胞内分枝杆菌临床分离株亚种组成及体外耐药性分析

doi:10.3969/j.issn.1000-6621.2021.02.009

中国防痨杂志 ›› 2021, Vol. 43 ›› Issue (2): 147-152.doi: 10.3969/j.issn.1000-6621.2021.02.009

胞内分枝杆菌临床分离株亚种组成及体外耐药性分析

李远春, 张越, 曾祥洁, 贺文从, 邱倩, 赵雁林, 李燕明()

100730 北京医院呼吸与危重症医学科国家老年医学中心国家卫生健康委北京老年医学研究所国家卫生健康委北京老年医学重点实验室中国医学科学院老年医学研究院(李远春、张越),呼吸与危重症医学科(李燕明);海南省疾病预防控制中心传染病预防控制所结核病防治室(曾祥洁);中国疾病预防控制中心传染病预防控制所结核病室(贺文从),结核病预防控制中心(赵雁林);重庆市公共卫生医疗救治中心歌乐山院区结核病研究室(邱倩)

收稿日期:2020-11-04 出版日期:2021-02-10 发布日期:2021-02-03
通信作者: 李远春,李燕明 E-mail:lymyl@263.net
基金资助:
国家自然科学基金(81870013)

Subspecies and in vitro drug sensitivity analysis of Mycobacterium intracellular clinical isolates

LI Yuan-chun, ZHANG Yue, ZENG Xiang-jie, HE Wen-cong, QIU Qian, ZHAO Yan-lin, LI Yan-ming()

The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China
The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission; The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China
The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China

Received:2020-11-04 Online:2021-02-10 Published:2021-02-03
Contact: LI Yuan-chun,LI Yan-ming E-mail:lymyl@263.net

摘要/Abstract

摘要：

目的分析深圳地区胞内分枝杆菌临床分离株的亚种组成及其耐药谱,为治疗胞内分枝杆菌肺病提供科学依据。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且初步鉴定为胞内分枝杆菌的临床分离株,共计97株。菌株均分离自深圳市第三人民医院2018年收集的疑似肺结核和非结核分枝杆菌(NTM)肺病患者的呼吸道标本。采用PCR及基因测序鉴定胞内分枝杆菌临床分离株亚种,使用Sensititre^? SLOWMYCO药敏板测定菌株对13种药品(克拉霉素、阿米卡星、莫西沙星、利奈唑胺、利福平、利福布汀、乙胺丁醇、链霉素、多西环素、环丙沙星、异烟肼、乙硫异烟胺及复方磺胺甲噁唑)的药物敏感性情况。结果 97株胞内分枝杆菌临床分离株以亚种胞内分枝杆菌最多[63.92%(62/97)],其次为奇美拉分枝杆菌[18.56%(18/97)]和副胞内分枝杆菌[17.53%(17/97)]。药物敏感性试验显示,胞内分枝杆菌的MIC₅₀和MIC₉₀值最低的是利福布汀(0.5和8μg/ml),最高的是链霉素(32和64μg/ml);亚种胞内分枝杆菌和副胞内分枝杆菌的MIC₉₀均为2μg/ml,而奇美拉分枝杆菌则高达64μg/ml。胞内分枝杆菌临床分离株对克拉霉素、乙胺丁醇、利福平、利福布汀、链霉素、阿米卡星、利奈唑胺和莫西沙星总的耐药率分别为9.28%(9/97)、44.33%(43/97)、42.27%(41/97)、15.46%(15/97)、36.08%(35/97)、11.34%(11/97)、38.14%(37/97)和46.39%(45/97);奇美拉分枝杆菌对克拉霉素的耐药率[27.78%(5/18)]明显高于亚种胞内分枝杆菌[4.84%(3/62)],差异有统计学意义(χ²=8.156, P=0.012)。结论深圳地区胞内分枝杆菌以亚种胞内分枝杆菌为主,对各药品的敏感性有差异,且各亚种对同一药品的敏感性也不同,建议临床治疗前应鉴定至亚种并行药物敏感性试验,以实现个性化治疗。

关键词: 分枝杆菌,非典型性, 分枝杆菌感染,鸟, 细菌分型技术, 微生物敏感性试验, 抗药性,细菌

Abstract:

Objective To analyze the subspecies composition and drug resistance profiles of Mycobacterium intracellular clinical isolates in Shenzhen, providing scientific basis for the treatment of Mycobacterium intracellular pulmonary disease. Methods A total of 97 Mycobacterium intracellular clinical strains stored in National Tuberculosis Reference Laboratory of Chinese Center for Disease Control and Prevention were finally included. All strains were isolated from respiratory specimens of suspected tuberculosis or NTM patients in Shenzhen Third People’s Hospital in 2018. Multi-target sequencing was applied to subspecies identification and minimum inhibitory concentrations (MIC) of strains to 13 drugs (clarithromycin, amikacin, moxifloxacin, linezolid, rifampin, rifabutin, ethambutol, streptomycin, doxycycline, ciprofloxacin, isoniazid, ethionamide and trimethoprim/sulfamethoxazole) was tested by Sensititre^? SLOWMYCO plate. Results The majority subspecies of Mycobacterium intracellular was Mycobacterium intracellular subsp,accounting for 63.92% (62/97), followed by Mycobacterium intracellular subsp.chimaera (18.56% (18/97)) and Mycobacterium intracellular subsp.paraintracellulare (17.53% (17/97)). Drug susceptibility testing (DST) indicated that the lowest values of MIC₅₀ and MIC₉₀ were 0.5 and 8 μg/ml (rifabutin); and the highest were 32 and 64 μg/ml (streptomycin) in Mycobacterium intracellular. The value of MIC₉₀ of clarithromycin in Mycobacterium intracellular and Mycobacterium paraintracellular both was 2 μg/ml while that in Mycobacterium chimaera was 64 μg/ml. The drug resistant rates of Mycobacterium intracellular to clarithromycin, ethambutol, rifampicin, rifabutin, streptomycin, amikacin, linezolid and moxifloxacin were 9.28% (9/97), 44.33% (43/97), 42.27% (41/97), 15.46% (15/97), 36.08% (35/97), 11.34% (11/97), 38.14% (37/97) and 46.39% (45/97), respectively. The resistance of Mycobacterium chimaera to clarithromycin was significantly higher than that of Mycobacterium paraintracellulare (27.78% (5/18) vs. 4.84% (3/62), χ²=8.156, P=0.012). Conclusion The dominant subspecies was Mycobacterium intracellular subsp. in Mycobacterium intracellular. Drug resistance profiles varied a lot within subspecies to different drugs, thus subspecies identification and DST should be conducted to guide tailored therapy.

Key words: Mycobacteria,atypical, Mycobacterium chelonae, Bacterial typing techniques, Microbial sensitivity tests, Drug resistance,bacterial

李远春, 张越, 曾祥洁, 贺文从, 邱倩, 赵雁林, 李燕明. 胞内分枝杆菌临床分离株亚种组成及体外耐药性分析[J]. 中国防痨杂志, 2021, 43(2): 147-152. doi: 10.3969/j.issn.1000-6621.2021.02.009

LI Yuan-chun, ZHANG Yue, ZENG Xiang-jie, HE Wen-cong, QIU Qian, ZHAO Yan-lin, LI Yan-ming. Subspecies and in vitro drug sensitivity analysis of Mycobacterium intracellular clinical isolates[J]. Chinese Journal of Antituberculosis, 2021, 43(2): 147-152. doi: 10.3969/j.issn.1000-6621.2021.02.009

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引物名称	引物序列(5'~3')	片段长度 (bp)
16S rRNA-F	AGAGTTTGATCMTGGCTCAG	1536
16S rRNA-R	CCGTCAATTCMTTTRAGTTT
rpoB-F	GGCAAGGTCACCCCGAAGGG	764
rpoB-R	AGCGGCTGCTGGGTGATCATC
hsp65-F	ATCGCCAAGGAGATCGAGCT	644
hsp65-R	AAGGTGCCGCGGATCTTGTT
16S-23S rRNA ITS-F	AAGTCGTAACAAGGTARCCG	约380
16S-23S rRNA ITS-R	TCGCCAAGGCATCCACC

药品名称	胞内分枝杆菌 (97株)		亚种胞内分枝杆菌 (62株)		副胞内分枝杆菌 (17株)		奇美拉分枝杆菌 (18株)		χ²值	P值
药品名称	耐药株数	耐药率(%)	耐药株数	耐药率(%)	耐药株数	耐药率(%)	耐药株数	耐药率(%)	χ²值	P值
克拉霉素	9	9.28	3	4.84	1	5.88	5	27.78	-	0.023^a
乙胺丁醇	43	44.33	28	45.16	5	29.41	10	55.56	2.470	0.311
利福布汀	15	15.46	8	12.90	5	29.41	2	11.11	-	0.249^a
利福平	41	42.27	23	37.10	9	52.94	9	50.00	1.914	0.402
链霉素	35	36.08	26	41.94	3	17.65	6	33.33	3.485	0.200
阿米卡星	11	11.34	9	14.52	0	0.00	2	11.11	-	0.333^a
利奈唑胺	37	38.14	25	40.32	7	41.18	5	27.78	1.011	0.626
莫西沙星	45	46.39	29	46.77	11	64.71	5	27.78	4.804	0.099

胞内分枝杆菌临床分离株亚种组成及体外耐药性分析

Subspecies and in vitro drug sensitivity analysis of Mycobacterium intracellular clinical isolates

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药品名称	MIC范围(μg/ml)	耐药临界浓度(μg/ml)
克拉霉素	0.06~64	32
乙胺丁醇	0.5~16	8
利福布汀	0.25~8	4
利福平	0.12~8	8
链霉素	0.5~64	64
阿米卡星	1~64	64
利奈唑胺	1~64	32
莫西沙星	0.12~8	4
多西环素	2~16	-
环丙沙星	0.12~32	-
异烟肼	0.25~8	-
乙硫异烟胺	0.30~20	-
复方磺胺甲噁唑	0.12/238~8/152	-

药品名称	胞内分枝杆菌 (μg/ml)		亚种胞内分枝杆菌 (μg/ml)		副胞内分枝杆菌 (μg/ml)		奇美拉分枝杆菌 (μg/ml)
药品名称	MIC₅₀	MIC₉₀	MIC₅₀	MIC₉₀	MIC₅₀	MIC₉₀	MIC₅₀	MIC₉₀
克拉霉素	1	4	2	2	1	2	2	64
乙胺丁醇	4	16	4	16	4	16	8	16
乙硫异烟胺	>20	>20	>20	>20	>20	>20	>20	>20
异烟肼	8	8	8	8	8	8	8	8
利福布汀	0.5	8	0.5	4	0.5	8	0.5	8
利奈唑胺	16	64	16	64	16	64	16	64
利福平	4	8	4	8	8	8	4	8
多西环素	16	16	16	16	16	16	16	16
环丙沙星	16	16	16	16	16	16	8	16
复方磺胺甲噁唑	2	8	2	8	2	4	2	4
莫西沙星	2	8	2	8	4	8	2	8
链霉素	32	64	32	64	16	64	32	64
阿米卡星	8	64	16	64	8	32	8	64

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