INTERNATIONAL J OURNAL OF LEPROSY Volume 45 . Number 2 Primed in (he U.S.A. A Scotochromogenic Slow-Growing Mycobacterium Probably the Etiologic Agent of Rat Leprosyl

Laszlo Kato and Muhammed Ishaque 2

Pioneering in the metabolism of nonculti­ a third group of molecules possessing SH vable mycobacteria, Gray ( 5), Hanks (6), groups as a common denominator (13). Oleic and Hanks and Gray ( 8) searched systemati­ acid, lanolin (oleum eucerini) and Tween 80 cally for s u bst ra tes as prospective e ne rgy were similarly oxidized by these cells. Some sources for M. leprae (MI) and M. lepraemu­ of these substrates are sources of energy rium (Mlm). After several years of investiga­ si nce substrate oxidation was coupled with tions, they concluded that MI and Mlm oxidative phosphorylation ( I). We concluded cannot derive energy from substrates which that Mlm has all the prerequisites for inde­ cultivable mycobacteria use as energy pendent growth outside the host and we ven­ sources for growth, multiplication and ex­ tured to predict that these "noncultivable pression of virulence. It was advocated, and mycobacteria" might be later recognized in quite generally accepted, that both of these vitro as fast-growing microorganisms ( 16). mycobacteria are metabolically deficient and We are now able to report that, in the pres­ depend on the host to supply most of the nec­ ence of yeast extract, succinate (N a) or cys­ essary molecules to build up their cellular teine, respectively as energy sources and constituents. This view was supported by the glycerol as the source of carbon, relatively findings by many investigators that MI and fa st-growing scotochromogenic acid-fast mi­ Mlm are deficient in enzymes which play an croorganisms resembling M. scrofulaceum important if not key role in energy metabo­ are regularly cultured from murine lepromas. li sm and respiration. Most of the studies When injected into albino rats, the cultures were made with Mlm because these cells are retain infectivity, though of a lowered patho­ readily available in large quantities from in­ genicity, but still provoking a limited disease fected rats. It was reported that a key en­ typical of murine leprosy. zyme of the tricarboxylic acid cycle, a-keto­ glutaric dehydrogenase, is absent in Mlm, MATERIALS AND METHODS that they also have low NADH oxidase Mycobacterium lepraemurium. The Ha­ activity and that cytochrome c is absent in waiian strain of Mlm has been passaged at these cells ( IX. 19 ). three to six month intervals for 25 years in In contrast to these findings, we advocated Sprague-Dawley female rats. A three month the antipode of this often accepted concept. old rat leproma was removed aseptically and We have shown that at least Mlm is meta­ cut into small pieces with scissors. The bolically competent, having a complete func­ minced tissue was washed with distilled wa­ tional tricarboxylic acid cycle and a well­ ter, saline solution and a pH 6.8, M j 15 phos­ functioning uninterrupted electron transport phate buffer solution to remove excess blood. chain; cytochrome c included (10. 16 ). With The tissue was homogenized in a Vortex improved methods, we have shown the pres­ homogenizer at 12,000 rpm, three times for ence of a-ketoglutarate dehydrogenase, twelve seconds. The homogenate was filtered NADH oxidase and cytochrome c in Mlm. through a sterile nylon stocking. The filtrate Our investigations clearly showed that host was centrifuged at 2,500 rpm for five min­ grown Mlm can oxidize at least four groups utes. The supernatant was centrifuged for ten of substrates. One is complex, heat stable: minutes at 6,000 rpm. The latter supernatant yeast extract. The other is succininc acid, and was then discarded and the sediment was resuspended in the PO 4 buffer solution and I Received for publicatio n 12 November 1976. the suspension was adjusted with the same 2L. Kato, M.D., Professor and Head; M. Ishaque, buffer to approximately 300 Klett units. Ph. D., Associate Professor, Hansen Chair of Research, Chemicals. Yeast extract "Difco" was Bacteri ology Research Center, Inst itut Armand-Frap­ pier, University of Quebec, Ville de Laval, P. Que., used . All chemicals, except the common Canada. salts, were purchased from Sigma Chemical

139 140 International Journal of Leprosy 1977

Company, N.J. Lanolin (oleum eucerini) was LOW ENS TEIN MEDI UM was prepared from kindly donated by Smith a nd Nephew Co. Lowenstein Medium Base (Difco). of Canada. Dextran was obtained through Media were inoculated with 0.5 ml of the the courtesy of Abbott Laboratories of standard bacillary suspension containing Canada. Mlm isolated from rat lepromas and 200 Phosphate buffer solution. High potassi­ units/ ml penicillin G. sodium were added to um, low sodium buffer was used to simulate the inoculated media. the intracellular environment. The 0.066 M Growth conditions. All cultures were in­ PO 4 buffer contained: K H 2 PO 4' 8.2 gm and cubated at 34° C. Tubes were shaken vigor­ Na 2 HP0 4 , 0.5 gm in one liter of distilled ously immediately after inoculation and once water. This solution at a pH of 5.5 was se­ daily. The inoculated liquid media were lected because previous experiments showed ~Iaced in a maximal possible inclined posi­ optimal endogenous res piration, substrate tIOn so as to assure the greatest possi ble con­ oxidation and growth at this acidic pH value. tact of the liquid surface with air. Preparation of the culture medium. The The hot Ziehl-Neelsen technic was used to following substrates were added respectively study morphology and acid-fastness. Coun­ to the culture media as expected sources of terstaining was done with methylene blue at energy. A constant concentration of 2 gm per pH 10 as by Skinsnes et al ( 22 ). liter was used: yeast extract powder "Difco"; Effect of polysaccharides and their constit­ sodium succinate; L-cysteine; penicillamine; uents on in vitro growth. A series of liquid Tween 80; oleic acid; lanolin (oleum euceri­ media were prepared containing connective ni). tissue mucopolysaccharides: heparin, hyal­ The oleic acid solution contained oleic acid uronic acid and chondroitin sulfate, respec­ (Merck), 48 ml and NaOH N / 20, 40 ml , ho­ tively. A highly branched bacterial muco­ mogenized in a mortar, adjusted to pH 5.6. polysaccharide, dextran, having an average Glycerol, 30 ml per liter was the only source molecular weight of 75;000, was also utilized. of carbon in all media. L-asparagin, 2 gm per Two polysaccharides of vegetable origin liter was the nitrogen source in some of the purified agar and galactomannan, were als~ media. inc.luded as well. as ovomucoid from egg Solid media contained 3% purified agar­ Whlt~. The folloWIng constituents of polysac­ agar (Difco). After sterilization, media were chandes were tested: D-glucuronic acid, D­ solidified in an inclined position at room tem­ galactosamine, N-acetyl-D-glucosamine and perature. Semisolid media contained 1.5% D-mannose. Each of these substances were agar-agar. added, respectively, to the yeast extract­ The media were distributed into test tubes glycerol medium in a constant concentration or flasks as desired. Nine milliliter amounts of 100 mg ~er 100 ml. Each 50 ml screw cap were distributed into 50 ml screw cap tubes tube contained 10 ml of the media. These or 100 ml into 400 ml flat culture-flasks. were sterilized in an autoclave for 25 min­ All media were sterilized in an autoclave utes. Media were kept at +4° C and were used for 30 minutes and those which contained within four weeks. hyaluronic acid for 20 minutes. After sterili­ Infectivity of the cultures. Female Sprague­ zation the final pH was 5.5 ± 0.2. The addi­ Dawley rats weighing 50 to 60 grams were tion of sera or serum albumin had an insignif­ infected with the bacilli from the third sub­ icant effect on the pH. culture. During the second week of the The sterlized media were cooled at room logarithmic growth phase, the culture was temperature and then 5% sterile serum albu­ vigorously shaken and 0.3 ml of the culture min fraction V or different animal sera were was ino~ula~ed subcutaneously into the scap­ added under aseptic conditions. Sera were ular regIOn In each rat. Animals were kept on sterilized by filtration. a standard Purina Chow diet and water ad D UBaS LIQUID MEDI UM was prepared from libitum at a constant temperature of 22° C Dubos Broth Base (Difco) with Tween 80 and with constant humidity. bovine serum albumin fraction V added. DUBas SOLID MEDIUM was prepared from RESULTS Dubos Oleic Agar Base (Difco) and con­ Growth in the presence of oxidizable sub­ tained serum albumin fraction V. strates. Seven substrates were oxidized by 45, 2 Kato & Ishaque: A Scotochromogenic Mycobacterium; Etiologic Agent 141 of Rat Leprosy

TABLE I. In vitro multiplication of acid-fast bacilli in a liquid medium containing different sources of energy, nitrogen and carbon in the presence or absence of sheep serum. Media were inoculated at 0 time with M . lepraemurium isolated from rat lepromas. Increasing turbidity was registered as +, ++, +++; heavy growth as ++++ and abundant growth as :tH:t. ------Source of carbon glycerol 31 gm/ I GI GI GI GI Source of nitrogen L-asparagin 2 gm/ I Asp Asp Asp Asp Sheep serum 10% v/ v Se Se Se Se Growth in six weeks with oxidizable substances as energy sources Yeast extract ++ ++ ++++ ++++ ++++ 2 gm/ I ± ± ± ++++ ++++ ++++ Succinate Na 2 gm/ I ± + + + ++ ++++ ++++ ++++ L-cysteine 2 gm/ I ± ± ± ± +++ ++++ ++ +++ Penicillamine 2 gm/ I ± ± ± ± ++ +++ + +++ Tween 80 2 gm/ I ± ± ± ± ± ± ± ± Oleic acid 2 gm/ I ± ± ± ± ± ± ± ± Lanolin 2 gm/ I ± + + + ++ ++++ ++++ ++++ PO 4 buffer control ± ± ± ± ± ± ± ± Lowenstein-Jensen medium No growth during 60 days Dubos liquid and solid media No growth during 60 days

Mlm isolated from rats. These were incorpo­ sheep serum and mainly when glycerol was rated respectively in a series of liquid culture added as a source of carbon. Table I also media as prospective sources of energy. shows that succinate, cysteine, penicillamine Some of the media were prepared with added and yeast extract provided some source of glycerol as carbon source and / or with added carbon and sheep serum, some source of L-asparagin as source of nitrogen. Each se­ nitrogen. It also became evident' that aspara­ ries of cultivation trials was performed with gin as an additional source of nitrogen did or without sheep serum added. Results are not improve growth in the media. No growth summarized in Table I. The turbidity which occurred during 60 days of incubation when developed in six weeks after incubation was Mlm isolated from the rat were inoculated recorded. These are estimated designations directly into Dubos medium, liquid or solid, of growth in order to visualize in vitro multi­ or on Lowenstein-Jensen media. plication of the acid-fast bacilli. I n each case, Table I summarizes the results after six Ziehl-Neelsen staining and phase contrast weeks of incubation; however, measur­ examination confirmed that turbidity was able growth already occurred within two due to bacillary multiplication and not to weeks in all the media which later showed precipitation. A sign ± designates a turbidity heavy turbidity. These will be presented In equal to the heat killed control. Increasing separate tables and in Figure I. turbidity was registered as +, ++, +++, heavy From the results we concluded that: growth as ++++ and extremely heavy growth asHH. Table I shows that no multiplication I. The medium of choice contained in one occurred in the presence of any of the oxi­ liter of distilled water: KH 2 P0 4 , 8.2 gm; dizable substrates with or without glycerol Na 2 HP0 4 , 0.5 gm; yeast extract "Difco," and/ or asparagin added. Tween 80, oleic 2 gm; glycerol, 30 gm; and \0% sheep serum acid and lanolin did not support growth when added aseptically. (Medium K I-I) sheep serum was added to the same varia­ 2. The medium of second choice contained tions of culture media. With succinate, L­ the same substances with Na succinate cysteine, penicillamine and yeast extract, added instead of yeast extract. (Medium KI- growth occurred only in the presence of 2) 142 International Journal 0/ Leprosy 1977

500 close to five weeks. This was followed by a seven day long positive growth acceleration phase (B). An abundant growth was ob­ 200 served at the end of the relatively short loga­

Ul rithmic growth phase (C). This was followed I-- 2 100 by a negative growth acceleration (D) and a ::J I-- maximum stationary phase (E). Accelerated I-- W death phase (F) and logarithmic death phase ;;! 50 (G) were of a relatively slow rate. The pre­ sented logarithmic growth curve is represent­ A ative of one of the several cultures. Varia­ tions of ± 20 days in the initial phase were measured with different inocula. 10 OR--'--3-2--'---;4!;--'---;:6--'---;8~-'--""'IO--'-1 2L--'-----'14 Bacilli freshly isolated from the rats were TIME IN WEEK S also inoculated into Dubos and Lowenstein FIG . I. Kinetics of in vitro growth of scotochro­ mogenic culture. The primary culture was inocu­ media. No growth occurred during two lated at 0 time with M. lepraemurium isolated months observation period. from rat leprotic nodules. Subcultures were trans­ During the third week of the logarithmic ferred into the homologue media from the primary growth phase, 0.2 ml of the culture was culture. A progressive shortening of the latency transferred into 10 ml homologue liquid me­ growth period in the subcultures indicates slow dium. Figure I also shows the obtained adaptation to extracellular substrates. logarithmic growth curves in the third, fifth and eighth subcultures. A short lasting laten­ cy period and initial growth acceleration 3. The medium of third choice contained phase was followed by a relatively sharp the same substances but had L-cysteine logarithmic growth phase. The growth curve added instead of yeast extract. (Medium KI- followed the known pattern of slow-growing 3) mycobacteria. Cultures are now regularly In the above three media, strong acid-fast­ transfe~red during the logarithmic growth ness was registered after three weeks of incu­ phase Into the homologue media. Subcul­ bation in the primary culture. tures are obtained regularly and with ease. With 3% agar-agar added to the KI-l, 2 Due to the adaptation to the extracellular ex­ and 3 media, visible but weak growth oc­ istence and to the artificial culture medium curred at the end of the sixth week. Bacilli period is shortened and the were strongly acid-fast and the surface t~e la~ency loga~ nthmlC growth rate is somewhat faster in the growth on the agar media showed the pres­ subcultures. ence of a scotochromogenic yellowish pig­ With 3% agar added to the KI-l medium ment. semisolid agar slants were prepared with From the third subculture and at the end 10% sheep serum added. Poor but visible of the sixth week, Lowenstein-Jensen and growth occurred after five to eight weeks in­ Dubos liquid and solid media were inoculated cubation. Growth was scotochromogenic and with bacilli grown on KI-l liquid and solid bacilli were strongly acid-fast. media. Visible scotochromogenic growth oc­ All cultures were scotochromogenic curred at 34° C after three weeks of incuba­ whether in the bottom of the liquid media or tion. on the surface of the solid media. Most of the Cultures obtained on KI-l as well as Lo­ bac.illi retai~ acid-fastness during the latency wenstein-Jensen and Dubos media are now penod. Dunng the growth acceleration phase regularly transferred and maintained on the and the first week of the logarithmic phase, homologue media. however, most of the bacilli were nonacid­ Growth in the yeast extract, glycerol, fast and relatively short. Full acid-fastness sheep serum media (KI-l) was measured in w~ s regained only at the end of the logarith­ a Klett spectrophotometer and the results for mIC growth phase. the primary culture are presented in Figure I. Changes in optical density were plotted Growth on media with polysaccharides. against time in days. The initial phase (A) In the second subculture, a heavy growth was was of a relatively long latency period lasting visible after three weeks incubation. The cul- 45, 2 KaT o & /shaque: A ScoTochromogenic MycobacTerium; ETiologic Agent 143 of RaT Leprosy

TABLE 2. Growth ofscoTochromogenic bacilli in media containing yeast extract (YE), glycerol (GI) with serum albumin (SA) and different polysaccharides or their constituenTs added respectively. Horse serum (HS). Media were inoculated from the second subculture of The scotochromogenic strain isolated from raT lepromas. EsTimation of growth as in Table f . Medium Growth in four weeks YE- GI ++++ YE- GI- HS ++++ YE- GI- Heparin ± YE- GI- Hya luroni c acid ± YE- GI- SA + YE- GI- SA- C hondroitin- S04 ++++ YE- GI- SA- Ovomucoid ++++ YE- GI- SA- D-glucuronic acid ++++ YE- GI- SA- D-galactosamine ++++ YE- GI- SA- D-glucosamine ++++ Y E- G I- SA- N -ac-D-gI ucosamine ++++ YE- GI- SA- D-mannose ++++ YE- GI- SA- Agar +++ YE- GI- SA- Dextran ++++ YE- GI- SA- Galactomannan ++++ Heat-killed M. lepraemurium in YE- GI ± ture was centrifuged, washed twice in phos­ constant 0.2% solution of yeast extract (Dif­ phate buffer and resuspended in yeast exact­ co) was prepared in the pH 5.6 phosphate glycerol medium. The suspension was buffer solution. A series of media were pre­ adjusted to 300 Klett units. From these pared containing Na succinate, I gm per suspensions, 0.2 ml was inoculated into each liter; penicillamine, I gm per liter; and glyc­ of the media containing the mucopolysac­ erol, 30 gm per liter, respectively. Media charides, their constituents and appropriate were autocIaved for 30 minutes in 15 ml controls. Tubes were incubated in a maxi­ scre~ cap tubes containing \0 ml of each mally inclined position at 34° C. They were medium. Tubes were inoculated from the shaken once daily. Results are shown sche­ third subculture, two weeks old, 0.2 ml into matically in Table 2. In four weeks, heavy each tube from a suspension adjusted to 300 growth occurred in the yeast extract-glycerol Klett units. media with or without horse serum added. Table 3 shows a sc hematic representation The growth was strongly inhibited by serum of the obtained growth after four weeks incu­ albumin, heparin and hyaluronic acid, re­ bation at 34° C. Yeast extract alone or sup­ spectively. The other tested mucopolysac­ plemented with horse serum did not promote chari des (chondroitin sulfate, ovomucoid, growth. Similarly, succinate and penicilla­ dextran, agar and galactomannan) and their. mine did not support the growth in the yeast constituents did not have an inhibitory effect extract-horse serum media. An abundant on the growth of the scotochromogenic cul­ growth was obtained in the yeast extract, tures. horse serum and glycerol containing media Glycerol, succinate and penicillamine as in four weeks. carbon sources. Since yeast extract was oxi­ The effect of different animal sera on the dized (13) by host grown Mlm, this heat sta­ bacillary growth. Horse, calf, sheep or goat ble complex can be considered as a prospec­ serum were added aseptically and respective­ tive energy source for in vitro growth. A ly to the yeast extract-glycerol medium. After 144 International Journal of Leprosy 1977

TABLE 3. Growth of scotochromogenic acid-fast bacilli in media containing yeast extract (YE) with or without horse serum (HS) and different substrates as prospective sources of carbon. Glycerol (Gl) was the only substrate which promoted multiplication. Media were inoculatedfrom the third subculture of a scotochromogenic strain isolatedfrom rat lepromas. Estimation of growth as in Table I. Medium Growth in six weeks YE :t YE- HS :t YE- HS- Na succinate + Y E- H S- Penicillamine + YE- HS- Glycerol ++++ Heat-killed M. lepraemurium ±

TABLE 4. The effect of d(fferent animal sera on the bacillary growth infa media containing yeast extract (YE) and glycerol (G/). Inoculum and estimation of growth as in Table J. Medium Growth in two weeks Growth in six weeks YE- Gl + ++++ YE- GI- Horse serum ++ ++++ YE- GI- Calf serum ++ ++++ YE- GI- Sheep serum ++++ ++++ YE- GI- Goat serum ++++ ++++ Heat-killed M. lepraemurium in YE- GI :t + two weeks of incubation, each of the four of the culture during the logarithmic growth animal sera potentiated bacillary growth in phase in KI-I medium. A scotochromogenic, the yeast extract-glycerol medium. Highest yellowish, smooth surface growth was visible growth rate was obtained with sheep and after 16 days of incubation at 34° C. The cul­ goat serum, somewhat less with calf, and tured cells were strongly acid-fast and easily even less with horse serum. After six weeks subcultured on Lowenstein-Jensen media. of incubation, however, the same abundant Infectivity of the cultures in rats. Bacilli growth was obtained with any of the .sera as from the third subculture in KI-I liquid me­ estimated by the naked eye. Results are dia during the logarithmic growth phase a nd shown in Table 4. also from the surface of the Lowenstein-Jen­ Reproducible cultivation. Over a period of sen medium subcultures were harvested. 12 months ten rats were sacrificed, one each Cells were washed once, resuspended in the from ten different colonies of infected rats. phosphate buffered solution and adjusted to Subcutaneous lepromas were removed from 200 Klett units. From this suspension, 0.5 ml each of the ten rats. From the isolated acid­ was injected subcutaneously into Sprague­ fast bacillary suspension, the same scoto­ Dawley rats weighing 60 to 70 grams. Ten chromogenic cultures were obtained in the rats were injected with each of the cultures KI-I liquid media. Only from three of the ten respectively. In four months, huge palpable lepromas was growth obtained on KI-I solid subcutaneous tumors developed in control media. rats at the site of injection with bacilli iso­ Growth on Lowenstein-Jensen media. lated from rat lepromas. At autopsy, the typi­ Lowenstein-Jensen media was inoculated cal visible "pathology" of rat leprosy was from the third subculture, transferring 0.2 ml observed. In rats which were injected subcu- 45, 2 KalO & Ishaque: A Scotochromogenic Mycobacterium; Etiologic Agent 145 of Rat Leprosy taneously with bacilli from cultures on K I-I tides. The addition of trace elements to the or Lowenstein media, the observed "pathol­ medium seems also to be unnecessary since ogy" was different in shape and size. Instead yeast extract contains most of the metals of the huge ovoid shaped lepromas, the flesh necessary for the growth of mycobacteria. colored lepromas were flat, non necrotic and Our theoretical considerations led to the f or­ had a diameter of two to six centimeters. mulation of a most simple medium contain­ The leprous tissue was less than one to two ing yeast extract and glycerol. Due to the millimeters thick. Impression smears showed heat stable ingredients, the medium can be connective tissue histiocytes parasitized with sterilized ·by autoclaving. Instead of yeast acid-fast rods. Very few lymphocytes and extract, other oxidizable substrates such as polymorphonuclear leucocytes were seen. No Na-succinate or L-cysteine permitted simi­ visible lesions were found in the organs. The larly excellent growth of the bacilli in the spleen was somewhat swollen. Six of ten rats media. A logarithmic growth rate was ob­ injected with bacilli from the KI-I medium tained by adding, in aseptic conditions, and eight of ten rats injected with the bacilli horse, bovine or goat, but preferentially from the Lowenstein medium subcultures sheep serum to the medium. developed similar "pathology" of murine lep­ It is safe to state that, in this simple medi­ rosy. Bacilli isolated from the subcutaneous um, yeast extract provides the source of ener­ lepromas were inoculated into Lowenstein­ gy and nitrogen as well as metals and trace Jensen media. No growth occurred in six elements. Glycerol is the source of carbon. weeks. Yeast extract might also contribute growth factors. DISCUSSION Gray ( 5), Hanks (7), and Hanks and Gray Hanks and Gray ( 8) and recently Pattyn ( 8) reported that, in addition to yeast extract, ( 21) strongly emphasized the need for meta­ bovine serum albumin preserved the hydro­ bolic studies leading to the cultivation of MI gen transfer capacity and infectivity of Mlm. and Mlm. Hanks ( 7) noted that the hydrogen Serum albumin is also a regular ingredient transfer capacity and infectivity of host­ of culture media for mycobacteria. The role grown murine leprosy bacilli was well-pre­ of serum albumin in the media is not clear. served in the presence of yeast extract. A sys­ It is believed to protect the cells from toxic tematic search for prospective substrates as ingredients or toxic metabolites ( 2). Based energy source for multiplication of Mlm led on Hanks' observations, Skinsnes and co­ to the finding that heat stable yeast extract is workers ( 22 ) also supplemented their LA-3 enzymatically oxidized by host-grown Mlm medium with serum albumin on which they (J3). The oxidation was coupled with oxida­ successfully cultured mycobacteria from tive phosphorylation (I). These findings human leprotic nodules. In our experiments, prompted large scale cultivation trials using bovine serum albumin definitely inhibited powdered, heat-resistant yeast extract as a growth of Mlm in the primary culture during source of energy. Using the Warburg mano­ 60 days of the acid-fast microorganisms metric technics and oxygen monitoring de­ grown on yeast extract-glycerol media. The vice, we found that optimal oxidation oc­ in vitro growth of cultivable species of myco­ curred at a pH value ranging from pH 5.5 to bacteria is enhanced, or at least not altered, pH 7.0 in a phosphate buffer solution with a by bovine serum albumin (9). It is known high potassium, low sodium ratio. The sim­ that all the scotochromogenic mycobacteria plest possible and most promising medium multiply abundantly in the presence of bo­ was with the addition of glycerol as the car­ vine serum albumin. bon source since glycerol is an excellent and Summarizing ten yea~s of work into the most widely used ingredient as a source of cultivation of M. lepraemurium, we reported carbon in most of the conventional media on ( II . 14) that heparin and a protein-bound mu­ which cultivable mycobacteria are cultured. copolysaccharide from rat tail tendon were Addition of any ingredient as a source of the only ingredients used in more than 8,000 nitrogen into the media seems to be unneces­ variations of culture media which permitted sary since yeast extract is a most abundant a limited mUltiplication and the prolonged source of nitrogen with its high content of preservation of the healthy morphology of amino acids and low molecular weight pep- these noncultivable microorganisms. These 146 International Journal of Leprosy 1977 observations led to a detailed search for pro­ lepromatous leprosy patient in Dakar (1 5). spective nutrients among more than 100 In cooperation with Mankiewicz (1 7), we mucopolysaccharides for growth, multiplica­ were able to confirm the findings of Pattyn tion, and retention of virulence of Mlm ( I I). that both the Honolulu and the Dakar strains Among the numerous polysaccharides listed, are slow-growing scotochromogenic myco­ Mlm multiplied in an alkaline medium con­ bacteria having all the characteristics of M . taining galactomannan (II). Though the scrofulaceum. We are, however, unable to multiplication was logarithmic for a short share the conclusion drawn by Pattyn (1 2.20 ). time, we were unable to subculture Mlm in In view of our present findings, we are rather the homologue media. In later experiments, inclined to debate that MI and Mlm once we were also unable to detect an increase of adapted for multiplication outside the host endogenous respiration or oxidative phos­ on artificial media, might belong to the phorylati0n in the presence of any of the scotochromogenic species probably resem­ mucopolysaccharides tested. Our present re­ bling M. scrofulaceum. In this respect, it is sults show that, in the yeast extract-glycerol interesting to note that both cultures, iso­ medium, none of the three tested mucopoly­ lated by the Skinsnes group and the Mon­ saccharides of vegetable origin, dextran, treal team from human leprotic nodules, are galactomannan or agar, enhanced or in­ scotochromogenic and so is M . marianum. hibited the growth of the bacilli. We are now able to culture regularly from In the experiments of Duran-Reynals, rat lepromas, strains of mycobacteria which hyaluronic acid inhibited the mUltiplication are scotochromogenic, thus forming a yellow of an intracellular parasite (3). In our pre­ pigment in the presence or absence of light. sented experiments, hyaluronic acid pre­ There is, however, a basic difference be­ pared from umbilical cord as well as umbili­ tween the scotochromogens isolated from cal cord extract and another connective tis­ rat lepromas. The human strains were iso­ sue mucopolysaccharide, heparin, inhibited lated as primary cultures in media containing growth and multiplication in the primary cul­ hyaluronic acid and serum albumin (1 5.22 ), ture of Mlm. Other connective tissue muco­ while both bovine serum albumin and hya­ polysaccharides, chondroitin sulfate and ovo­ luronic acid are extremely toxic for the rat mucoid, had no inhibitory effect. This is in bacilli in the primary culture. Already, our sharp contrast with our working theory pre­ earlier studies indicated the toxicity of serum sented earlier. We advocated at that time albumin for Mlm freshly isolated from the that, since human and murine leprosy bacilli host (4). Animal sera or serum albumin were grow abundantly in the connective tissue his­ the sine qua non and the absolute necessity tiocytes, they must utilize for growth, multi­ for the growth of primary cultivation of the plication and virulence, substrates which are human strains ( 15.22 ). The rat strain is easily present in the connective tissue matrix (14). cultured from the host on artificial media in We suspected, at that time, the protein­ the absence of animal sera but supplemented bound carbohydrates, acid mucopolysac­ with yeast extract. Once adapted to life out­ charides, the biological amines present in side the host, both the human and the rat mast cells as histamine, serotonin and cate­ strains grow abundantly on conventional cul­ cholamines as prospective substrates for in ture media such as the Lowenstein-Jensen vitro growth of Mlm on media enriched with medium used for the cultivation of other my­ connective tissue constituents (14). Then, cobacteria. Both the human and the rat Skinsnes and his co-workers discovered that strains are scotochromogenic and biological­ Ml isolated from the host is provided by the ly closely related and identifiable as belong­ enzyme J3-glucuronidase for the cleavage of ing to the M. scrofulaceum subspecies hyaluronic acid. Subsequently, they reported ( 17. 20 ). (22) the successful cultivation of MI on a hy­ Once adapted to growth outside the host, aluronic acid-based medium. The obtained the Mlm cultures also grow in the presence culture was scotochromogenic and a slow­ of hyaluronic acid, heparin or serum al­ growing species of mycobacteria identified bumin, substances which were toxic in the by Pattyn ( 20 ) as M . scrofulaceum. Using a primary culture. modified LA-3 medium, we isolated a similar When the cultures isolated from the rat scotochromogenic strain from an untreated lepromas on KI-I, 2, or 3 media and also 45,2 Kalo & Ishaque: A Scotochromogenic Mycobacterium; Etiologic Agent 147 0/ Rat Leprosy TABLE 5. Sensitivity of/our scotochromogenic cultures to different antituberculosis drugs. The scotochromogenic cultures were isolated/rom ralleproma (RAT)./rom human lepromatous leprosy nodules in our laboratories (DAKAR). and isolated by Skinsnes et al (HI-75) and an authentic culture o/M. scrofulaceum. (R= Resistant; S= Sensitive)

PAS p.g / ml 5 p.g / ml 10 p.g/ ml Mycobacteria R 90% R 60% R RAT R R R DAKAR R R 90% R HI-75 R R R SCROFULACEUM Streptomycin p.g/ ml 5 p.g / ml 10 p.g/ ml R 90% R 75% R RAT R 95% R 95% R DAKAR R R 95% R HI-75 R R R SCROFULACEUM INH .2 p.g / ml p.g / ml 5 p.g/ ml R R 90% R RAT R R R DAKAR R R R HI-75 R R R SCROFULACEUM Ethanbutol 4 p.g / ml 8 p.g / ml 16 p.g/ ml S S S RAT R R R DAKAR R R R HI-75 R R R SCROFULACEUM Rifampicin .2 p.g / ml .5 p.g/ ml p.g/ ml S S S RAT S S S DAKAR S S S HI-75 S S S SCROFULACEUM when adapted to the Lowenstein-Jensen me­ fully grown and can be easily and ad irifini­ dium, were reinjected into rats, a limited tum subcultured in the homologue media. disease was reproduced in the animals re­ 4. When adapted they can be easily sub­ sembling the "pathology" of rat leprosy. The cultured on conventional culture media for final identification and classification of the mycobacteria, i.e., Lowenstein-Jensen medi­ obtained cultures remain to be investigated um. and confirmed. It can be stated, however, 5. The cultures grown on KI-I medium, that the described mycobacteria: . once adapted to the Lowenstein-Jensen me­ I. Can be regularly cultured from rat lep­ dium, reproduce the specific disease known romas. as murine leprosy. The subcultures retain 2. They grow at a logarithmic rate on a infectivity with considerably reduced patho­ simple medium which provides one of the genicity. specific sources of energy, nitrogen, carbon Table 5 shows the sensitivity and resist­ and probably growth promoting factors. ance of the obtained cultures to antibacterial 3. The bacilli are strongly acid-fast when agents (1 7). The scotochromogenic cultures 148 International Journal of Leprosy 1977 isolated from rat lepromas, the cultures ob­ cultures on the homologue media retained tained from human leprous tissue (Dakar and specific infectivity for rats but lost their path­ HI-75) as well as an authentic strain of M. ogenicity and virulence considerably. scrofulaceum showed rdistance to strepto­ mycin. The same antibiotic is inactive in vivo RESUMEN against human and murine leprosy. The cul­ tures are resistant to IN H and PAS, s ub­ Se prepararon medios de cultivo que contenfan, stances which have no therapeutic effect in respectivamente, extracto de levaduras, succi nato human and murine leprosy. All the cultures o cistefna, como unica fuente de energfa. Emple­ were extremely se nsitive in vitro to rifampi­ ando tecnicas manometricas se encontro que el cin, the most effective drug known so far for M. lepraemurium fue capaz de utilizar todos estos substratos: La unica fuente de carbo no en el leprosy. Sensitivity to antibacterial agents is medio fue el glicerol. Los bacilos se aislaron de los a basic genetic characteristic of microorga­ lepromas subcutaneos de ratas infectadas. nisms. The parallelism found between in Despues de 5 semanas de latencia se desarrollo un vitro se nsitivity of the four scotochromogenic crecimiento abundante durante una fase de creci­ cultures and the effect of the drugs on the miento logarftmico que duro aproximadamente 10 disease merits special attention. dfas. Esto sucedio en los medios conteniendo cual­ quiera de los substratos energeticos y glicerol. En SUMMARY una solucion reguladora de fosfatos, pH 5.5, el crecimiento optimo ocurrio cuando la incubacion Culture media were prepared in which se hizo a 34° C. Los sueros de bovino, de caballo, yeast extract, succinate and L-cysteine, re­ de cabra, 0 de camero, incrementaron la multipli­ spectively, served as a source of energy. cacion del bacilo in vitro. EI acido hialuronico, la These substrates were oxidized by M . leprae­ heparina y la seroalbumina resultaron toxicos e murium as measured with manometric tech­ inhibieron el crecimiento de los cultivos primarios nics. Glycerol was the only source of carbon del M. lepraemurium. No se obtuvo crecimiento in the media. Bacilli were isolated from sub­ cuando los bacilos de la lepra murina crecidos en su huesped se inocularon en los medios de Lowen­ cutaneous lepromas of infected rats. After stein 0 de Dubos. Los cultivos fueron escotocro­ five weeks of latency, a heavy growth devel­ mogenos y produjeron un pigmento amarillo. Los oped during a logarithmic growth phase last­ cultivos jovenes no fueron resistentes al alcohol ing about ten days in media containing any acido pero esta resistencia al alcohol acidulado se of the substrates for energy generation with desarrollo plenamente durante la fa se de creci­ glycerol added. In phosphate buffer solution, miento logarftmico. at pH 5.5, optimal growth occurred when in­ Las cepas se pudieron subcultivar con facilidad cubated at 34° C. Bovine, horse, goat and en el medio homologo y tam bien se adaptaron sheep serum respectively enhanced in vitro rapidamente a crecer en presencia de otros sub­ multiplication. Hyaluronic acid, heparin and stratos (acido hialuronico, heparina y seroalbu­ mina), y aun en otros medios (Lowenstein y serum albumin were toxic and inhibited Dubos). En los subcultivos posteriores el periodo growth in the primary M. lepraemurium cul­ de latencia se acorto mucho (llegando a ser hasta tures. When host-grown murine leprosy ba­ de 2 dfas) y fue seguido por 304 dias de creci­ cilli were inoculated into Lowenstein or miento logari'tmico. Los cultivos primarios y sus Dubos media no growth occurred. subcultivos en el medio homologo retuvieron su The cultures were scotochromogenic and infectividad especi'fica en las ratas pero perdieron produced a yellow pigment. Young cultures considerablemente su patogenicidad y virulencia. were nonacid-fast. Full acid-fastness devel­ oped during the logarithmic growth phase. The strains were easily subcultured not only in the homologue media but became Des milieux de culture ont ete prepares dans rapidly adapted to new substrates. They then lesquels l'extrait de levure, Ie succinate et la L­ cysteine ont servi respectivement comme source became adapted to and grew in the presence d'energie. Ces substrats furent oxydes par M. lep­ of hyaluronic acid, heparin and serum albu­ raemurium tels que mesures par des techniques min as well as on Lowenstein and Dubos manometriques. Le glycerol etait la seule source media. With subsequent subculturing, the de carbone dans les milieux. Les bacilles etaient latency period became as short as two days isoles a partir de lepromes sous-cutanes des rats followed by three to four days of logarithmic infectes. Apn!s cinq semaines de latence, une growth. The primary cultures and their sub- bonne poussee s'est develop pee au cours de la 45,2 Kato & Ishaque: A Scotochromogenic Mycobacterium; Etiologic Agent 149 of Rat Leprosy phase logarithmique de croissance qui a dun! en­ 5. GRAY, C. T. the res piratory metabolism of viron dix jours. Ceci avait lieu dans les milieux murine leprosy bacilli. J . Bacteriol. 64 (1952) contenant n'importe lequel des substrats source 305-315. d'energie accompagne de glycerol. Dans Ie tam­ 6. HANKS, J . H. Influence of physical and chem­ pon phosphate, pH 5.5, la croissance optimale ical factors on the hydrogen transfer capacity avait lieu lorsqu'incube a 34° C. Les serums de of murine leprosy bacilli. Int. J . Lepr. 22 boeuf, de cheval, de chhre et de mouton de fat;on (1954) 162-173. respective stimulaient la multiplication in vitro. 7. HANKS, J . H. Relationship between the meta­ L'acide hyaluronique, l'heparine, I'albumine bolic capacity and the infectiousness of My­ serique etaient toxiques et inhibaient la crois­ cobacterium lepraemurium: refrigeration sance dans les cultures primaires de M. leprae­ studies. Int. J . Lepr. 22 (1954) 450-460. murium. Lorsque les bacilles de la lepre murine 8. HANKS, J . H . and GRAY , C. T. The applica­ cultives dans I'hl'>te etaient inocules sur les milieux tion of metabolic studies to leprosy research. Lowenstein ou Dubos, aucune poussee n'avait Int. J. Lepr. 22 (1954) 147-161. lieu. 9. HANKS, J . H. and PRATT, D. B. Species adap­ Les cultures etaient scotochromogenes et pro­ tation of acid-fast bacteria as revealed by duisaient un pigment jaune. Les cultures jeunes their growth in serum. Soc. Am. Bacteriol. etaient non acido-resistantes. L'acido-resistance Proc. of Meetings 1(1948) 90. etait maximale au cours de la phase logarithmique. 10. ISHAQ UE, M . and KATO, L. Occurrence of c Les souches etaient facilement sous-cultivables type cytochrome in Mycobacterium leprae­ non seulement sur milieu homologue, mais murium. Can. J. Biochem. 52 (1974) 991-996. s'adaptaient rapidement a de nouveaux substrats. II . KATO, L. Attefnpts to cultivate Mycobacteri­ Les souches se sont adaptees et ont pousse en um lepraemurium in cell-free media. Int. J. presence d' acide hyaluronique, d'heparine et Lepr. 33 (1965) 509-521. d'albumine serique et aussi sur les milieux Lo­ 12. KATO, L. In vitro grown M. leprae probably wenstein et Dubos. Avec les sous-cultures, la a member of the M. scrofulaceum species. peri ode de latence diminuait jusqu'a deux jours, Int. J . Lepr. 44 (1976) 385-386. suivie par trois a quatre jours de croissance loga­ 13. KATO, L. , ADAPOE, C. and ISHAQUE, M . The rithmique. Les cultures primaires et leurs sous­ respiratory metabolism of M. lepraemurium. cultures sur milieu homologue avaient conserve Can. J. Microbiol. 22 (1976) 1293-1299. une infectiosite specifique pour les rats, rna is 14. KATO, L. and GOZSY , B. Attempts to cultivate avaient perdu considerablement de leur patho­ Mycobacterium lepraemurium . Int. J . Lepr. genicite et de leur virulence. 31 (1963) 344-347. 15. KATO, L. and ISHAQUE, M. A simplified hy­ Acknowledgments. This work was supported by aluronic acid-based culture for· mycobacteria grants from the Institute Fame Pereo and the isolated from human lepromata. I nt. J. Lepr. !'v1erieux Foundation (Lyon, France). The Hansen 44 (1976) 431-434. Chair of Research of the I nstitute Armand-Frap­ 16. KATO, L. and ISHAQUE, M . The proof of the pier is generously supported by Le Secours aux pudding is in the eating. Widely circulated Lepreux (Canada) Inc. memo of November, 1974. 17. MANKIEWI CZ, E. and KATO, L. 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