Abstract: Objective To confirm the species of the clinical isolates suspected Mycobacterium massiliense (M. massiliense) which could not be identified by the single gene comparison, and test their drug-sensitivity spectrum to provide the basis of clinical diagnosis and treatment.  Methods In 2013，we collected 47 clinical samples of the patients suspected with non-tuberculous Mycobacteria (NTM) infection in Tuberculosis Laboratory of National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The Mycobacteria isolates were determined with the media containing p-nitrobenzoic acid (PNB) and 2-thiophene carboxy-lic acid hydrazide (TCH) respectively and multilocus PCR. Five house-keeping genes (16S rRNA, hsp65, rpoB, sodaand recA) were analyzed by DNA sequencing, and compared by Basic Local Alignment Search Tool (BLAST) to identifying the species of NTM, in which the species of 6 M. massiliense isolates were confirmed by means of multilocus sequence analysis (MLSA). The drug-sensitivities of M. massiliense isolates to 36 antibiotics were tested with the minimal inhibitory concentrations (MICs) by Microplate AlamarBlue Assay(MABA).  Results Of 47 clinical samples from the patients suspected with NTM infection, 34 NTM strains were isolated, in which 6 strains could not be identified by DNA sequencing and BLAST using the single gene sequence comparison with five house-keeping genes. These strains were confirmed as M. massiliense by MLSA with a Neighbor-joining tree. The drug sensitive spectrum to 36 antibiotics showed that M. massiliense isolates were sensitive to Amikacin (16 μg/ml or 32 μg/ml), resistant to 12 kinds of anti-tuberculosis drugs, rifampin, isoniazid, ethambutol, ciprofloxacin, ethionamide, capreomycin, para-aminosalicylic acid, ofloxacin, kanamycin, cycloserine, meropenem, and minocycline; and extremely insensitive to isoniazid aminosalicylate, cefoperazone and thioacetazone.  Conclusion The MLSA method is effective in identification of M. massiliense strain. M. massiliense strains had a higher degree of drug resistance.

Key words: Mycobacterium, Multilocus sequence typing, Microbial sensitivity tests