M U T A T I O N OF M Y C O B A C T E R I A TO P R O A C T I N O M Y C E S BY RADIOACTIVE COBALT

by

MAHMOUD ~AMAL MUFTIC

(Mycological Laboratory-Middle Euphrat Hospital; Ku//a; Iraq) (1. 9. 1955)

(With plate IV)

FROUIN and GUILLAUMIE (1923) estimated that mycobacteria are not schizomycetes but pure fungi. LANGEI~ON (1945) believed that tubercle bacilli are with no doubt actinomycetes, and tuber- culosis is the most grave and generalised mycosis. There is also an intermediate form between non ramified mycobacteria and aboun- dantly ramified actinomyces, in genus: Proactinomyces. They are filamentous microbes with traces of ramification. According to TURFITT (1944) and ARNAU])I (1949) both mycobacteria and pro- actinomyces can use cholesterol as the only carbohydrate source.

Mycobacteria being capable of adaptation and mutation with comparative ease, the synthetic and catabolic functions of its strain are susceptible to very wide variations, some of which are direct and fairly rapid adjustments to the environment and others only produced by considerable alteration to the "genes" or their nucleic acids. Nucleoproteids appear to be the self-reproducing units of living mat ter and an alteration to their nucleic acid results in a permanent alteration to the reproducing units so this affects the properties of the cell's progeny.

METALNIKOV, YAXlMACtt and GELELOVITCH (1940) succeeded to obtain hereditary mutants in sarcinae b y radon. NADSON (1935), RENAUD (1943), GUYENOT (1942) and SARTORY and MEYER (1926) obtained different irreversible mutants by action of radium salts in various fungi. RENON (1913), BEQUEREL (1913) and FROUIN (1920) have examined action of radioactive salts on tubercle bacilli. Their reports proved that low concentration of uranium and thorium salts exert a favourable action on rapidity and intensity of development of surface growth.

RICE, ORR, REEl), and GARDINER (1931) used ultraviolet rays for separation of "S" (smooth) form from "R" (rough) form of tubercle bacilli. Form "R" is more sensitive to ultraviolet and rapidly perishes and form "S", go obtained, is less virulent than usual.

In our essays on mutat ion of mycobacteria we used "S" form of mycobacteria tuberculosis hominis, cultivated in DUBOS Tween- Albumin liquid media with soluble salt of radioactive isotope of

122 M. 142. MUFTIC

cobalt (Co 6°) which has a radiation energy in gamma-rays of 1.3 MEV. We chose cobalt isotope only because it has soft gamma radia- tion of long life, and "S" form of tubercle bacilli, because it is less sensitive to the lethal effect of short rays.

Experimental A strain of tubercle bacilli form "S", obtained from pulmonary

lesion at necropsy, was studied in about ten reinoculations in Loewenstein eggs-medium and by inoculation in guinea pigs.

We prepared three series of liquid Dubos Tween-Albumin medium.

A - Medium with different concentration of radioactive cobalt chloride.

B - Medium with different concentration of non-active cobalt chloride.

C - Medium serving as control without cobalt salts.

After incubation of three weeks we inoculated M1 media with 0.1 mg of moist weight of fresh bacillary culture from Loewenstein medium. Cultivation was carried on for four weeks and the colonies were reinoculated on Loewenstein medium.

In media "A" with 100--200 #Cu per ml we obtained a strain, different from the mother strains, so that we can estimate it as a mutat ive type, with hereditary reproduction of all characteristics. We designed this type as Mycobacterium tuberculosis hominis (isotope Co 6°, chromogene, virulent) H~0 Chv.

The new mutant strain is polydynamic and dysgonic. Its colonies have a thick, creamy, rough surface growth, pigmented oker- purple and occasionaly okerviotet. I t rises on walls of bottles, covers surface completly in a very short time. These polydynamic proper- ties of the new strain are rare in pathogenic mycobacteria and are comparable only to the avian type. After only five days of in- cubation on Loewenstein media all surface was covered. The quant i ty of growth was about sixty times greater than that of mother strains obtained from culture "B" and "C" after 12 days of incubation.

Microscopically the mutant looked like coenotic rarely ramified mycelium of very weak acid-fastness and with a fine iodophylic granulation. Also a typical Proactinomyces.

I t consumed: glucose, maltose, trehalose, inosit, glycerol, choleste- rol, paraffine and naphta-petrol. I t reduced methylene blue, and decomposed: amygdalin, salicin and arbutin.

The guinea pigs inoculated intraperitoneal by H6o Chv die in the third week. At the end of the second week we can isolate the germs from blood. Necropsy showed many mycetomes in lymphatic glands and lung. A week after inoculation the animals were sensitive to

M U T A T I O N O F M Y C O B A C T E R I A 123

tuberculin. The rabbits are of the same sensitivity to inoculation. They perished in the fourth week with formation of mycetome in mesenteric glands. The fowels are refractery to the infection.

Comparative pathogenetic study 30 guinea pigs were intraperitoneally inoculated by 0.1 mg of

original strains of tubercle bacilli. They died in between 8--14 weeks. Necropsy showed general granular tuberculosis in abdominal viscera, thoracic ganglions with engorgement and caseification of retrosternal glands and formation of tuberculous nodules on the omentum. The rabbits were resistent to the infection.

Another group of 30 guinea pigs, inoculated by strains developed in presence of non-active cobalt (0.2 mg per ml), has shown the same pathological characters as above. Survival time was the same. In both groups of animals the spleen and liver were enlarged.

The third group of 30 guinea pigs was inoculated by mutant strain. They succumbed in the third week. Animals showed tumefaction of visceral and mesenteric glands, formation of mycetomes in neck glands, omentum and mucous membranes. Lung, spleen and liver were only slightly enlarged and without tubercle formation. In table No I and II the quantitative amount of infection found at necropsy time in some animals is represented.

Discussion I t is well known that the chromogen form of tubercle bacilli by

their antigen structure is nearer to the smooth form than to the rough. At the same time they are less virulent than S form, which is less virulent than R form. I t is for this reason more characteristic that the new type, inspite of its chromogene characters, is more virulent than any other strain of mycobacteria including HaT Rv.

Photodynamic action of soft gamma-rays of Co ~° on mycobacteria can be interpreted or by Roussel effect (FRANCIS, MADINAVEITIA, MACTURK and SNow, 1949) (LAcASSAGNE, LATARJET, 1934) due to the presence either photodynamic dyes as vitamin K 2 (FIESER, CAMPBELL and FRY, 1939) (PARsHIN 1946) a naphtoquinon pigment in membrane of bacilli or as absorption effect on nucleoprotides or nucleic acids, the absorption coefficient of which is very strong in short ultraviolet and soft X-ray (VLES, 1946).

As results of radiation shocks SUBRAMANIAM, RANGANATHAN and KnlSHNA (1948) observed gene mutation, PRIMA BAI (1947) chromosomal translocation, and SUBRAMANIAM, RANGANATHAN (1948b) tetraploidy. I t is doubtless that in our experiments the reducing effect on "genes" of mutant stabilised the mycobacteria in the parent form of Proactinomyces. The process during our observation was irreversible. The radiation power of mutant strain is preserved during many generations. Picture 1 shows an autoradio- graphy of the 12th generation culture H60Chv on Loewenstein

1 2 4 M . K . MUFTIC

medium. The amount of radiation diminished by time. There is no sign of mutat ion in other cultures treated by the same concentration of non radioactive cobalt. Thus, it is not the polydynamic effect of cobalt as iron-like metal which is responsible for the mutation. Mutation of Mycobacteria to Proactinomyces by radioactive cobalt.

TABLE I

No of animal

Weight \¥eight pre-infect, post-

Gm infect. Gm I

Spleen Gm

Liver Gm

Survival W,

7 9

10 15

400 370 360 400 420 400 500 I 475

3,5 3,9 4 4

10,5 18 23 11

3 weeks 2 3 3

Some of guinea pigs inoculated intraperitoneally by 0.1 mg of fresh culture of H6oChv from Loewenstein medium.

TABLE II

No of anim.

Weight pre-infect.

Gm

Weight Spleen post- Gm

infect. Gm I

Liver Gm

Survival W.

~ B ~ ~

6 " B " 7"C"

28 "C" 29 "B"

450 500 500 360 420

375 I 9 360 ! 7,5 510 I 4 [ 350 6 400 11

31 46 30 31 26

9 weeks 8

11 14 13

Some of guinea pigs inoculated by: Group "B" (cobalt non radioactive culture of tubercle bacilli 0.1 mg intraperitoneally). Group "C" (control culture of tubercle bacilli without cobalt 0.t mg intraperitoneMly).

The new mutant strain of tubercle bacilli is a fairly convincing proof that mycobacteria are really fungi, and also tuberculosis a mycosis.

But as LANGERON (1945) writes, tuberculosis is not studied from this point of view. It became a social disease, that means a disease which let nearly the same number of individuals live as it kills. This is the biological equilibrium and that satisfies the law of moderation.

S u m m a r y

A smooth strain of htmmn tubercle bacilli exposed in liquid me- dium to the gamma irradiation of radioactive cobalt was transmu-

M U T A T I O N OF M Y C O B A C T E R I A 125

tated in a new type ressembling genus Proactinomyces. Morphologi- cally it showed a rarely ramified mycelium of very weak acid- fastness, and high virulence for guinea pigs and rabbits. I t was designated as H6oChv.

Resum6

Une souche de bacilles tuberculeux humains le type "S" (glabre), cultiv6e dans un milieu liquide, 6tait expos6e au rayonnement gamma du cobalt radioactif, provoquant une transmutation dans un type nouvel ressemblant au genre proacfinomyces. Morphologiqne- ment il se repr&ent comme un mycel microsiphon6, avec des ramifications en trace et d'une acido-resistence tr~s faible. I1 est tr6s virulent pour les cobayes et les lapins. L'abr6viation pour cette souche est: H60 Chv.

Acknowledge

The supply of radioactive cobalt salts (Co G°) was made by PHILIPS-ROXANE, Pharmaceut.-Chemisch. Industr. "DueRAn", Amsterdam, the Netherlands, to whom we due our best thanks.

Kuffa-Iraq, End of July 1955.

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