脓肿分枝杆菌复合群对克拉霉素耐药基因的初步研究

doi:10.3969/j.issn.1000-6621.2021.12.013

摘要/Abstract

摘要：

目的分析脓肿分枝杆菌复合群对克拉霉素的耐药特征,为临床正确选用克拉霉素及有效控制其耐药提供依据和新思路。方法选取中国疾病预防控制中心国家结核病参比实验室保存的2016—2017年全国耐药监测菌株中,鉴定为脓肿分枝杆菌复合群的385株临床分离株作为研究对象。采用多靶位基因测序法进行菌种鉴定和耐药基因检测;采用微孔板稀释法对菌株进行药物敏感性试验(简称“药敏试验”),检测其对克拉霉素和阿米卡星的耐药情况,并分析耐药菌株的耐药基因突变情况。结果 385株脓肿分枝杆菌复合群中,218株为脓肿分枝杆菌[包括185株为erm(41)T28基因型和33株erm (41)C28基因型],163株为马赛分枝杆菌,4株为博莱分枝杆菌。脓肿分枝杆菌、马赛分枝杆菌对阿米卡星和克拉霉素的耐药率分别为2.8%(6/218)和6.4%(14/218)、4.3%(7/163)和10.4%(17/163)。其中,erm(41)C28基因型脓肿分枝杆菌培养3d和14d对克拉毒素的耐药率(均为9.1%,3/33)不变,未发生诱导耐药,而erm(41)T28基因型脓肿分枝杆菌的耐药率则由培养3d时的5.9%(11/185)上升为培养14d的98.4%(182/185),171株发生诱导耐药。31株获得性耐药菌株中,10株脓肿分枝杆菌及10株马赛分枝杆菌均在rrl基因的2058/2059位点突变,最常见突变类型分别为A2058C(6株脓肿分枝杆菌,3株马赛分枝杆菌)和A2058G (3株脓肿分枝杆菌,5株马赛分枝杆菌);另有9株对阿米卡星耐药的菌株在rrs基因的A1408G位点发生突变,其中7株同时对克拉霉素耐药。结论 erm(41)T28基因型脓肿分枝杆菌更易诱导克拉霉素耐药。脓肿分枝杆菌和马赛分枝杆菌最常见的获得性耐药突变位点是rrl基因的A2058C和A2058G。

关键词: 分枝杆菌,非典型性, 基因组,细菌, 抗菌药, 抗药性, 药物评价

Abstract:

Objective To analyze the characteristics of clarithromycin resistance against Mycobacterium abscessus complex, and further to provide the basis for the adequate application and effective control of clarithromycin resistance in clinical. Methods A total of 385 Mycobacterium abscessus complex clinical isolates strains stored in National Tuberculosis Reference Laboratory of Chinese Center for Disease Control and Prevention for the national drug resistance surveillance from 2016 to 2017 were finally collected. Isolates were characterized using multilocus sequence analysis of various genes. Meanwhile, the drug susceptibility testing was determined using the broth microdilution method and the drug resistance genes were analyzed. Results Of all the 385 Mycobacterium abscessus complex isolates, 185 possessed the erm(41) T28 sequevar, 33 possessed the erm(41) C28 sequevar, 163 were M.massiliense, while the other four isolates belonged to M.bolletii. The resistance rate of amikacin against M.abscessus and M.massiliense was 2.8% (6/218) and 6.4% (14/218), respectively. For clarithromycin, the resistance rate was 4.3% (7/163) and 10.4% (17/163) for M.abscessus and M.massiliense, respectively.The resistance rate of clarithromycin against erm(41) C28 sequevar isolates was 9.1% (3/33) on day 3 and day 14 and no inducible resistance was occurred, however, the resistance rate was increased from 5.9% (11/185) on day 3 to 98.4% (182/185) on day 14 with 171 inducible resistant isolates of erm(41) T28 sequevar. Among the 31 acquired resistant isolates, 10 M.abscessus and 10 M.massiliense isolates possed an rrl 2058/2059 mutation with A2058C and A2058G the most common mutation type. Of the 9 amikacin resistant isolates with an A1408G rrs mutation, there were 7 isolates resistant to both clarithromycin and amikacin. Conclusion erm(41) T28 sequevar was more likely to induce clarithromycin resistance. The most common acquired resistance mutation site was A2058C for M.abscessus, which was A2058G in M.massiliense.

Key words: Mycobacteria,atypical, Genome,bacterial, Antimicrobial agent, Drug resistance, Drug evaluation

王怡婷, 郑惠文, 赵冰, 夏辉, 王胜芬, 欧喜超, 周杨, 宋媛媛, 郑扬, 赵雁林, 申阿东. 脓肿分枝杆菌复合群对克拉霉素耐药基因的初步研究[J]. 中国防痨杂志, 2021, 43(12): 1302-1307. doi: 10.3969/j.issn.1000-6621.2021.12.013

WANG Yi-ting, ZHENG Hui-wen, ZHAO Bing, XIA Hui, WANG Sheng-fen, OU Xi-chao, ZHOU Yang, SONG Yuan-yuan, ZHENG Yang, ZHAO Yan-lin, SHEN A-dong. Preliminary study on clarithromycin resistance gene of Mycobacterium abscessus complex[J]. Chinese Journal of Antituberculosis, 2021, 43(12): 1302-1307. doi: 10.3969/j.issn.1000-6621.2021.12.013

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引物名称	引物序列(5'~3')
16S rRNA-F	AGAGTTTGATCMTGGCTCAG
16S rRNA-R	CCGTCAATTCMTTTRAGTTT
rpoB-F	GGCAAGGTCACCCCGAAGGG
rpoB-R	AGCGGCTGCTGGGTGATCATC
hsp65-F	ATCGCCAAGGAGATCGAGCT
hsp65-R	AAGGTGCCGCGGATCTTGTT
转录间隔区-F	AAGTCGTAACAAGGTARCCG
转录间隔区-R	TCGCCAAGGCATCCACC
erm(41)-F	GACCGGGGCCTTCTTCGTGAT
erm(41)-R	GACTTCCCCGCACCGATTCC
rrl-F	GTAGCGAAATTCCTTGTCGG
rrl-R	TTCCCGCTTAGATGCTTTCAG
rrs-F	ATGACGTCAAGTCATCATGCC
rrs-R	AGGTGATCCAGCCGCACCTTC

药品	MIC范围(μg/ml)			耐药菌株[株(耐药率,%)]
药品	敏感	中度	耐药	脓肿分枝杆菌(218株)	马赛分枝杆菌(163株)
阿米卡星	≤16	32	≥64	6(2.8)	7(4.3)
克拉霉素	≤2	4	≥8	14(6.4)	17(10.4)
阿奇霉素	≤16	-	≥32	33(15.1)	21(12.9)
利奈唑胺	≤8	16	≥32	34(15.6)	36(22.1)
替加环素	≤1	2~4	≥8	50(22.9)	56(34.3)
头孢西丁	≤16	32~64	≥128	56(25.7)	51(31.3)
加替沙星	≤1	2	≥4	99(45.4)	102(62.6)
莫西沙星	≤1	2	≥4	120(55.0)	108(66.3)
妥布霉素	≤2	4	≥8	135(61.9)	148(90.8)
亚胺培南	≤4	8~16	≥32	181(83.0)	126(77.3)
米诺环素	≤1	2~4	≥8	198(90.8)	83(50.9)
利福布汀	≤0.5	-	≥1	212(97.2)	154(94.5)
美罗培南	≤4	8~16	≥32	213(97.7)	159(97.5)
左氧氟沙星	≤1	2	≥4	216(99.1)	155(95.1)

菌株	rrl 基因突变(株)					rrs基因A1408G 突变(株)
菌株	A2058C	A2058G	A2059C	合计	突变率(%)	rrs基因A1408G 突变(株)
脓肿分枝杆菌
erm(41)T28型	4	2	1	7	22.6	3
erm(41)T28型	-	-	-	-	-	2
erm(41)C28型	2	1	0	3	9.7	2
马赛分枝杆菌	3	5	2	10	32.3	2