Comparison Of Molecular Methods For Rapid Identification Of Mycobacterium And Its Analysis Of Drug-resistane

Objective:To investigate and evaluate the application of four molecular methods for rapid identification of mycobacterium, and compare two antimicrobial susceptibility test for the drug-resistance analysis of Mycobacterium.Methods:Two hundred and four clinical mycobacterium isolates were collected, inoculated to Lowenstein-Jensen media and incubated at37degree, single colone was picked for acid fast staining when growth was detected. Meanwhile, colloidal gold immunochromatography (Chuangxin, China) was used for rapid discrimination of Mycobacterium tuberculosis and Non-tuberculosis mycobacterium (NTM). Method of16S rRNA gene sequencing was used as the standard method for Mycobacterium identification, meanwhile, heat shock protein65gene restriction fragment length polymorphism (hsp65PCR-RFLP), hsp65gene sequencing and GenoType Mycobacterium CM/AS assay (HAIN Lifescience, Germany) were used to identify Mycobacterium strains and compare with16S rRNA gene sequencing. Tube proportion method and sub-grid plate agar proportion method were used to detect the antimicrobial susceptibility of Mycobacterium.Results:All of the two hundred and four clinical mycobacterium isolates were positive for acid fast staining, and grew well on Lowenstein-Jensen media. One hundred and thirteen M. tuberculosis strains and ninty-one NTM strains were detected with colloidal gold immunochromatography.Result of16S rRNA gene sequencing showed that there were one hundred and thirteen M. tuberculosis strains, forty M. intracellulrae, fifteen Mycobacterium chelonae complex strains, eight M. fortuitum strains, six Mycobacterium kansasii complex strains, five M. avium strains, four M. smegmatis strains, three M. phlei strains, two M. scrofulaceum strains, two M. nonchromogenicum strains, two M. terrae strains, two M. gordonae strains, one M. aurum strain and one M. neoaurum strain.Compared with16S rRNA gene sequencing, three M. phlei, one M. fortuitum strain and one M. nonchromogenicum strain by hsp65PCR-RFLP conflicted, others got the same results with16S rRNA gene sequencing and the coincidence was97.5%; one M. intracellulrae strain, one M. hiberniae strain and one M. terrae strain by hsp65gene sequencing conflicted with16S rRNA gene sequencing and the coincidence was98.5%; except for six strains identified to Mycobacterium by GenoType Mycobacterium CM/AS assay, others got the same results as16S rRNA gene sequencing and the coincidence was97.1%.The results of tube proportion method showed that the resistance rate of isoniazid, rifampicin, ethambutol and streptomycin was78.76%(89/113),65.49%(74/113),41.59%(47/113) and57.52%(65/113), respectively. The percent of extensively drug-resistant (XDR) strains and multidrug-resistant (MDR) strains were54.87%(62/113)and16.81%(19/113), respectively; However, the numbers of full-susceptible strains were only14with the sensitivity of12.39%(14/113). Each Mycobacterium was resistant to the first anti-tuberculosis drugs at different degree.The results of two isoniazid drug-resistant strains, two rifampicin drug-resistant strains and one ethambutol drug-resistant strains by sub-grid plate agar proportion method conflicted with the tube proportion method for M. tuberculosis strains, and the coincidence was96.63%(86/89),97.29%(72/74),97.87%(46/47) and100%(65/65) for isoniazid, rifampicin ethambutol, and streptomycin, respectively. Only one M. intracellulrae strain and one M. kansasii strain by sub-grid plate agar proportion method conflicted with tube proportion method for NTM strains, and the coincidence was97.80%(89/91).Conclusions:1. Method of colloidal gold immunochromatography could differentiate M. tuberculosis and NTM strains preliminary rapidly.2. Method of16S rRNA gene sequencing couldn't identify some most common clinical Mycobacterium strains like Mycobacterium chelonae complexes and Mycobacterium kansasii complexes, and it wasn't advised to be the fisrt choice for Mycobacterium identification clinically.3. GenoType Mycobacterium CM/AS assay is advised to be the fisrt choice for Mycobacterium identification clinically. For those rare isolates of Mycobacterium,16S rRNA and hsp65gene sequencing are advised. Hsp65PCR-RFLP should be the secondary verification method and supplement of restriction enzymes database.4. The method of tube proportion method should be the first choice for drug-resistance method of Mycobacterium; the coincidence of sub-grid plate agar proportion is relatively high and the opperation is very easy. So it could be applied at clinical tuberculosis laboratories for batch production.