Species identification of mycobacteria by sequencing of amplified 16S rDNA from hemocultures

Abstract

PCR sequencing of the gene coding for 16S rRNA (16S rDNA) is a well established method used to identify mycobacteria in clinical samples. A common technique problem with PCR is amplification failure due to the presence of PCR inhibitor (s). Initial attempt to amplify mycobacterial 16S rDNA from hemocultures failed because of this reason. Five DNA extraction methods were used for purification of DNA and removal of inhibitor (s) from hemoculture. Alkali wash and heat lysis was found to be the best suit method for preparation of mycobacterial DNA from hemoculture. The results of sequencing of amplified 16S rDNA were compared with those of conventional method and AccuProbe (Gen-Probe, Inc., San Diego, Calif.). Out of 381 hemoculture in MB/BacT instrument, 73 samples (19.16%) were flagged positive. Sixty-nine flagged positive hemocultures were acid-fast bacilli (AFB) positive and 4 samples were acid-fast bacilli (AFB) negative. Of these 69 AFB positive samples, 66 grew 67AFB and 3 could not grow AFB on solid media. Identification by conventional method and AccuProbe revealed 4 different species as follows: M. tuberculosis, M. avium complex, M. scrofulaceum and M. simiae. Four isolates from 4 samples were unidentified and one isolate was mis-identified. Identification by 16S rDNA sequencing demonstrated 9 different species as follows: M. tuberculosis, M. avium. M. intracellulare, M. scrofulaceum. M. simiae, M. ulcerans, M. haemophilum. M. interjectum, and M. triplex. The results of species identification by these methods were concordant except one isolate identified as M. scrofulaceum with 16S rDNA analysis was identified as M. xenopi with conventional methods. This was not uncommon as M. scrofulaceum phenotypically resembles M. xenopi . This study concludes that direct sequence analysis of amplified 16S rDNA is a promising, rapid (within 3 days) and accurate method for species determination of mycobacteria. This method might also be applicable for routine identification of mycobacteria from hemocultures in advanced laboratory.