The Sequence Analyses Of 16S RRNA Genes And 16S-23S RRNA Gene Internal Transcribed Spacers Of Mycobacteria

Mycobacteria include Mycobacterium tuberculosis complex (MTC, including M. tuberculosis, M.microti, M. bovis, M. africanum), M. leprae and nontuberculous mycobacteria (NTM) which include all mycobacterial organisms, except for MTC and M. leprae. NTM agents are more than 100 species and may be pathogenic, opportunistic, or nonpathogenic. NTM agents infect both immune-competent and immune-compromised patients, especially AIDS patients; they primarily cause pulmonary disease, cervical lymphadenopathy and localized skin and soft tissue lesions. With the increasing of infection caused by M. tuberculosis and NTM agents worldwide, quickly to identify the pathogenic or opportunistic mycobacterial strains isolated from the patients is becoming more clinically important. The identification of the pathogenic or opportunistic mycobacterial agents from the patients is not only epidemiological implications but is also relevant to the demand of patient treatments. Identification of mycobacterial strains by conventional biochemical methods is cumbersome and time-consuming and the phenotype tests sometimes fail to discriminate between some closely species. Genotype methods for the identification of mycobacteria have been developed in recent years. Currently, a selected target gene-based sequence analysis is a now widely accepted strategy used for rapid and accurate identification of mycobacteria.In this study, the sequence analyses of 16S rDNA and 16S-23S rDNA internal transcribed spacer (ITS) of mycobacteria were established and 29 mycobacterial strains isolated in a hospital of ChongQing city were identified by the sequence analyses. The 16S rDNA sequences from 14 strains were identical to that of MTC, M.gordonae, M.neoaurum, M.chlorophenolicum, and M.abscessus/ M.chelonae, respectivelyï¼›the ITS sequences of 10 strains were identical to that of MTC, M.gordonae, and M.abscessu, respectively. Therefore, these strains may correctly be identified based on the identical sequences. The ITS sequences of mycobacteria are much more variant compared with the 16S rDNA sequences and ITS sequence are used in identifying the closely related species and the strains of the species. In this study, one strain considered to be M.abscessus orM.chelonae was identified to be M.abscessus. The 16S rDNA sequences of 4 strains of M.neoaurum were identical, however, their ITS sequences were different at 1.7%~23.7%., suggesting they are different subspecies or types of M.neoaurum.Some of the 16S rDNA or ITS sequences of the isolated strains were not identical to any sequence in the GenBank. The similar levels of the different sequences were 86.7%~ 99.7% for 16S rDNA sequences and 30.4%~99.2% for ITS sequences. The sequence comparison of 16S rDNA and ITS sequences with the members of genus mycobacteria reveals that certain strains are new species or subspecies. The PCR primers used in thist studies were designed based on the genus-specific sequences of mycobacteria and they may exclude the DNA contamination of other bacteria in samples. The costs of DNA sequencing of the PCR products are low because the fragment sizes of 16S rDNA and 16S-23S rDNA ITS amplified in PCR are ~500bp and they may be completely sequenced in only one reaction. It is more important that the PCR and sequencing in this study may be completed in 1~2 days; therefore, the 16S rDNA and 16S-23S rDNA sequence analyses may be a reliable, rapid, and low costs way for identification of mycobacterial strains.