Species Identification of Mycobacterium avium Complex Isolates by a Variety of Molecular Techniques

MAC isolates.A patient profile sheet was obtained for each human isolate. The strains used in this study were obtained from the following sources: Walter Pace, University of Arkansas for Medical Sciences clinical laboratory and the John L. McClellan Memorial Veteran's Hospital clinical laboratory; Richard Wallace and Barbara Brown, Department of Microbiology, University of Texas Health Center at Tyler; Leonid Heifets and Gwen Huitt, National Jewish Center for Immunology and Respiratory Medicine; Richard J. Blinkhorn, MetroHealth Medical Center Clinical Laboratory, Cleveland, Ohio; Hiroe Shiratsuchi, Case Western Reserve University, Cleveland, Ohio; and William Stead, Arkansas State Health Laboratory. The isolates from the United Kingdom were received from Jack Crawford, Centers for Disease Control and Prevention, Atlanta, Ga., and were isolated by Tobin Hellyer, Becton-Dickinson Corp., Baltimore, Md.

In some cases the isolates had been streaked for isolation and single colonies had been selected before being sent to us. All other isolates, upon receipt, were streaked onto 7H11 plates and were examined for colonial morphology in our laboratory. Single colonies were selected and cultivated in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI), two aliquots were frozen, and the remainder of the culture was used for DNA isolation and pulsed-field gel electrophoresis (PFGE).

Testing with Gen-Probe kits.Culture confirmation kits (AccuProbe; Gen-Probe) were used to determine the species of each isolate. The M. avium probe kit detects M. aviumisolates, the M. intracellulare probe kit detects M. intracellulare isolates, and the MAC probe kit detects M. avium, M. intracellulare, and MAC isolates which are designated X or nonspecific MAC and which are not detected by either species-specific probe (3). Each test was performed by following the manufacturer's instructions and with the inclusion of controls.

Each isolate was tested with the MAC AccuProbe kit to confirm that the isolates belonged to MAC. Isolates were then tested with the M. avium-specific AccuProbe, and all positive isolates were given a number preceded by an “A” to identify them as M. aviumisolates. Isolates that were positive with the MAC-specific AccuProbe but negative with the M. avium-specific probe were tested with the M. intracellulare-specific AccuProbe. Isolates that were positive with this probe were given a number preceded by “I” to identify them as M. intracellulare isolates. Isolates that were positive with the MAC-specific probe but that were negative with the M. avium-specific or the M. intracellulare-specific probe were given a number preceded by “X” to identify them as nonspecific MAC isolates.

Preparation of genomic DNAs from mycobacteria.The following large-scale DNA preparation method was developed in our laboratory. For DNA preparation, mycobacterial cells (10 to 100 ml) were incubated to a Klett reading of 100 to 150, which represents logarithmic-phase growth, and were treated with d-cycloserine (1 mg/ml) for 24 h. After sedimentation in the centrifuge, the cell pellets were heat killed, resuspended in 1 ml of lysing buffer (4 mM Tris HCl [pH 8.0], 4% sodium dodecyl sulfate, 10 mM NaCl, 0.001 mM EDTA, 0.1 mg of proteinase K per ml), and incubated at 37°C overnight or at 68°C for 2 h. The lysate was cooled to room temperature, and the debris was pelleted by centrifugation. The supernatant was extracted two times with phenol-chloroform in phase-lock gel tubes (Five Prime to Three Prime Inc., Boulder, Colo.). After a final extraction in chloroform the aqueous phase was precipitated with 0.3 M sodium acetate (pH 5.5) and 2 volumes of ethanol for 15 min at −70°C. Preparations were centrifuged for 20 min at 4°C, washed one time with 70% ethanol, dried, and reconstituted in TE (Tris-EDTA) buffer. Yields were approximately 200 to 1,000 ng/μl.

PCR assays.DNA amplification was performed on a GeneAmp PCR system 9600 (Perkin-Elmer, Norwalk, Conn.) in a 50-μl reaction mixture containing 10 pmol of each primer, 1 ng of genomic DNA, 200 μM deoxyribonucleoside triphosphates, 1× PCR buffer (1.5 mM MgCl₂ [pH 8.3]; Perkin-Elmer), and 1 U of TaqDNA polymerase (Perkin-Elmer). Occasionally, the organisms were tested directly in the PCR without prior DNA extraction. In this case, a sterile toothpick was used to obtain cells from a colony, and the cells were added directly to 49 μl of the complete PCR mixture. Primers and conditions for IS1245 PCR were as reported and amplify IS1245 and the closely related element IS1311(6, 20). The primers specific for the mig gene were designed to amplify a 737-bp fragment within the coding region of the gene (16). The mig-specific primer sequences were as follows: mig upper, 5′-CCC GTT CAA CGT CAA CTT CC-3′; mig lower, 5′-GGG CTC GCC GGT CAT CAG GT-3′. The cycling conditions were as follows: initial denaturation at 95°C for 5 min and 30 cycles of 30 s of denaturation at 95°C and 2.0 min of annealing and extension at 68°C, followed by a single 5-min extension at 72°C. The PCR products were electrophoresed on a 2% agarose gel, with a 1-kb DNA ladder used for size determination (Gibco Life Technologies, Grand Island, N.Y.), stained with ethidium bromide, and photographed by using the Eagle Eye II gel documentation system (Stratagene, La Jolla, Calif.).

Fingerprinting with IS1245. For fingerprinting with IS1245 (6), isolated genomic DNA (500 ng) was digested with the restriction enzyme PvuII, electrophoresed on a 0.7% gel, alkaline transferred to Hybond (Amersham Pharmacia Biotech, Piscataway, N.J.), probed with a 427-bp fragment of IS1245 produced by PCR, and labeled with [³²P]dCTP with the Random Primed DNA Labeling Kit (Boehringer Mannheim, Mannheim, Germany). The gel images were saved in tif format and were analyzed by using the PFGE program described below.

PFGE and analysis.Whole cellular DNA for the experiments was prepared essentially as described by Zhang et al. (26). Bacterial DNA slices were restricted with the low-frequency-cleavage restriction endonuclease XbaI and in some cases withSpeI (New England Biolabs, Beverly, Mass.). In order to detect large plasmids, whole cellular DNA was electrophoresed without prior digestion with a restriction endonuclease. PFGE of the digested or undigested DNA samples was performed in a contour-clamped homogeneous electric field on the CHEF-DR II system (Bio-Rad Laboratories, Richmond, Calif.) in 0.8% low-endosmosis agarose gels (Pulsed Field Certified Agarose; Bio-Rad Laboratories) at a ramped pulse time from 5 to 30 s for 24 h. DNA size standards consisting of concatemers of bacteriophage lambda DNA (Bio-Rad Laboratories) were included in each gel.

After electrophoresis, the gels were stained with ethidium bromide, destained in distilled water, and visualized on a UV transilluminator (Stratagene), and a tif image of each gel was stored on a diskette. The profiles were scanned and analyzed with the Molecular Analyst Fingerprinting software (Bio-Rad Laboratories). This package enables conversion of the gel image, normalization to a universal standard, analysis of the macrorestriction patterns, and cluster analysis of the PFGE or IS1245 patterns. By using the hierarchic unweighted pair group average method as a clustering algorithm and the Jaccard coefficient of similarity, dendrograms for the isolates from each subtype were created.

hsp65 sequencing.The hsp65 gene encodes a 65-kDa heat shock protein and has been used to identify species in the MAC complex (11, 21-23). Amplification and sequencing of the 441-bp segment of the hsp65 gene were performed for selected strains, as described elsewhere (23).