55, 2^ Correspondence^ 361

II. SHEPARD, C. C. and McRAE, P. H. Hereditarycharacteristic that varies among isolates of My-cobacterium leprae. Infect. Immun. 3 (1971) 121126.

12. VAISIINAVI, C., KAUR, S., KUMAR, 13., GANGULY,N. K. and CHAKRAVARTI, R. N. Trophic changesand extensive dissemination in normal mice in-fected with human Mycobacterium leprae. IndianJ. Lepr. 56 (1984) 742-747.

13. VAISHNAVI, C., KUMAR, B., GANGULY, N. K. andKAUR, S. Phagocytic and bactericidal activities ofmacrophages from Mycobacterium leprae infectednormal and immunosuppressed mice. Int. J. Lepr.53 (1985) 233-237.

14. WEMAMBU, S. N. C., TURK, J. L., WATERS, M. F.R. and REES, R. J. W. Erythema nodosum lepro-sum: a clinical manifestation of the Arthus phe-nomenon. Lancet 2 (1969) 933-935.

Mycobacterium leprae, Probably aMicrobe-dependent Microorganism

To THE EDITOR:Experimental evidence supports the re-

cently advanced concept that Mycobacte-rium leprae is probably a unique microbe-dependent microbe ( 2 4 ' 6 ). Results led to theformulation of a new culture medium, per-mitting in vitro multiplication of bacilli de-rived from armadillos infected with M. lep-rae (3 4 ). The proposed multifactorial culturemedium (MFM) was based on the recentlyacquired knowledge that getting M. lepraeto grow is a multifactorial problem (I 3 ).The slow growth of the bacilli in a deepliquid medium, however, was difficult toassess and not satisfactory for practical pur-poses as a tool for diagnosis, drug sensitiv-ity, metabolic studies, and as a pharmaco-logical model for detecting new antileprosydrugs. Experiments were aimed at achievingvisible growth on solid media, acceleratinggrowth rate, increasing yield, and providingfurther evidence for the validity of the claimthat getting M. leprae to grow is dependenton a supply of growth factors (mycobactinand exochelin) from secondary mycobac-teria. Results just available now bring fur-ther evidence that growth of those bacillifrom armadillos infected with M. leprae isdependent on the above growth factors, butthat a still-unidentified third growth factormight contribute to considerable yield ofcell mass, and finally that a visible growthcan be achieved on the surface of agar slantsof the multifactorial media.

An iron-poor Sauton-Tween liquid me-dium contained asparagine 4 g, citric acid

2 g, K,HPO, 0.5 g, ZnSO, 0.04 g, MgS0,0.4 g, glycerol 40 ml, and Tween 80 10 mlin 1 liter of distilled water. It was adjustedto pH 7.0 with NH 2OH. The solution wasdistributed 200 ml/flask and autoclaved for30 min. The media were inoculated with M.phlei and incubated at 37C until a thick,heavy growth developed on the surface ofthe medium. The flasks were sonicated in aBronsonic sonicator (20-90 kilocycles fre-quency) for 10 min. The cultures were au-toclaved for 30 min, and filtered while hotthrough Whatman 802 filter paper. The fil-trate was adjusted to pH 5.8 with KH,PO 4 .Na thioglycolate 1.5 g, thioctic acid 0.05 g,and 0-cyclodextrin 1.5 g were collected in amortar, mixed, and ground for 5 min. Thefine powder was dissolved in the mortar withsmall amounts from the filtrate and addedto the collected filtrate. Ferric ammoniumcitrate 0.05 g and MgS0, 0.1 g were alsodissolved in the filtrate. The solution wasreadjusted to pH 5.8 and distilled wateradded to the volume of 1 liter. Readjust-ment to pH 5.8 was required; 500 ml of theliquid MFM was autoclaved 30 min. In 500ml of the medium 15 g agar (Difco) wasdissolved and 20 ml was distributed intoeach 50-m1 screw-cap tube. Autoclaved for30 min, the media were cooled to make agarslants.

Armadillo-grown M. leprae cells werecollected, prepared, and semi-purified asep-tically, using the liquid MFM. The cell sus-pension was centrifuged at 3000 rpm for 15min. Two 5-mm diameter loopsful of the

362^ International Journal of Leprosy^ 1987

thick sediment was evenly spread on thecenter of the agar slant in an area of about5 cm'. The heavy inoculum was needed toobtain growth in the primary culture.Growth became visible in 2 to 3 weeks ofincubation at 34C. The inoculum increasedin thickness during the following 3 monthsas a light beige, creamy, semiglossy mass.Subsequent subcultures were obtained onthe same medium, but a heavy transfer wasneeded to obtain comparable growth. Thecells were strongly acid-fast, arranged inclumps resembling globi in smears of lep-romatous leprosy lesions. Pyridine extrac-tion abolished acid-fastness but not gram-positivity of the cells. The cells did not growon LOwenstein, in Dubos, or 7H10 liquidmedia. No growth was obtained either onthe MFM agar slants if media were preparedwithout sonication of the Al. phlei culturein the iron-poor Sauton-Tween media.

It is known that both mycobactins andexochelins are secreted by mycobacteria intoSauton-Twcen 80 liquid media ( 5). Undermicroaerophilic conditions, these two fac-tors of iron acquisition and transport weresufficient to support multiplication of bacilliderived from armadillos infected with Al.leprae ( 3 ' 4). However, prolonged sonicationof the growth factor donor Al. phlei culturewas probably necessary to release either in-creased amounts of mycobactin and/or exo-chelin, or eventually other growth factors tosupport growth under strictly aerobic con-ditions on the solid multifactorial medium,as described in the present communication.

Results are not sufficient to claim the suc-cessful cultivation of AI. leprae, neither arethe cultures sufficiently identified as au-thentic M. leprae. It is therefore proposedto designate the obtained strains as Al. lep-rae(?). Results however do strengthen theconcept that M. leprae might be a microbe-

dependent microbe and that donor cells, ifsonicated, supply growth factors probablyother than mycobactin and exochelin. Thereare indications that these bacilli are not mi-croaerophilic as often suggested for AI. lep-roe. Further cultivation trials are warrantedand proposed on the above-described mul-tifactorial medium agar slants.

Laszlo Kato, M.D.Department of ResearchThe Salvation Army

Catherine Booth Hospital4375 Avenue MontclairMontreal, Canada 114B 2J5

Acknowledgments. This study was supported by

grants from Deutsches Aussatzigen Hilfswcrk, Institut

Fame l'ero, and Secours aux Lepreux, Canada.

REFERENCESI. HALL, R. M., WHEELER, P. R. and RATLEDGE, C.

Exochelin-mediated iron uptake into Mycobacte-rium leprae. Int. J. Lepr. 51 (1983) 490-494.

2. KATO, L. A culture medium for cultivation of my-

cobacteria, probably ;Vivo/meter/um leprae, from

Mycobacterium leprae infected tissues. Indian J.Lepr. 57 (1985) 58-70.

3. KATO, L. A multifactorial culture medium with

growth factors from leprosy-derived mycobacteriaproposed in cultivation trials for Mycobacterium

leprae. Int. J. Lepr. 54 (1986) 310-311.

4. KATE), L. Investigations into the cultivation of My-

cobacteriztoi leprae; a multifactorial approach. Proc.

Symposium on Multidrug Therapy in Leprosy, 24-26 April 1986, Wiirzburg, West Germany. Lepr.

Rev. 57 Suppl. 3 (1986) 209-219.

5. ....rviAcilAst, L. P., RATLEDGE, C. and NOCTON, J. C.

Extracellular iron acquisition by mycobacteria; roleof the exochelins and evidence against the partici-

pation of mycobactin. Infect. Immun. 12 (1975)1242-1251.

6. TWORT, F. W. A method for isolating and growing

the lepra bacillus of man (preliminary note). Proc.R. Soc. London [B] 83 (1910) 156-158.

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