Investigations into cultivation of M. leprae under low oxygen tension
To the Editor:
A critical review of the literature strongly suggests that Mycobacterium leprae is a microaerophilic organism. Growth of this mycobacterium in the foot pads of mice (3) may not necessarily be due to lower temperature but most likely because of low oxygen tension. The concentration of oxygen in the host tissues where M. leprae multiplies abundantly has been reported to be 2.5% (2). It was thus postulated that low oxygen tension of the culture medium might facilitate the multiplication of M. leprae. Attempts were thus made to cultivate M. leprae in vitro under low oxygen tension.
M. leprae cells were isolated from nonirradiated livers removed aseptically from armadillos previously infected with M. leprae. Both liquid and solid media were used: The liquid medium contained (NH4),SO40.2 g; KH2 PO4 2.0 g; glycerol 2.5 g; MgSO47 H2O 0.2 g; Na thioglycolate 0.8 g; hemin 0.002 g and water 100 ml. The solid medium, in addition, contained 200 ml of egg yolk.
Several tubes containing 9 ml of liquid medium were inoculated with 1 ml of a bacillary suspension which contained 1 x 109 bacilli. To inoculate the solid medium, 2.0 g of armadillo liver was homogenized in 4.0 ml of 2% NaOH and then neutralized. Several tubes containing solid medium were inoculated with 0.2 ml of bacillary paste. The inoculated tubes were placed in a jar. The jar was closed and excess air was removed from the jar by vacuum. The jar was then flushed with a gas mixture containing 2.5% O2, 5% CO2, and 92.5% N2. Air served as the control. The cultures were incubated at 34ºC. At various time intervals aliquots were taken from the liquid medium for microscopic counts. To assess the in vitro growth on solid medium, the growth from the surface of the solid medium was removed and a homogenous suspension was made in 100 ml of 0.05 M phosphate buffered saline solution. Appropriate aliquots were then used from microscopic counts.
It was observed that no multiplication of acid-alcohol fast bacilli (AAFB) occurred when incubated under air. However, twofold and fourfold increases in AAFB were observed, respectively, when incubated in liquid and solid medium under a gas mixture of 2.5% O2 and 5% CO2, between 16- 24 weeks. The morphology of the bacilli was well maintained. The bacilli did not show any growth on Löwenstein-Jensen or Dubos medium. The number of AAFB started to decline rapidly after about 26 weeks of incubation under a gas mixture, and no bacilli could be detected after about 36 weeks of incubation.
Further studies are in progress under different gas mixtures. During these studies, in addition to microscopic counts, determinations of ATP and DNA will be done and identification of the cultivated AAFB will be carried out.
- M. Ishaque, Ph.D.
Professor
Department of Applied Microbiology
Institut, Armand-Frarppier
Universitty of Quebec
C.P. 100 Laval, Quebec
Canada H7N 4Z3.
REFERENCES
1. Mori, T., Miyta, Y., Yoneda, K., and Ito, T. Collection method for Micobacterium leprae from infeeted armadillo liver. Int. J. Lepr. 52 (1984) 41-43.
2. Seever, M. H. O2. and CO2, tensions in the subcutaneous tissues of normal subjects. Am. J. Physiol.115 (1936) 38-42.
3. Shepard, C. C. The experimental discase that follows the injection of human leprosy bacilli into foot pads of mice. J. Exp. Med. 112 (1960) 445-454.
4. Stevens, K. M. Cultivation requirements for Trepnoema pallidium, Mycobacteriun leprae and other microbial and mammalian microaerophilic cells.Med. Hypotheses 5 (1979) 1091-1104.