广州市非结核分枝杆菌临床分离株菌种鉴定及药物敏感性试验结果分析

doi:10.3969/j.issn.1000-6621.2019.05.012

摘要/Abstract

摘要：

目的对广州市非结核分枝杆菌(NTM)临床分离株进行菌种鉴定与药物敏感性试验(简称“药敏试验”),为临床诊断和治疗NTM病提供依据。方法从2016年广州市胸科医院行分枝杆菌分离培养的1250份标本中搜集培养阳性的717株临床分离株,采用水溶性对硝基苯甲酸(PNB)和噻吩-2-羧酸肼(TCH)对其进行培养,以鉴别MTB与NTM,通过多靶位基因聚合酶链反应 (PCR)测序,对PNB和TCH培养确定为NTM的菌株进一步行菌种鉴定,并采用最低抑菌浓度法(MIC)检测98株NTM对18种抗结核药物的耐药性。结果717株分枝杆菌中,检出106株NTM菌株,检出率为14.4%;其中103株传代成功,传代率为97.2%(103/106)。经NTM菌种鉴定,按照构成比高低依次为脓肿分枝杆菌[34.0%(35/103)]、胞内分枝杆菌[25.2%(26/103)]、马赛分枝杆菌[20.4%(21/103)]、鸟分枝杆菌[9.7%(10/103)]、堪萨斯分枝杆菌[5.8%(6/103)]、偶发分枝杆菌[1.9%(2/103)],以及1株缓黄分枝杆菌、1株猪分枝杆菌和1株阿加普尔分枝杆菌[均占0.9%(1/103)]。98株NTM菌株的药敏试验结果显示,敏感者前3位依次为阿米卡星[93.9%(92/98)]、克拉霉素[91.8%(90/98)]和阿奇霉素[71.4%(70/98)],耐药者前3位依次为利福平[89.8%(88/98)]、左氧氟沙星[83.7%(82/98)]和亚胺培南[77.6%(76/98)]。并且NTM不同菌种对部分药物的耐药性存在种间差异。结论广州市NTM的检出率高于我国其他地区,流行的菌种以脓肿分枝杆菌和胞内分枝杆菌为主;克拉霉素和阿米卡星是临床治疗NTM患者的优选抗生素,应重视对利福平和左氧氟沙星的耐药性。

关键词: 分枝杆菌, 非典型性, 细菌学技术, 微生物敏感性试验, 抗药性, 细菌, 结果评价(卫生保健)

Abstract:

Objective To evaluate the species of non-tuberculous mycobacterium (NTM) isolated from Guangzhou Chest Hospital and perform drug susceptibility testing on these strains, to provide scientific basis for the diagnosis and treatment of NTM disease. Methods In 2016, 717 clinical mycobacterium isolates were collected from Guangzhou Chest Hospital, obtained from 1250 sputum species. All isolates were subjected to pnitrobenzoic acid (PNB) and 2 thiophene carborylic acid hydrazide (TCH) testing to distinguish Mycobacterium tuberculosis (MTB) from NTM. Then multi-locus polymerase chain reaction sequencing analysis was carried out to identify the NTM strains (confirmed by PNB and TCH) to species level. Furthermore, microporous plate method contained 18 antimicrobials was conducted to confirm the minimal inhibitory concentrations (MICs) of different NTM species. Results Among the 717 clinical mycobacterium isolates, 106 (14.4%, 106/717) NTM isolates were confirmed, and 103 isolates were available. According to the species identification assay, the most frequently isolated NTM species was Mycobacterium abscessus (34.0%, 35/103), followed by Mycobacterium intracellulare (25.2%, 26/103), Mycobacterium massiliense (20.4%, 21/103), Mycobacterium avium (9.7%, 10/103), Mycobacterium Kansasii (5.8%, 6/103), Mycobacterium fortuitum (1.9%, 2/103), Mycobacterium flavobacteriae (0.9%, 1/103), Mycobacterium porcine (0.9%, 1/103) and Mycobacterium acapulcensis (0.9%, 1/103) in Guangzhou. According to the drug susceptibility testing results of 98 NTM strains, amikacin (93.9%, 92/98), clarithromycin (91.8%, 90/98) and azithromycin (71.4%, 70/98) were the three most effective agents for NTM infection. However,rifampicin (89.8%,88/98), levofloxacin (83.7%,82/98) and imipenem (77.6%,76/98) were the most resistant drugs to NTM. Furthermore, the different drug susceptibility profiles of different NTM species was also characterized. Conclusion The NTM incidence in Guangzhou is much higher than other regions in China, Mycobacterium abscessus and Mycobacterium intracellular are the two most prevalent NTM species. Clarithromycin and amikacin are the preferred antimicrobials for treatment of NTM disease, doctors should pay more attention to the resistante of rifampicin and levofloxacin in clinical.

Key words: Mycobacteria, atypical, Bacteriological techniques, Microbial sensitivity tests, Drug resistance, bacterial, Outcome assessment (health care)

刘东鑫,郑惠文,贺文从,宋怡蒙,何萍,赵冰,欧喜超,刘春法,赵雁林. 广州市非结核分枝杆菌临床分离株菌种鉴定及药物敏感性试验结果分析[J]. 中国防痨杂志, 2019, 41(5): 534-540. doi: 10.3969/j.issn.1000-6621.2019.05.012

Dong-xin LIU,Hui-wen ZHENG,Wen-cong HE,Yi-meng SONG,Ping HE,Bing ZHAO,Xi-chao OU,Chun-fa LIU,Yan-Lin ZHAO. Analysis of strains identification and drug susceptibility characteristics of non-tuberculous mycobacterium isolated in Guangzhou[J]. Chinese Journal of Antituberculosis, 2019, 41(5): 534-540. doi: 10.3969/j.issn.1000-6621.2019.05.012

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

药名	溶剂	药物储存液浓度(μg/ml)	浓度范围 (μg/ml)
克拉霉素	DMSO	1280	0.0625~64
阿奇霉素	无水乙醇	1280	0.0625~64
阿米卡星	水	1280	0.0625~64
卷曲霉素	水	1280	0.0625~64
左氧氟沙星	1%NaOH	1280	0.0625~64
莫西沙星	水	1280	0.0625~64
加替沙星	水	1280	0.0625~64
替加环素	水	1280	0.0625~64
氯法齐明	DMSO	1280	0.0625~64
利福平	DMSO	1280	0.0625~64
利福布汀	甲醇	1280	0.0625~64
利奈唑胺	DMSO	1280	0.0625~64
乙胺丁醇	水	1280	0.0625~64
米诺环素	水	5120	0.25~256
妥布霉素	水	5120	0.25~256
新诺明	1%NaOH	5120	0.25~256
亚胺培南	水	5120	0.25~256
头孢西丁	水	5120	0.25~256

菌种药物	敏感临界值 (μg/ml)	中度敏感临界值(μg/ml)	耐药临界值 (μg/ml)
快生长分枝杆菌
阿米卡星	≤16	32	≥64
克拉霉素	≤16	32~64	≥128
亚胺培南	≤4	8~16	≥32
利奈唑胺	≤8	16	≥32
米诺环素	≤4	8~16	≥32
莫西沙星	≤1	2	≥4
新诺明	≤2/38	-	≥4/76
妥布霉素	≤2	4	≥8
鸟-胞内分枝杆菌
克拉霉素	≤8	16	≥32
莫西沙星	≤1	2	≥4
利奈唑胺	≤8	16	≥32
堪萨斯分枝杆菌
克拉霉素	-	-	>16
利福平	-	-	>1
阿米卡星	-	-	>32
乙胺丁醇	-	-	>4
利奈唑胺	-	-	>16
莫西沙星	-	-	>2
利福布汀	-	-	>2
新诺明	-	-	>2/38

抗生素	药敏试验结果[株(构成比,%)]
抗生素	敏感	耐药
克拉霉素	90(91.8)	8(8.2)
利福平	10(10.2)	88(89.8)
利福布汀	39(39.8)	59(60.2)
利奈唑胺	66(67.3)	32(32.7)
乙胺丁醇	32(32.7)	66(67.3)
米诺环素	24(24.5)	74(75.5)
妥布霉素	41(41.8)	57(58.2)
新诺明	45(45.9)	53(54.1)
头孢西丁	47(48.0)	51(52.0)
亚胺培南	22(22.4)	76(77.6)
阿奇霉素	70(71.4)	28(28.6)
阿米卡星	92(93.9)	6(6.1)
卷曲霉素	34(34.7)	64(65.3)
左氧氟沙星	16(16.3)	82(83.7)
莫西沙星	57(58.2)	41(41.8)
加替沙星	49(50.0)	49(50.0)
替加环素	46(46.9)	52(53.1)
氯法齐明	64(65.3)	34(34.7)

抗生素	脓肿分枝杆菌(35株)		马赛分枝杆菌(21株)		鸟分枝杆菌(10株)		胞内分枝杆菌(26株)		堪萨斯分枝杆菌(6株)		χ²值	P值
抗生素	敏感	耐药	敏感	耐药	敏感	耐药	敏感	耐药	敏感	耐药	χ²值	P值
克拉霉素	33(94.3)	2(5.7)	19(90.5)	2(9.5)	8(80.0)	2(20.0)	25(96.1)	1(3.9)	5(83.3)	1(16.7)	-	0.341
阿奇霉素	30(85.7)	5(14.3)	19(90.5)	2(9.5)	3(30.0)	7(70.0)	15(57.7)	11(42.3)	3(50.0)	3(50.0)	-	<0.001
阿米卡星	34(97.1)	1(2.9)	20(95.2)	1(4.8)	8(80.0)	2(20.0)	25(96.1)	1(3.9)	5(83.3)	1(16.7)	-	0.161
卷曲霉素	5(14.3)	30(85.7)	2(9.5)	19(90.5)	6(60.0)	4(40.0)	16(61.6)	10(38.4)	5(83.3)	1(16.7)	-	<0.001
左氧氟沙星	3(8.6)	32(91.4)	5(23.8)	16(76.2)	2(20.0)	8(80.0)	2(7.7)	24(92.3)	4(66.7)	2(33.3)	-	0.009
莫西沙星	18(51.4)	17(48.6)	9(42.9)	12(57.1)	7(70.0)	3(30.0)	18(69.2)	8(30.8)	5(83.3)	1(16.7)	-	0.213
加替沙星	20(57.1)	15(42.9)	9(42.9)	12(57.1)	6(60.0)	4(40.0)	9(34.6)	17(65.4)	5(83.3)	1(16.7)	6.671	0.154
替加环素	28(80.0)	7(20.0)	15(71.4)	6(28.6)	1(10.0)	9(90.0)	1(3.8)	25(96.2)	1(16.7)	5(83.3)	-	<0.001
氯法齐明	25(71.4)	10(28.6)	11(52.4)	10(47.6)	6(60.0)	4(40.0)	18(69.2)	8(30.8)	4(66.7)	2(33.3)	-	0.651
利福平	0(0.0)	35(100.0)	1(4.8)	20(95.2)	3(30.0)	7(70.0)	4(15.4)	22(84.6)	2(33.3)	4(66.7)	-	0.003
利福布汀	4(11.4)	31(88.6)	3(14.3)	18(85.7)	7(70.0)	3(30.0)	21(80.8)	5(19.2)	4(66.7)	2(33.3)	-	<0.001
利奈唑胺	30(85.7)	5(14.3)	17(81.0)	4(19.0)	5(50.0)	5(50.0)	10(38.5)	16(61.5)	4(66.7)	2(33.3)	-	0.001
乙胺丁醇	2(5.7)	33(94.3)	2(9.5)	19(90.5)	6(60.0)	4(40.0)	17(65.4)	9(34.6)	5(83.3)	1(16.7)	-	<0.001
米诺环素	5(14.3)	30(85.7)	10(47.6)	11(52.4)	3(30.0)	7(70.0)	3(11.5)	23(88.5)	3(50.0)	3(50.0)	-	0.011
妥布霉素	14(40.0)	21(60.0)	3(14.3)	18(85.7)	6(60.0)	4(40.0)	16(61.5)	10(38.5)	2(33.3)	4(66.7)	-	0.011
新诺明	22(62.9)	13(37.1)	4(19.1)	17(80.9)	5(50.0)	5(50.0)	9(34.6)	17(65.4)	5(83.3)	1(16.7)	-	0.004
头孢西丁	27(77.1)	8(22.9)	15(71.4)	6(28.6)	3(30.0)	7(70.0)	1(3.8)	25(96.2)	1(16.7)	5(83.3)	-	<0.001
亚胺培南	10(28.6)	25(71.4)	8(38.1)	13(61.9)	2(20.0)	8(80.0)	1(3.8)	25(96.2)	1(16.7)	5(83.3)	-	0.032