Stimulating effect of pyruvate on the growth of Mycobacterium leprae in cellfree, semisynthetic, soft agar medium*

Bull. Org. mond. Sante 1973, 48, 571-579 Bull. Wid Hlth Org. Stimulating effect of pyruvate on the growth of Mycobacterium leprae in cell-free, semisynthetic, soft agar medium * TOYOHO MUROHASHI 1 & KONOSUKE YOSHIDA 2 A bacterial suspension preparedfrom a nodule from a patient with lepromatous leprosy was inoculated into the cell-free, semisynthetic, semiliquid agar medium M-Y ld-modified from the authors' medium M-Y Ic by omitting glycerol and adding pyruvate-and incu- bated at 37°C. The growth of the organism was stimulated so markedly that after about 50 weeks' incubation of the primary culture microcolonies could be seen floating in the agar layer as tiny white particles, while the medium fluid assumed a yellow tinge. These macroscopic colonies were sucked up by pipette and subjected to microscopic examination. A bacterial suspension prepared from the first subculture of this strain by centrifugation elicited the same skin reactions in leprosy patients as did the standard Mitsuda's antigen. The organism in the culture was therefore identified as M. leprae. Since 1967, attempts have been carried out by the present authors to cultivate Mycobacterium leprae in cell-free, semisynthetic soft agar (M-Y) media, and it has been repeatedly demonstrated that micro- colonies of various sizes and shapes increased in number, although very slowly, by incubation at 37°C, and that subcultures on new media could be successfully made for several culture generations (Murohashi & Yoshida, 1969, 1971, 1972; Muro- hashi et al., 1971). In most cases, however, the size of microcolonies remained microscopic, and it was hardly possible to make them macroscopic even by incubation for more than one year. Such limited and poor multiplication suggested that the restriction of the organism's metabolic activity, and hence its failure to acquire energy for biosynthesis, might be attributable to the inadequate composition of the basic media. Even though the basic media employed in part of the present experiments contained glycerol * From the Department of Tuberculosis, National Insti- tute of Health, 10-35, Kamiosaki 2-chome, Shinagawa-ku, Tokyo, Japan. This investigation was carried out with financial assistance from the World Health Organization. A report on it was presented at the annual meeting of the Japanese Society for Leprologists in April 1972. A preliminary communication was published in Leprosy Scientific Memo- randa, November 1972 (Memo. L-358). Chief. 'Technical Officer. and/or glucose as main carbon sources, the very poor results obtained suggested that the glycolytic processes of this organism were inefficient. Pyruvate, an important intermediate product from d-glucose by glycolysis that is indispensable for biosynthesis, was accordingly added to the basic M-Y Ic medium to investigate its possible stimulating effect on the multiplication of M. leprae, as suggested by culture experiments using extremely dysgonic bovine tubercle bacilli (Boissvain, 1943; Schaeffer, 1952; Kato, 1971; Kato & Murohashi, 1972). MATERIALS AND METHODS Pathological material A bacterial suspension was prepared as described previously (Murohashi & Yoshida, 1969, 1971, 1972; Murohashi et al., 1971) from a nodule removed aseptically from a patient with lepromatous leprosy at the National Suruga Leprosarium in October 1970. After confirmation had been obtained by cultivation using nutrient broth, nutrient agar, and Sabouraud agar that the bacterial suspension was uncontami- nated, 0.1-ml amounts of the suspension were inoculated into test tubes each containing 10 ml of the soft agar medium M-Y lId (see below), without previous alkali treatment, mixed well, and incubated at 370C. A parallel series of cultures was 3051 - 571 - T. MUROHASHI & K. YOSHIDA prepared as a control using medium M-Y 1g. The strain of M. leprae was named L-Oct-70. Culture media The composition of M-Y lId medium was modi- fied from that of the basic M-Y Ic medium (Muro- hashi & Yoshida, 1969, 1971, 1972; Murohashi et al., 1971) by omitting glycerol, adding pyruvate, and increasing the quantities of phosphates and magne- sium sulfate. The composition of M-Y lId medium was as follows: potassium dihydrogenphosphate disodium hydrogenphosphate (12H20) asparagine sodium citrate magnesium sulfate (7HO) calcium chloride sodium pyruvate glucose yeast RNA bovine serum albumin Fraction V powdered agar distilled water to The pH of the solution was adjusted to I 10% sodium hydroxide. The control medium M-Y Ig was medium M-Y lid except that it contain vate, so that glucose was the main car] Subcultures After incubation at 37°C for 30 and 1-ml amounts of the primary culture wer into fresh medium to make two series subculture. A second subculture was ma first subculture in the same way. Examination of cultures For microscopic examination, 0.1-ml the cultures were smeared on to slides, methanol, stained by acid-fast staining, au as described previously (Murohashi d 1969, 1971, 1972; Murohashi et al., ascertain the viability of organisms to I smears prepared immediately after inoci media were stained by the malachite gr method (Murohashi & Yoshida, 1957) to acid-fast staining. Identification of organisms cultured One culture tube each of the two series subculture was heat-killed, an equal volui was added to dilute the agar, about 1 r solution of nitric acid was added, and the 4g 3 g 3 g 2g 0.1 g 0.0025 g 2g II a heated in a water bath to liquefy the agar. Bacterial cells were collected by centrifugation at 3 000 rev/ min for about 30 min, and the sediment was neutra- lized with sodium hydroxide. These bacterial suspen- sions were sent to Dr S. Ishihara, National Suruga Leprosarium, who carried out skin tests in leprosy patients to compare the skin reactions to the suspen- sions and to Mitsuda's antigen. Electron microscopy One particle of the bacterial mass measuring nearly 200 ,um in size floating in the soft agar in the first subculture was sucked up in a capillary pipette, and subjected to electron microscope exami- nation by Dr Y. Suzuki of the Tokyo University Medical School. RESULTS 100 zg The increase in the number of bacterial masses g according to size is shown in Table 1. The growth 1 g rate in M-Y lid medium, which contained both 1 000 ml glucose and pyruvate, was faster than in M-Y Ig i.6-6.8 with medium, which contained glucose alone. It can be seen that the addition of pyruvate promoted the identical to growth of the organism so much that similar numbers ed no pyru- of bacterial masses in each size group were obtained bon source. about 20 weeks earlier, and the maximum size ofbacterial mass was clearly larger. Bacterial masses at different stages of culture are 1 50 weeks, illustrated in Fig. 1-6. After 30 weeks' incubation einoculated in M-Y lId medium, fairly large, compact, and of the first variously shaped bacterial masses measuring 50 ,um de from the to more than 100 ,m were detected. After 50 weeks the number of masses had increased, and some measured over 200 ,um. At this stage of culture, tiny white particles could be seen by naked eye amounts of floating in the soft agar layer in every culture tube fixed with by lighting against a dark background (Fig. 7). It id examined proved simple to suck some of these particles into k Yoshida, a capillary pipette to make smears, and fairly large, 1971). To compact, and variously shaped bacterial masses be cultured, measuring 200-300 ,um were repeatedly obtained. ilation into In the first subcultures, in which the effect of een-fuchsin Tween 80 as a carbon source in addition to pyruvate in addition was also examined, tiny white particles were detect- able by naked eye floating in the medium after about 20 weeks' incubation. They increased in number gradually as incubation continued, and were confirm- of the first ed as microcolonies by both light and electron me of water microscopy. In the second subcultures too, the nl of a 1% findings were the same after about 20 weeks' tubes were incubation. 572 Fig. 1. Bacterial masses at time of inoculation in M-Y 11 d medium. O0 ymm- . 1 rn Fig. 2. Bacterial masses after 30 weeks' incubation (primary culture). Fig. 3. Bacterial masses after 50 weeks' incubation (primary culture). Fig. 4. A bacterial mass after 50 weeks' incubation (primary culture). 10mPML-- 100 elM Fig. 5. A bacterial mass after 50 weeks' incubation (primary culture). Fig. 6. A bacterial mass after 27 weeks' incubation (first subculture). Fig. 7. M-Y 11 d medium, 73-week-old primary culture. Very tiny, white particles are visible. Fig. 8. Electron micrograph of a bacterial mass taken from first subculture. CULTURE OF M. LEPRAE Table 1. Number and size of bacterial masses per 0.1 ml of culture, according to medium and incubation period Incubation Number of bacterial masses according to size (,&m) Medium period (weeks) 2.5-5.0 5.1-10 10.1-25 >25 Total M-Ylld 0 352a 61 a 48a 12a 437a(8132)b (536)b (83)b (17)b (8 768)b 30 14066 1 062 271 46 15445 50 49 485 3 682 511 70c 53 748 M-Ylg 0 352a 61 a 48a 12a 437fr 50 21 355 1 399 210 34d 22918 a Number of bacterial masses stained green by malachite green-fuchsin staining immediately after inoculation, and so expected to be viable. Viability at 0 week was therefore assumed to be almost 5 %. b Total number of bacterial masses detected by acid-fast staining. c Largest bacterial mass detected measured 285 ,um. d Largest bacterial mass detected measured 108 Mm. The bacterial suspension prepared from the first subcultures of this strain elicited positive skin reactions in patients with tuberculoid leprosy and negative reactions in those with lepromatous leprosy; these reactions were closely similar to those caused by Mitsuda's antigen (Table 2). The acid-fast organ- isms present in the first subcultures were therefore identified immunologically as M. leprae. Examination by electron microscope revealed that the bacterial masses that were visible to the naked eye and sucked up in a pipette were composed of numerous rods arranged in bundles pointing in various directions, and that a single rod was likely to be enveloped by a membranous structure (Fig. 8). It was noteworthy that the culture media, which were colourless immediately after the organism was inoculated, took on a slight yellow tinge after about 20 weeks' incubation, and this yellowness increased very gradually as incubation at 37°C continued. On the other hand, the media in cultures in control tubes, either inoculated with heat-killed organisms and kept at 370C, or inoculated with living organ- isms but kept at 5°C, showed no yellow tinge. This suggested that the colour change was due to the metabolic activity of the inoculated organism, and may serve as an indicator of the success of a culture of this organism. DISCUSSION Pyruvate was shown to have a striking growth- promoting effect on cultures of M. leprae, L-Oct-70 strain, using the cell-free, semisynthetic, soft agar medium M-Y lId. This result was impressive, because a very low viability of some 5% had been predicted from the number of bacterial masses stained green by malachite green-fuchsin staining (Murohashi & Yoshida, 1957) in smears of the bac- terial suspension to be cultured (Table 1), and accordingly poor culture results were anticipated. Indeed, the growth of the organisms in the new medium containing pyruvate as a carbon source was so conspicuous that the size of the bacterial masses became macroscopic after about 50 weeks' incu- bation, and we were able to observe them with the naked eye as white, very tiny particles floating in the agar layer. Most of them were distributed over a fairly wide area some 10-40 mm from the surface of the medium. They were easily sucked into a capil- lary pipette, smeared, and confirmed as microcolonies by light microscopy. The acid-fast microorganisms present in the first subcultures were indentified immunologically as M. leprae by skin tests in leprosy patients. In this experiment pyruvate played a very import- ant role as a trigger, and the organisms seemed to reproduce themselves much more speedily than hitherto through increased biosynthetic activity, possibly resulting from the addition of sufficient pyruvate. The added pyruvate supposedly resulted primarily in an increase in both oxaloacetate and acetyl-CoA, which are indispensable for further biosynthesis. The addition of Tween 80, a derivative of oleic acid, as a carbon source was attempted because of suggestions of its stimulating effect on the growth 577 T. MUROHASHI & K. YOSHIDA Table 2. Reactions of leprosy patients to intradermal skin tests with bacterial suspen- sions from first subcultures of the L-Oct-70 strain (Murohashi) and with Mitsuda's antigen a Mean size (mm) of skin reaction Type after 48 hours b after 15 days c after 21 days cof disease Mitsuda Murohashi Mitsuda Murohashi Mitsuda Murohashi tuberculoid 14 15 17 13 11 (4)d 11 (3)d (6 patients) 27 13 12 13 11 10 23 13 15 8 76 9 20 8 16 10 14 10 26 11 9 7 10 9 13 11 11 9 9 10 lepromatousf 7 6 7 6 3 4 0 (5 patients) 6 5 4 5 3 3 6 6 0 0 4 39 5 5 0 0 3o 0 5 7 3 3 6 6a a Number of bacterial cells per field: Mitsuda, 351; Murohashi, 74. b Read on the basis of erythema. c Read on the basis of induration. d Ulcer was present; figure in parentheses shows its size. 6 Pustule. f Some of these patients might have been classified as - non-tuberculoid -. U Pigmentation. of extremely dysgonic bovine strains of mycobacteria (Schaeffer, 1952; Kato, 1971; Kato & Murohashi, 1972). It was added to medium M-Y lId in a concentration of 0.01% in combination with pyru- vate, and fairly good results have already been obtained in nearly 30-week-old first subcultures, indicating that the size of bacterial masses is likely to be a little larger than without it. The effect of L-glutamate is also under observation using the second subcultures of this strain and other new isolates of M. leprae, in the expectation that it will further promote biosynthesis. The results will be reported later. The culture results described above appear to show that, contrary to belief in the past, M. leprae has the ability to metabolize by itself, at its own speed, in cell-free, semisynthetic media and indepen- dently of host cells, following the same principles as other microorganisms, even though the detailed course of metabolism may not be the same. The results therefore suggest that the organism is likely to have the full system of enzymes necessary for metabolic activity, although not in sufficient quan- tities. Hence it may be concluded that M. leprae is not an exception among microorganisms with regard to metabolism, and that cultivation in cell- free media can be successfully achieved along the lines indicated in the present studies. ACKNOWLEDGEMENTS The authors are indebted to Dr S. Ishihara, National Suruga Leprosarium, Japan, who carried out the identification test using our cultures, and to Dr Y. Suzuki, Tokyo University Medical School, Tokyo, who carried out the electron microscope examination of microcolonies. 578 CULTURE OF M. LEPRAE 579 R, SUMmt ACTION STIMULANTE DU PYRUVATE SUR LA CROISSANCE DE MYCOBACTERIUM LEPRAE DANS UN MILIEU A LA GtLOSE MOLLE, SEMI-SYNTHETIQUE ET DtPOURVU D'ALPMENTS CELLULAIRES Une suspension bacterienne obtenue a partir d'un nodule pr6lev6 sur un malade atteint de lepre l6proma- teuse a 6t6 inocule au milieu M-Y lId (milieu M-Y Ic modifi6 par suppression du glycerol, addition de pyruvate et augmentation de la teneur en phosphates et en sulfate de magn6sium) et incub6e A 37°C. Aprls 50 semaines environ d'incubation, on a constat6 a l'aeil nu la pr6sence dans le milieu de microcolonies for- m&es de minuscules particules blanches. Ces 6lements ont ete retrouves dans les premieres et les deuxiemes sous- cultures. Une suspension bacterienne prepar6e A partir des premieres sous-cultures a provoqu6 chez des malades atteints de 1Mpre tuberculoIde ou l6promateuse des r6actions tr6s semblables a celles suscit6es par 1'antig6ne de Mitsuda, ce qui a permis d'identifier les micro-orga- nismes acido-r6sistants contenus dans les premieres sous- cultures comme 6tant Mycobacterium leprae. L'examen au microscope electronique a montr6 que chaque microcolonie etait form6e d'un grand nombre de batonnets dispos6s en faisceaux irreguliers, chaque bitonnet 6tant probablement recouvert d'une structure membraneuse. REFERENCES Boissvain, C. H. (1943) Proc. Soc. exp. Biol. (N.Y.), 54, 344-345 Kato, M. (1971) Jap. J. med. TechwoL, 20, 650-656 Kato, M. & Murohashi, T. (1972) Kekkaku, 47, 443-448 Murohashi, T. & Yoshida, K. (1957) Acta tuberc. scand., 34, 208-225 Murohashi, T. & Yoshida, K. (1969) Jap. J. Bact., 24, 202-211 Murohashi, T. & Yoshida, K. (1971) Int. J. Leprosy, 39, 308-319 Murohashi, T. et al. (1971) Med. Bio., 83, 29-34 Murohashi, T. & Yoshida, K. (1972) Bull. Wld Hlth Org., 47, 195-210 Schaeffer, W. B. (1952) J. exp. Med., 96, 207-219