Identification On Species Of Nontuberculosis Mycobacterium Clinical Isolates From8Provinces Of China

Objectives: To investigatethe species of nontuberculosis mycobacteria (NTM) inChina, preliminarily, for providing the important basis of NTM diseases clinicaldiagnosis, treatment, control and prevention. Evaluate the methods for speciesidentification of NTM, such as PCR-restriction fragment-length polymorphismanalysis (PRA) and gene sequencing.Methods:1. From2005to2012, we collected610NTM clinical strains isolated from thepatients with suspected with TB in8provinces (including Anhui, Fujian, Gansu,Hunan, Jiangxi, Inner Mongolia, Sichuan and Xijiang) of China, preliminarilyidentified by local province’s TB hospital or TB institute with p-nitrobenzoicacid/2-thiophene carboxylic acid hydrazide (PNB/TCH) medium, and cultured andpreserved with conventional methods.2. The strains were identified by means of PNB/TCH medium and multi-locuspolymerase chain reaction (Multi-locus PCR) in this study again.3. The strains of NTM were identified to species or sub-species by the differentmolecular methods including PRA-rpoB and PRA-hsp65and multiple genesequencing including sequencing hsp65, rpoB,16S–23S internal transcribed spacerregion (ITS), gnd, glpK, secA and sodA.Results:1. We collected a total of610isolates of NTM identified in the local labs werefrom the8provinces of China, and identified and confirmed them to beMycobacterium (M.) species, including53Mycobacterium tuberculosis (M.tuberculosis) and557NTM strains.2. Of above557clinical NTM strains identified with PRA and analysis of multiple gene sequencing, the results showed that there were4Gordonia bronchialis,46Nocardia farcinica and507NTM strains. There were28species of NTM,including66M. abscessus,106M. avium,17M. chelonae,3M. colombiense,12M.fortuitum,1M. genavense34M. gordonae,1M. holsaticum,209M. intracellulare,3M. kansasii,2M. kumamotonense,6M. lentiflavum,1M. mantenii,5M. marseillense,24M. massiliense,1M. monacense,2M. neoaurum,3M. parascrofulaceum,1M.phocaicum,2M. saskatchewanense,1M. senegalense,2M. seoulense,2M. septicum,1M. setense,2M. shimoidei,1M. stomatepiae,3M. szulgai and2M. triplex..3. Of507clinical NTM strains the first nine species of NTM are M.intracellulare, M. avium, M. abscessus, M. gordonae, M. massiliense, M. chelonae, M.fortuitum, M. lentiflavum and M. marseillense, respectively.Conclusions:1. A total of28species of NTM were found causing pulmonary infection ofhuman being in8provinces of China. In China, we firstly identified6new NTMspecies, including M. marseillense, M. saskatchewanense, M. seoulense, M.kumamotonense, M. stomatepiae, M. mantenii, and Gordonia bronchialis in thepatients clinically diagnosed with tuberculosis. It is confirmed that there are greatvariety species of NTM causing pulmonary infection of human being in China.2. PRA-rpoB and PRA-hsp65are good methods for identifying NTM strains tospecies.3. Combining multilocus PCR, PRA-hsp65, PRA-rpoB and multiple genesequencing methods can improve the accuracy and efficacy for identifying NTM tospecies/subspecies.