Inactivation of Cultured Mycobacterium tuberculosis Organisms Prior to DNA Extraction

Abstract

Somerville and his colleagues have made an important point that current protocols for DNA extraction from cultured Mycobacterium tuberculosis organisms do not ensure complete inactivation of the microorganism (9). While diagnosis samples of tuberculosis can be manipulated under biosafety level 2 (BSL2) conditions, live cultured M. tuberculosis organisms have to be manipulated under BSL3 conditions. Accordingly, M. tuberculosis organisms have to be inactivated prior to release outside a BSL3 laboratory for further molecular biology manipulation, for example. Indeed, M. tuberculosis poses potential risks for the laboratory personnel working with this microorganism (6). There are several reports of laboratory-acquired tuberculosis infections, with aerosols and skin punctures being the most common reported routes of transmission (8). This emphasizes the need for complete inactivation of M. tuberculosis before extraction of DNA from this microorganism. We surveyed the 2004 issues of the Journal of Clinical Microbiology for published reports addressing this issue. To our surprise, in only 6 of a total of 64 reports that we surveyed were cultured M. tuberculosis organisms inactivated before DNA was extracted from them. This prompted us to evaluate a simple protocol for the inactivation of and DNA extraction from cultured M. tuberculosis. Discussed herein are 10 isolates of mycobacterium belonging to the M. tuberculosis complex. Their identities were based on a 16S rRNA gene probing assay (BioMerieux, Marcy l'Etoile, France). The identification of species within the complex was based on the resistance of the isolates to pyrazinamide in order to distinguish M. tuberculosis from Mycobacterium bovis (5). The mycobacterial isolates had been stored at −20°C by freezing the beads on which they were placed (Dominique Dutscher, Brumath, France) before they were used. For inactivation, one bead was held (about 2 h) in 100 μl of 90% ethanol (Carlo Erba Reactifs SA, Val de Reuil, France) at room temperature until complete evaporation of the ethanol was achieved before the organisms were incubated at 96°C in 20% Chelex for an hour (2). The efficacy of the inactivation procedure was assessed by inoculating BACTEC9000 MB broth (BD Diagnostic Systems, Le Pont de Claix, France) with the bead according to the manufacturer's instructions and, in parallel, scraping the bead onto 5% sheep blood agar at 37°C (4). As a positive control, a bead of the same isolate not inactivated as described above (i.e., the noninactivated bead) was cultured in parallel under the same conditions. After 3 weeks of incubation, M. tuberculosis was cultured in broth and blood agar from only the noninactivated beads, not the beads that had been inactivated (10 of 10 versus 0 of 10 tested; P < 0.0001 by the chi-square test with Yates' correction). Furthermore, the supernatant was subjected to PCR for the amplification of the rpoB gene (7) and hybridization using 16S rRNA probes (GenProbe, San Diego, CA). A positive amplicon was obtained with all 10 isolates tested, and sequencing was easily performed in every case. Like-wise, all DNA extracts yielded positive hybridization with the 16S rRNA gene-derived probe. These data show that the inactivation-extraction protocol that we evaluated was suitable for providing M. tuberculosis DNA for hybridization and PCR-based experiments. This protocol may be helpful since the issue of M. tuberculosis organisms' inactivation remains problematic, as no protocol until now had proved effective. Heating the microorganism at 80°C is the most popular protocol according to favorable evaluations in several reports (1, 3). However, the reproducible effectiveness of this simple protocol remains controversial (10). Likewise, further incubation in the presence of proteinase K and lysozyme failed to inactivate M. tuberculosis (1). Somerville and collaborators found that as many as 77.1% of 35 M. tuberculosis isolates heated at 80°C remained viable, despite further chemical treatments (9). The protocol presented herein takes 3 h, uses nontoxic substances, and requires only basic laboratory equipment, thus offering a convenient way to effectively inactivate M. tuberculosis prior to PCR amplification and hybridization. Assessment of DNA suitability for other molecular biology works, such as restriction profiling, warrants further studies. We are using this protocol routinely for the inactivation of M. tuberculosis isolates before their release from a level 3 containment facility for further molecular workup.