码子的碱基替换是最常见的突变类型,占87.2%(34/39)。2株Cfz-Bdq交叉耐药菌株均在Rv0678基因中存在突变。4株Lzd耐药菌株中有2株存在rplC基因突变。4株Dlm耐药菌株中,1株突变发生在fbiC基因的第318位密码子,2株发生在ddn基因的第81位密码子。结论北京市昌平区耐多药结核分枝杆菌对Lfx或Mfx耐药率较高,对Bdq的耐药率较低;应根据本地区耐多药结核病患者的特点选择耐药率较低的药品。

关键词: 分枝杆菌, 结核,结核, 抗多种药物性, 突变, 基因, 小地区分析

Abstract:

Objective To explore the drug susceptibility of levofloxacin (Lfx), moxifloxacin (Mfx), bedaquiline (Bdq), linezolid (Lzd), clofazimine (Cfz) and delamanid (Dlm) against multidrug-resistant tuberculosis (MDR-TB) isolates from Changping District, and to illustrate the genetic characteristics of MDR-TB isolates with acquired drug resistance, further to provide the basis for clinical drug use, the results of drug resistance gene sequencing were compared with the standard strain. Methods A total of 83 MDR-TB isolates were collected from 2011—2015 in Beijing Changping Center for Tuberculosis Control after culture and strain identification. The minimum inhibitory concentrations (MICs) were used to detect the drug sensitivity of MDR-TB strains, and genes sequence related to drug resistance related genes were amplified and compared with the standard strains. Results Of the 83 MDR-TB isolates, Lfx and Mfx both had the highest resistance rate (41.0%, 34/83). The resistance rate to Bdq was the lowest (2.4%, 2/83). The resistance rate to Lzd, Cfz, and Dlm was 4.8% (4/83), 3.6% (3/83), and 4.8% (4/83), respectively. Among the 39 isolates resistant to Lfx and Mfx, the most prevalent resistance mutation was found in gyrA with the substitution of codon 94 (87.2%, 34/39). All 2 Cfz-Bdq cross resistant strains had a mutation in the Rv0678 gene, 2 of 4 Lzd resistant isolates carried mutations in rplC gene. Of the 4 isolates resistant to Dlm, 1 isolate with mutation in codon 318 of fbiC gene and 2 isolates in codon 81 of ddn gene. Conclusion The resistance of MDR-TB isolates to Lfx/Mfx was high while the lowest resistance rate was observed in Bdq in Beijing Changping District. The anti-TB drug with a low resistance rate should be selected according to the characteristics of MDR-TB patients in this region.

Key words: Mycobacterium tuberculosis, Tuberculosis, multidrug-resistant, Mutation, Genes, Small-area analysis

刘新宇, 张倩, 孙倩. 北京市昌平区耐多药结核分枝杆菌对新抗结核药品的耐药情况分析[J]. 结核与肺部疾病杂志, 2021, 2(2): 169-173. doi: 10.3969/j.issn.2096-8493.2021.02.014

LIU Xin-yu, ZHANG Qian, SUN Qian. Analysis of drug resistance of multidrug-resistant Mycobacterium tuberculosis to multiple drugs in Changping District of Beijing[J]. Journal of Tuberculosis and Lung Disease, 2021, 2(2): 169-173. doi: 10.3969/j.issn.2096-8493.2021.02.014

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参考文献 17

[1]	World Health Organization. Rapid communication: key changes to treatment of multidrug- and rifampicin-resistant tuberculosis (MDR/RR-TB). Geneva: World Health Organization, 2018.
[2]	Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardia, and other aerobic actinomycetes; approved standard. Wayne: Clinical and Laboratory Standards Institute, 2018.
[3]	Wang G, Jiang G, Jing W, et al. Prevalence and molecular characterizations of seven additional drug resistance among multidrug-resistant tuberculosis in China: A subsequent study of a national survey. J Infect, 2021,82(3):371-377. doi: 10.1016/j.jinf.2021.02.004. doi: 10.1016/j.jinf.2021.02.004 URL
[4]	Zhang Z, Li T, Qu G, et al. In vitro synergistic activity of clofazimine and other antituberculous drugs against multidrug-resistant Mycobacterium tuberculosis isolates. Int J Antimicrob Agents, 2015,45(1):71-75. doi: 10.1016/j.ijantimicag.2014.09.012. doi: 10.1016/j.ijantimicag.2014.09.012 URL
[5]	Schena E, Nedialkova L, Borroni E, et al. Delamanid susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTEC MGIT 960 system. J Antimicrob Chemother, 2016,71(6):1532-1539. doi: 10.1093/jac/dkw044. doi: 10.1093/jac/dkw044 URL
[6]	Huo F, Luo J, Shi J, et al. A 10-Year Comparative Analysis Shows that Increasing Prevalence of Rifampin-Resistant Mycobacterium tuberculosis in China Is Associated with the Transmission of Strains Harboring Compensatory Mutations. Antimicrob Agents Chemother, 2018,62(4):e02303-17. doi: 10.1128/AAC.02303-17. doi: 10.1128/AAC.02303-17
[7]	Che Y, Song Q, Yang T, et al. Fluoroquinolone resistance in multidrug-resistant Mycobacterium tuberculosis independent of fluoroquinolone use. Eur Respir J, 2017,50(6):1701633. doi: 10.1183/13993003.01633-2017. doi: 10.1183/13993003.01633-2017 URL
[8]	Hameed HMA, Tan Y, Islam MM, et al. Phenotypic and genotypic characterization of levofloxacin- and moxifloxacin-resistant Mycobacterium tuberculosis clinical isolates in southern China. J Thorac Dis, 2019,11(11):4613-4625. doi: 10.21037/jtd.2019.11.03. doi: 10.21037/jtd.2019.11.03 pmid: 31903250
[9]	Zhu C, Zhang Y, Shen Y, et al. Molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates from Shanghai, China. Diagn Microbiol Infect Dis, 2012,73(3):260-263. doi: 10.1016/j.diagmicrobio.2012.03.025. doi: 10.1016/j.diagmicrobio.2012.03.025 URL
[10]	Mokrousov I, Otten T, Manicheva O, et al. Molecular characterization of ofloxacin-resistant Mycobacterium tuberculosis strains from Russia. Antimicrob Agents Chemother, 2008,52(8):2937-2939. doi: 10.1128/AAC.00036-08. doi: 10.1128/AAC.00036-08 URL
[11]	Chan RC, Hui M, Chan EW, et al. Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong. J Antimicrob Chemother, 2007,59(5):866-873. doi: 10.1093/jac/dkm054. doi: 10.1093/jac/dkm054 URL
[12]	Umubyeyi AN, Rigouts L, Shamputa IC, et al. Limited fluoroquinolone resistance among Mycobacterium tuberculosis isolates from Rwanda: results of a national survey. J Antimicrob Chemother, 2007,59(5):1031-1033. doi: 10.1093/jac/dkm038. doi: 10.1093/jac/dkm038 URL
[13]	Andries K, Villellas C, Coeck N, et al. Acquired resistance of Mycobacterium tuberculosis to bedaquiline. PLoS One, 2014,9(7):e102135. doi: 10.1371/journal.pone.0102135. doi: 10.1371/journal.pone.0102135 URL
[14]	孙晴, 黄海荣, 王桂荣. 贝达喹啉、氯法齐明和德拉马尼对常见致病性非结核分枝杆菌体外抑菌活性及耐药机制的研究进展. 中国防痨杂志, 2020,42(8):880-884. doi: 10.3969/j.issn.1000-6621.2020.08.019. doi: 10.3969/j.issn.1000-6621.2020.08.019
[15]	Beckert P, Hillemann D, Kohl TA, et al. rplC T460C identified as a dominant mutation in linezolid-resistant Mycobacterium tuberculosis strains. Antimicrob Agents Chemother, 2012,56(5):2743-2745. doi: 10.1128/AAC.06227-11. doi: 10.1128/AAC.06227-11 URL
[16]	Liu Y, Matsumoto M, Ishida H, et al. Delamanid: From discovery to its use for pulmonary multidrug-resistant tuberculosis (MDR-TB). Tuberculosis (Edinb), 2018,111:20-30. doi: 10.1016/j.tube.2018.04.008. doi: 10.1016/j.tube.2018.04.008 URL
[17]	Fujiwara M, Kawasaki M, Hariguchi N, et al. Mechanisms of resistance to delamanid, a drug for Mycobacterium tuberculosis. Tuberculosis (Edinb), 2018,108:186-194. doi: 10.1016/j.tube.2017.12.006. doi: S1472-9792(17)30271-8 pmid: 29523322

药品	MIC值范围 (mg/L)	MIC值菌株分布(株)										临界浓度 (mg/L)
药品	MIC值范围 (mg/L)	≤0.015 mg/L	0.03 mg/L	0.06 mg/L	0.12 mg/L	0.25 mg/L	0.5 mg/L	1 mg/L	2 mg/L	4 mg/L	8 mg/L	临界浓度 (mg/L)
Lfx	0.12~8	0	0	0	16	22	2	9	10	20	4	1
Mfx	0.06~4	0	0	18	14	6	11	9	15	10	0	0.5
Bdq	0.015~2	62	10	5	2	2	1	1	0	0	0	0.25
Cfz	0.06~4	0	0	57	15	3	5	0	2	1	0	1
Lzd	0.03~2	0	9	7	31	26	5	1	4	0	0	1
Dlm	0.015~1	78	1	0	0	0	0	4	0	0	0	0.125

药品基因名称	引物序列(5'~3')	片断长度(bp)
Lfx/Mfx
gyrA	上游 TGACATCGAGCAGGAGATGC	320
	下游 GGGCTTCGGTGTACCTCATC
gyrB	上游 GTGGAAATATGTTGGCCGTC	413
	下游 GTCGTTGTGAACAACGCTGTG
Lzd
23S rRNA	上游 GGTTGAAGACTGAGGGGATGAG	2422
	下游 CATCGGCGCTGGCAGGCTTAG
rplC	上游 GCTGCGGCTGGACGACTC	420
	下游 CTCTTGCGCAGCCATCACTTC
rplD	上游 CCGGGCGGATGGGCAATGACC	936
	下游 GGAATCCGGGCGCACCAAAAAC
Bdq
atpE	上游 TGTACTTCAGCCAAGCGATGG	454
	下游 CCGTTGGGAATGAGGAAGTTG
pepQ	上游 ATCAATGCCCCCTGGAAC	1371
	下游 GCACGTTCTTCAACTTGGTG
Cfz
rv1979c	上游 GCGGCGGAAATGAGTGT	1647
	下游 ATGCACGACGGCTTTATCA
Bdq/Cfz
rv0678	上游 TGCCTTCGGAACCAAAGAA	795
	下游 GACAACACGGTCACCTACAA
Dlm
fbiA	上游 CGGTTCTGTTGTGGTTGGG	1136
	下游 CCGATGACGGGCAGGATC
fbiB	上游 GCCGCTGCTGATGACCGA	1494
	下游 TCGGGAGGTTGATGGTTGG
fbiC	上游 GTCCACCGCTCTGCCGAGTC	2386
	下游 GCCACCTTCGAGCATCACC
fgd1	上游 TCGCGTTTATGGCATAGGAGT	1090
	下游 ACTTACCCGTCTGCGATTCTG
ddn	上游 CACCATCATCGAGCGGATTT	765
	下游 CAAGGGCGTGAAATGGGAT

基因碱基改变	氨基酸改变	药品	菌株数	突变率(%)^a
gyrA
G262T	Gly88Cys	Lfx	1	2.6(1/39)
		Mfx	1	2.6(1/39)
G265A	Asp89Asn	Lfx	1	2.6(1/39)
		Mfx	1	2.6(1/39)
C269T	Ala90Val	Lfx	8	20.5(8/39)
		Mfx	6	15.4(6/39)
T271C	Ser91Pro	Lfx	1	2.6(1/39)
		Mfx	1	2.6(1/39)
G280T	Asp94Tyr	Lfx	11	28.2(11/39)
		Mfx	11	28.2(11/39)
G280A	Asp94Asn	Lfx	4	10.3(4/39)
		Mfx	4	10.3(4/39)
A281C	Asp94Ala	Lfx	6	15.4(6/39)
		Mfx	5	12.8(5/39)
A281G	Asp94Gly	Lfx	13	33.3(13/39)
		Mfx	9	23.1(9/39)
G284C	Ser95Thr	Lfx	34	87.2(34/39)
		Mfx	34	87.2(34/39)
Rv0678
A152G	Gln31Arg	Bdq	1	1/2
		Cfz	1	1/3
T157C	Ser53Pro	Bdq	1	1/2
		Cfz	1	1/3
rplC
T460C	Cys154Arg	Lzd	2	2/4
ddn
G241A	Gly81Ser	Dlm	2	2/4
fbiC
G952A	Val318Ile	Dlm	1	1/4