Analysis of strains identification and drug susceptibility characteristics of non-tuberculous mycobacterium isolated in Guangzhou

doi:10.3969/j.issn.1000-6621.2019.05.012

Abstract

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)

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

Figures/Tables 7

References 30

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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