Evaluation of methods for isolation of DNA from slowly and rapidly growing mycobacteria

International Journal of Leprosy and Other Mycobacterial Diseases, Dec 1997 by Zhang, Zheng-Qing, Ishaque, Muhammad

Evaluation of Methods for Isolation of DNA from Slowly and Rapidly Growing Mycobacteria1

Zheng-Qing Zhang and Muhammad Ishaque2

At present, 71 recognized or proposed species meet the standards for inclusion in the genus Mycobacterium. These species are usually grouped into two major divisions, "rapidly growing" and "slowly growing," based on the time required for visible colonies to appear on a solid medium. The appearance of colonies requires less than 7 days for the rapidly growing species and more than 7 days for the slowly growing species. These divisions are very important clinically and in identification schemes. The slowly growing species may cause disease in humans and animals; whereas the rapidly growing species do not (18,19, 20). More than 25 species in the Mycobacterium genus, including M. leprae and M. tuberculosis, may cause disease in humans, and more than six species may cause disease in animals.

Mycobacterial cell walls have been shown to contain type-specific antigenic glycolipids, such as phenolic glycolipids (PGL), glycopeptidolipids (GPL), and trehalose-containing lipo-oligosaccharides (LOS). The compositions of these waxy lipids, mycolic acid-containing cell walls vary depending upon the species of Mycobacterium (1, 2, 7, 21). Various methods have been described for the extraction of DNA from mycobacteria (3, 6, 9, 22). The critical step is lysis of the cells to release DNA, while avoiding its degradation or shearing. Due to the cell-wall thickness, mycobacteria are resistant to enzyme lysis by the commonly described methods. The ability to identify and to type mycobacteria from environmental and clinical sources is limited by the lack of a simple, rapid and efficient method of DNA preparation by which many samples can safely be processed simultaneously (22). Since the morphology and the biosynthetic capabilities of mycobacteria vary, depending on cultural conditions and strain differences within a species, the most reliable method of identification would be at the DNA level (15, 19).

Although research in molecular biology and recombinant DNA technology has advanced markedly in recent years, the lack of a simple and efficient method for extracting DNA from mycobacteria has lagged behind the need of research. During this study, five methods were used to extract DNA from slowly growing mycobacteria such as M. leprae and M. lepraemurium, the causative agents of human and murine leprosy, respectively, as well as M. bovis BCG, an etiologic agent of tuberculosis. M. phlei, a nonpathogen and rapidly growing mycobacterium, was used as a control. The quantity and the quality of these DNAs were analyzed by electrophoresis and restriction endonuclease digestion.

MATERIALS AND METHODS

Sources and growth of mycobacteria

M. bovis BCG substrain Laval was kindly provided by Dr. P. Rousseau (BioVac, Institut Armand-Frappier, Laval, QC, Canada), after being grown on Sauton medium. M. phlei was grown on the Lowenstein-Jensen medium at 37 deg C for a period of 5-7 days (15). M. leprae were obtained from infected foot pads of nude mice maintained in our laboratory (12). M. leprae from infected armadillo liver tissue was a gift kindly provided by Dr. A. M. Dhople, Florida Institute of Technology, Melbourne, Florida, U.S.A. M. lepraemurium Hawaiian strain used in this study was obtained from Dr. P. Greenburg (La Roche Research Laboratory, Nutley, New Jersey, U.S.A.). The strain was maintained in C3H mice by subcutaneous serial passages every 4-6 months in our laboratory (13).

Reagents

Reagents used for isolation of M. leprae and M. lepraemurium cells from infected animals were those recommended by Draper (10) and Clark-Curtiss, et al. (8). Buffer A contained 150 mM NaCl, 1 mM MgSO^sub 4^o and 15mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.2. Buffer B contained 150 mM NaCl, 200 mM Tris buffer, and 1 mM MgSO^sub 4^, pH 7.2. Buffer C contained 1 ml 10% Tween 80, 0.2 ml of 10% 2-(N-morpholino)ethanesulfonic acid (MES), and 100 ml distilled water, pH was adjusted to 7.2. All three buffers contained 1 mM benzamidine as an inhibitor of animal cellular proteases. The 30% Percoll gradient contained 30 ml Percoll (Sigma Chemical Co., St. Louis, Missouri U.S.A.) and 70 ml buffer C.

Reagents for DNA extraction and preservation were described by Sambrook, et al. (17). NET buffer contained 100 mM NaCl, 10 mM EDTA and 10 mM Tris-HCl, pH 7.5. TE buffer contained 1 mM EDTA and 10 mM Tris-HCl, pH 7.5.

The enzymes, such as proteinase K, pronase, lysozyme, DNase, and RNase A were purchased from Boehringer Mannheim (Laval, Canada); the other enzymes, such as subtilisin (protease VIII) and collagenase were purchased from Sigma; Pfu DNA polymerase was purchased from Stratagene (La Jolla, California, U.S.A.); Pst I was purchased from Pharmacia Biotech, (Baie d'Urfe, Canada).

Isolation and preparation of mycobacterial bacillary suspensions

Isolation of M. leprae bacilli from infected nude mouse foot pads and preparation of bacillary suspensions. The method for quantitative recovery of M. leprae cells from infected nude mouse foot pads was adapted from a method developed by Franzblau and Hastings (11) with some modifications. Briefly 10 g of foot pads were surface decontaminated by using ultraviolet (UV) irradiation, 1% iodine and 70% ethanol. Foot pads were then cut into small pieces with a pair of scissors and triturated thoroughly in a mortar in buffer A. The mixture was passed through a sterile nylon filter into a sterile 250-ml beaker to remove tissue strands, and then the filtrate was centrifuged (Beckman J21 B) at 1200 x g x 10 min at 4 deg C to remove the tissue debris. The tissue debris was triturated and filtered through a nylon filter once more as described above to further recover the bacilli. The pooled filtrate was centrifuged at 10,000 x g x 15 min at 4 deg C. The resulting supernatant was carefully removed, without disturbing the pellets, and discarded. The pellets were suspended in 30 ml NET buffer.