Aeration, temperature and presence of glycerol modifying the multiplication of Mycobacterium leprae.

The multiplication of 8 strains of M. leprae on medium NM6 + DOPA, described by Olitzki (1976) in this journal, was enhanced by maximal aeration, by the presence of 1.0% glycerol and by an optimal incubation temperature of 30 degrees C as compared with 37 degrees C. It took place in the presence of several organic acids and N-acetyl-glucosamine.

Further potential sources of energy modifying the multiplication of Mycobacterium leprae.

The multiplication of Mycobacterium leprae was modified by graded dilutions of organic acids. 0.01%-0.05% gluconic acid inhibited its multiplication. 0.005% of it promoted the growth of 2 out of 6 strains. 0.2% - 1.0% glucuronic acid promoted the multiplication of the majority of strains. 2.0% inhibited their multiplication, and 0.05% promoted the growth of one strain. Galacturonic and pyruvic acids were active in 0.2 - 2.0% concentrations, while the activity of citric acid was mainly noted at 1.0 and 2.0% concentrations.

The effect of dioxyphenylalanine (DOPA), amides and some potential sources of energy on the multiplication of Mycobacterium leprae.

The multiplication of 2 out of 3 M. leprae strains on a medium containing substances from digested non-acid-fast microorganisms or even free of them was significally promoted to D-3,4-dihydroxyphenylalanine (DOPA). The following organic substances exerted growth-promoting effects on several strains: 0.02-0.10% concentrations of succinate greater than fumarate greater than alpha-ketoglutarate and acetate greater than glycerol; 0.2% concentrations of citrate and pyruvate greater than isonicotinamide and benzamide greater than lecithin. 0.5% concentrations of oleate greater than citrate greater than pyruvate greater than acetate greater than fumarate greater than succinate; 5.0% concentrations of butanol and butandiol greater than propanol greater than sorbitol greater than ethanol. However, these effects were variable and strains of various origins acted differently. On media containing DOPA, malachite-green (MG) and at least 0.12 x 10(6) microorganisms/ml the following oxidation-reduction reaction was observed: DOPA was oxidized to a brown compound and malachite-green reduced to an almost colourless product. Consequently, the blue-colour of the cultures turned from blue to brown. This DOPA-MG reaction and the inability to grow on conventional media were used for the identification of 10 cultures (inocula directly from patients) as M. leprae, while a DOPA-MG negative patient-strain grew on media employed for cultivable mycobacteria and was not identical with M. leprae, as proved by the foot-pad test in mice.

Effect of microbial substances of different origins on the growth of Mycobacterium leprae.

Mycobacterium leprae multiplied in media enriched with substances originating from other mycobacteria, from non-acid-fast Actinomycetales or from gram-positive or gram-negative Eubacteriales. Most of the M. leprae strains did not grow on a synthetic medium containing the amino acids present in M. smegmatis, but the growth-promoting effect of sonic extracts of this organism indicated that substances of bacterial origin, other than amino acids, do act as growth factors. The identification of the growing, acid-fast microorganisms with M. leprae was verified by their ability to oxidize D-3,4-dihydroxyphenylalanine (D-dopa test). M. leprae did not multiply on Nakamura medium unless at least 40% of medium NM7 enriched by bacterial substances was present. Adequate aeration was essential for multiplication of M. leprae in enriched NM7 and No. 3 media.

Further investigations on M. leprae. Growth promotion and inhibition by organic substances and observations on antagonistic and syntergistic effects.

The multiplication of 2 strains of M. leprae on a medium containing a sonic extract (SE), prepared from M. smegmatis, was promoted by cysteine, tryptophane and dimethylasulfoxide (DMSO), while glutamic acid, glutamine and histidine exerted variable effects. The final effects of glutamic acid and glutamine were determined by the total concentration of both compounds together. The presence of cysteine and glutamic acid alone or together with DMSO abolished all inhibitory effects. Desferal did not enable the multiplication of M. leprae on media devoid of SE prepared from M. smegmatis. However with SE and 0.005 per cent and 0.002 per cent concentrations of Desferal its initial growth was accelerated. Its final counts, noted after an 8-month incubation, did not exceed those observed without Desferal. Puring and pyrimidine compounds promoted markekly the multiplication of M. leprae (counts greater than 3 times 10-7/ml). The highest counts were observed with pyrimidines (thymine, thymidine, cytosine) applied single or combined.

In vitro effects of some chemotherapeutic agents on mycobacteria.

Examination of the bactericidal and bacteriostatic effects of nine antibiotics on atypical mycobacteria revealed that streptovaricin complex and streptovaricin C exerted bactericidal effects on several strains in concentrations lower than 1.0 mug/ml. An exposure to the drug for 48 hr at 37 C was necessary to effect a complete inactivation of more than 99.9% of the exposed microorganisms. The appearance of strepto-varicin-resistant mutants was observed. However, these mutants were unstable, and reversion to streptovaricin susceptibility occurred. Celesticetin salicylate, added in a concentration of 100.0 mug/ml to the medium of Olitzki and Gershon inoculated with Mycobacterium leprae, effected a complete change of the uniformly stained mycobacteria to bipolarity, which indicates the devitalization of this microorganism.