[Application of DNA-DNA hybridization, DDH MYCOBACTERIA 'Kyokuto' to species-identification of mycobacteria grown in Middlebrook 7H9 broth].

ABSTRACT

A colorimetric DNA-DNA hybridization for the genetic identification of mycobacteria, DDH MYCOBACTERIA 'KYOKUTO' (Kyokuto Pharmaceuticals, Tokyo) was evaluated for the clinical isolates of mycobacteria grown in Middlebrook 7H9 broth of MB/BacT (Organon Teknika, Durham, NC, U.S.A.). When the MB/BacT gave the positive interpretation, 10 ml of Middlebrook 7H9 broth was collected from the bottle. After centrifugation at 3,000 rpm for 10 min, two drops of acetone were added to the pellet, then let it stand for one hour at the room temperature. The air-dried pellet was resuspended in a small volume of distilled water, and proceeded to the identification described. Of 136 clinical isolates of mycobacteria, comprising of 76 M. tuberculosis complex and 60 nontuberculous mycobacteria (NTM), ninety-five (70%) were correctly identified when compared to the reference identification. Thirty (22%) isolates resulted in the unidentified due to negative reaction throughout the test wells, and the remaining 11 (8%) were also unidentified due to low likelihoods. According to the package insert, the DDH MYCOBACTERIA may not be applicable to the isolates grown in Middlebrook broth. However, our test procedure using acetone prior to the extraction of bacterial DNA enables us to directly identify the isolates of mycobacteria grown in Middlebrook 7H9 broth of MB/BacT.