J. Med. Microbiol. - Vol. 21 (1986), 251-255 0 1986 The Pathological Society of Great Britain and Ireland Phenotypic changes in mycobacteria grown in oxygen-limited conditions JANE GILLESPIE, L. L. BARTON* and E. W. RYPKAt Department of Biology, University of New Mexico, Albuquerque, NM 87737 and tSection of Microbiology, 1o velace Medical Center, Albuquerque, NM 87 7 08 USA Summary. Laboratory strains of Mycobacterium phlei, M. smegmatis, M.fortuitum, M.gordonae, M.kansasi, M.bovis, M. tuberculosis and M. intracellulare were adapted to grow in an anaerobic environment. Concomitant with the transition to anaerobic growth was loss of acid-fastness, loss or modification of colonial pigmentation, and loss of ability to grow on a malachite green-containing medium. The mycobacteria grown anaerobically produced acid from a greater range of carbohydrates than aerobically grown cultures, lost iron-uptake activity, and showed a reduction of urease, catalase and nitratase activity. Back adaption of mycobacteria from an anaerobic to an aerobic environment resulted in the acquisition of acid-fastness, pigmentation, and other characteristics used in the taxonomy of mycobacteria. These results suggest that mycobacterial cultures, if grown in an anaerobic environment, may be erroneously identified in clinical laboratories. Introduction ATCC 354, M. smegmatis ATCC 14468, M. ,fortuitum TMC 1529, M. gordonae TMC 1318, M. kansasi TMC Mycobacteria are considered to be obligate aer- 1201, M. intracellulare TMC 1403, M. tuberculosis obes that can grow in oxygen tensions that range H37Ra TMC 201, M.bovis BCG Pasteur TMC 101 1 and from atmospheric to microaerophilic (Jenkins, et M.phlei HMS, a smooth strain supplied by Dr J. Hanks, al., 1982; Moore and James, 1982). Research and Harvard Medical School. TMC strains were from the clinical laboratories routinely observe that myco- Trudeau Mycobacterial Culture Collection, obtained bacteria in broth cultures will clump, sink to the from the National Jewish Hospital and Research Center, bottom of the tube and continue to grow in the Denver, CO, USA. pellet where oxygen levels would be low because of oxygen consumption by the cytochrome system and Media lactate oxidase (Ratledge, 1976). The report of ' survival of Mycobacterium tuberculosis in anaerobic M. phlei and M. smegmatis were maintained on Trypticase Agar (Baltimore Biological Laboratories, Bal- conditions (Wayne and Lin, 1982) and the demonst- timore, MD) slants containing glycerol 0.2%. All othcr ration of ferredoxin in M. smegmatis (Imai et af., strains were maintained on Lowenstein Jensen slants 1983) gave impetus to our investigation of anaero- containing malachite green (Difco) 0.025%. For routine bic growth of mycobacteria. maintenance in anaerobic conditions, a glycerol-trypti- Laboratory strains of mycobacteria were adapted case medium consisting of glycerol 0-2%, Tween 80 0.1';& to grow anaerobically and their physiological ac- trypticase (BBL) 0.8% and sodium thioglycollate 0.05(>/, tivities compared with those of strains grown was used. A I-ml inoculum from an actively growing aerobically. We describe procedures for the adap- culture was added to 10 mi of freshly steamed medium in a tion of mycobacteria to different oxygen tensions 16 mm x 125 mm tube. Inoculated tubes were placed in and record the unique phenotypic characteristics of anaerobic jars containing Gas Pak Hz + COz generators MD). mycobacteria grown anaerobically. (Becton Dickinson, Cockeysville, Adaptation of cultures Materials and methods The nine strains of mycobacteria were adapted to anaerobic growth by inoculation into Thioglycollate Bnc teria Broth without dextrose (Difco) supplemented with gly- Nine strains of mycobacteria were used: M. phlei cerol 0.2%and Tween 80 0.1 %. After 9-1 2 serial transfers from the bottom of the micro-aerophilic growth zone, * Requests for reprints should be addressed to L. L. Barton. dense growth occurred throughout the bottom half of the Received 7 Aug. 1984; revised version accepted 30 Jun. 1985. tube. Transfers were made at approximately 4-week 25 I 252 JANE GILLESPIE, L. L. BARTON AND E. W. RYPKA intervals. Following the eighth transfer, the tubes were extract 0-4%, and glycerol 0.2%. When growth was incubated in anaerobic jars with a Hz +COz atmosphere visible, ferric ammonium citrate 20% was overlaid on the to eliminate the possibility that cultures could be growing slant. The appearance of rusty brown colonies after in micro-aerophilic regions of the medium and cells incubation for a further 21 days indicated a positive result sinking into the anaerobic zone. (Wayne and Doubek, 1968). To adapt the anaerobically growing mycobacteria to Niacin test. Niacin production was detected in strains aerobic conditions, the cultures were inoculated into grown for 6 weeks on Dubos' agar enriched with thioglycollate broth containing glycerol and Tween 80 asparagine 0.5% by the spot-plate test (Runyon, et al., and incubated in atmospheric conditions. After 4 weeks a 1959). subculture was made of growth from the uppermost level Nitrate reduction. The colorimetric tube test for nitrite of the tube. After 8-10 serial transfers the organisms were production (Vestal, 1973) was used to test strains grown transferred to Lowenstein Jensen slants and their physio- on nutrient agar 2.3%, yeast extract 0-4%, and glycerol logical characteristics determined. 0.2%. Rapidly growing strains were incubated for 1 week and slowly growing strains for 2 weeks before cells were tested for nitrate reduction. Cultural Characters Tween hydrolysis. Hydrolysis of Tween 80 was detected Cojonial morphology and pigmentation. Colonial according to the procedure of Wayne, et al. (1964). characteristics were recorded after growth for 3 weeks on Urease. The procedure employed for hydrolysis of urea a medium consisting of Nutrient Agar (Difco) 2.3%, was a modification (Runyon et al., 1980) of the method of Yeast Extract (Difco) 0.4% and glycerol 0.2%. Toda et al. (1 960). Scotochromogenicity and photochromogenicity were Acid production from carbohydrates. Cystine Tryptic determined according to the procedures recommended by Broth (Difco) was used as the nutrient base for the Runyon et al. (1980). After inoculation the strains of evaluation ofcarbohydrate metabolism. After addition of mycobacteria were incubated in ambient conditions or in carbohydrate 0.5% and 0.1 ml of the OD 0.4 suspension, anaerobic jars containing H2 + C02 generators. tubes were incubated in aerobic or anaerobic conditions Growth characteristics. Strains were divided into for 10 days. Acid production in aerobic cultures was rapidly or slowly growing groups on the basis of visible evaluated in the aerobic zone of the medium. growth after incubation for 3 days in thioglycollate broth containing glycerol and Tween 80. Sensitivity to mala- Results chite green 0.025% was revealed by failure of growth on Lowenstein Jensen slants after incubation for 28 days. The adaptation of mycobacteria to anaerobic Growth of rapidly growing mycobacteria in the presence growth produced cultures with characteristics dis- of NaCl 5% was determined after incubation for 10 days tinct from their aerobic counterparts. Colonies in Kirchner's broth containing NaCl5% (Hedgecock and formed in anaerobic conditions were smooth, moist Costello, 1962). Growth at 25"C, 33"C, 45°C and 54°C and colourless. Most significant was the loss of acid- was determined with the glycerol-trypticase medium fastness by cells grown anaerobically and stained by incubated for 10 days for rapidly growing and 6 weeks for the Ziehl-Neelsen method. They would not grow in slowly growing strains. Each of these characteristics was the presence of malachite green. Anaerobic cultures determined for the nine strains cultivated in aerobic and did not display unique cell morphologies and were anaerobic conditions. characteristically composed of gram-positive rods. In broth culture, mycobacteria grew anaerobically Biochemical tests without cell aggregation. Transition from aerobic to anaerobic or anaerobic to aerobic metablism was Preparation of cell suspensions. Aerobic and anaerobic cultures of mycobacteria were grown in a medium through an obligate microaerophilic state. Con- consisting of glycerol 0.2%,Nutrient Broth (Difco) 0.8%, siderable sensitivity to oxygen was displayed by the Yeast Extract (Difco) 0.4%, and Tween 80 0.1%. Cells mycobacteria gown anaerobically and inoculation were collected by centrirugation, washed twice with 0.02 of aerobic media resulted in cell death. With M phosphate buffer, pH 7.0, containing NaCl 0-8%, and adaptation of anaerobic cultures to aerobic growth, resuspended in phosphate buffered saline to an optical the colonies became rough and regained pigmen- density of 0.4 at 540 nm. For each test an inoculum of 0-1 tation and cells displayed acid-fastness and the ml was used. ability to grow in the presence of malachite green. Catalase. The semiquantitative catalase test described The temperature range in which growth occurred by Runyon et al. (1980) was used on strains grown for 2 was the same for anaerobic and aerobic cultures of weeks on Dubos' agar enriched with asparagine (Difco) 0.5%. The spot test for catalase was performed by adding all the strains. Those strains that grew in the presence of NaCl 5y4 in aerobic conditions also H202 to a colony from Dubos' agar (Finegold, et al., 1978). grew anaerobically with the exception of M..fortui- Irdn uptake. Rapidly growing mycobacteria were tum. Strains that failed to grow in the presence of inoculated on to slopes of nutrient agar 2.3%, yeast NaCl 5% in aerobic conditions also failed to grow ANAEROBIC GROWTH OF MYCOBACTERIA 253 Table I. Physiological characteristics of rapidly growing mycobacteria 1 M.phlei 354 M.phlei HMS M. smegmatis M.fortuitum Character aerobic anaerobic aerobic anaerobic aerobic anaerobic aerobic anaerobic Pigmentation* S N S N N N N N Growth on Lowenstein Jensen plus malachite green Catalase production Nitrate reduction Urease production Tween hydrolysis Iron uptake * S = Scotochromogen, P = photochromogen, N = no pigment. + =Positive result in test; - =negative result in test. Table 11. Physiological characteristics of slowly growing mycobacteria M.