湖南省51株疑似非结核分枝杆菌临床分离株的菌种鉴定

Identification of 51 clinical isolates suspicious of nontuberculosis mycobacteria in Hunan province, China

目的对2011年湖南51株非结核分枝杆菌(NTM)临床分离株进行菌种鉴定。方法从湖南省结核病防治所接收到分枝杆菌临床分离株后,通过罗氏培养基(L-J)、对硝基苯甲酸(p-nitrobenzoic acid,PNB)和噻吩-2-羧酸肼(2-thiophene carboxylic acid hydrazide,TCH)鉴别培养基和多位点PCR方法鉴别出非结核分枝杆菌后,对其进行基于rpoB基因的限制性酶切片段长度多态性(PRA)和基因测序方法的鉴定。结果从2011年湖南省51份临床分离菌株中培养出51份抗酸杆菌,通过初步鉴别(即L-J、PNB/TCH鉴别培养基培养)鉴别出NTM有51株,经rpoB-PRA及进一步的测序鉴别出50株(98.04%)NTM,分别为胞内分枝杆菌(Mycobacterium intracellμlare)22株(43.14%),鸟分枝杆菌(M.avium)13株(25.49%),脓肿分枝杆菌(M.abscessus)6株(11.76%),偶发分枝杆菌(M.fortuitum),戈登分枝杆菌(M.gordonae),新产色分枝杆菌(M.neoaurum)各2株(均占3.92%),堪萨斯分枝杆菌(M.kansasii),马赛分枝杆菌(M.massiliense),福西亚分枝杆菌(M.phocaicum)各1株(分别占1.96%);以及鼻疽诺卡菌(Nocardia farcinica)1株(1.96%)。结论抗酸染色阳性、PNB/TCH鉴定为非结核分枝杆菌临床分离株中包含不同种类的非结核分枝杆菌和诺卡菌。湖南非结核分枝杆菌病的病原至少有9种。使用分子生物学方法能够快速、准确地对其鉴别。

Objective To identify 51 clinical isolates suspicious of nontuberculosis mycobacteria（NTM）in Hunan province,China.Methods Sputum samples were collected from suspected tuberculosis patients and cultured using L-J and PNB/TCH media.Nontuberculosis mycobacteriawere determined by PCR,and PCR-restriction fragment length polymorphism analysis（PRA）of the rpoB gene and DNA sequencing were applied for the identification of species.Results 51 acid-fast bacilli were cultured from 51 samples which were found to be NTM by initial L-J and PNB/TCH differential media culture.Further identification by rpoB-PRA and PCR-sequencing revealed there were only 50（98.04%）NTM including 22（44.00%）of Mycobacterium intracellular,13（26.00%）of M.avium,6（11.76%）of M.abscessus,2（3.92%）of M.fortuitum,M.gordonae and M.neoaurum,1（1.96%）of M.kansasii,M.massiliense and M.phocaicum,and 1（1.96%）of Nocardia farcinica.Conclusions There are as many as 9 NTM species found in clinical isolates from patients with suspicious tuberculosis in Hunan,which can be rapidly identified from acid fast bacilli by molecular methods.