Role of sulfhydryls in in vitro growth of Mycobacterium lepraemurium.

In an attempt to evaluate various factors that influence the growth of Mycobacterium lepraemurium in NC-5 medium, the effects of sulfur and --SH compounds were investigated. Cysteine could be replaced by equimolar concentrations of other --SH compounds containing carboxyl group, and at lower concentrations by nonpolar sulfhydryl compounds. The oxidized form of sulfhydryls, as well as certain organic and inorganic reducing agents, did not support growth. The results suggest that the function of sulfhydryl compounds is to maintain low reducing potential in the medium and also to participate in metabolic or biosynthetic pathways or both. A combination of dithiothreitol and thioglycolate gave better results than when these compounds were incorporated individually in the medium. This suggests the protective action of dithiothreitol in preventing oxidation of monothiols.

Pedigreed stocks of Mycobacterium lepraemurium for cultivation and metabolic studies.

Experience has shown that a prime requirement for investigations with Mycobacterium lepraemurium is pedigreed stocks of cells which provide constant baselines over a long period of time. Biochemical indicator, ATP, was employed to devise a method for preservation of metabolic pools of M. lepraemurium during storage. ATP assays were made by the luciferin-luciferase bioluminescent method. Several cryoprotective agents were compared at -76 and 4 degrees C. The essential steps have been found to be (i) for prolonged storage and constant supply of material, to freeze the intracellular bacteria within infected cells containing 28% proteins, i.e., to freeze the infected tissue; and (ii) when a large number of diverse experiments are to be undertaken on a single suspension, to stabilize working stocks of refrigerated cells for 12-16 weeks by using bovine serum albumin, fraction V, and Difco yeast supplement B to compensate for the leaching of intracellular cofactors, metabolites, nucleotides, etc.

In vitro growth of Mycobacterium lepraemurium, an obligate intracellular microbe.

By using an ultrasensitive technique to measure adenosine triphosphate in terms of functional biomass, we have confirmed that Mycobacterium lepraemurium (the agent of rat leprosy and a classical obligate intracellular microbe) grows in vitro in the Nakamura system. By using a sulfhydryl-containing medium that ocupies 65 to 75% of the culture tube volume, together with the five supplements recommended by Nakamura, we have obtained growth rates some eight times above the original. The new physicochemical environment and the use of adenosine triphosphate as an index of energy status in the presence and absence of growth provide a basis for investigating the physiology and growth of other noncultivated microbes.

Energetics (adenosine 5'-triphosphate) of Mycobacterium lepraremurium in diffusion chambers incubated in vitro and in mice.

Adenosine 5'-triphosphate (ATP) measurements and the processing of samples have been refined to a point where the energetics and growth potential of microscopic samples of unwashed host-grown, host-dependent microbes can be investigated. Mycobacterium lepraemurium, the noncultivated agent of murine leprosy, was employed to examine three reports of the slow microscopic growth of this organism in the absence of host cells. A few million bacterial cells were enclosed in Rightsel- and Ito-type diffusion chambers, which were incubated in vitro and in the peritoneal cavities of mice. In the in vitro experiments, a complex medium containing bovine serum and mouse brain extracts, renewed three times a week, did not sustain the energetics of the bacilli. The microscopic counts declined to 72% and the ATP per culture to 9% of the original values. Very different results were obtained from chambers incubated in the peritoneal cavities of mice. The bacterial biomass increased 2.7-fold and the ATP per culture increased 2.5-fold. Because the ATP per cell was 93% of the original, this system is regarded as the first to permit the extracellular growth of a so-called "obligate intracellular microbe." The results obtained with only 1 x 10(6) host-grown cells per assay demonstrate a significant biochemical tool for investigating the growth potential of host-grown microbes during the progression, regression, and therapy of disease.

Quantitative extraction of adenosine triphosphate from cultivable and host-grown microbes: calculation of adenosine triphosphate pools.

EXISTING DATA ON ADENOSINE TRIPHOSPHATE (ATP) POOLS IN MICROBES ARE DEFICIENT FOR TWO REASONS: (i) incomplete extractions of ATP, and (ii) the failure to take into account that the adverse effects of extracting procedures on standard ATP exert analogous effects on the ATP released from bacterial cells. Methods for correcting observed yields and calculating ATP pools have been demonstrated. Three bacterial species were used in the studies on extraction of ATP: Escherichia coli, Mycobacterium phlei, and Mycobacterium lepraemurium. Perchloric acid and n-butanol were disqualified because of their failure to extract total bacterial ATP even from E. coli and because of inconvenient procedures. The new extraction procedure had minimal effects on standard ATP, liberated 100% of the ATP pools from the three representative species of microbes, and caused no ionic imbalance or quenching of bioluminescence. This method involves vortexing of cell suspensions for 10 s with 23% chloroform (vol/vol), heating at 98 C for the required time (E. coli, 3 min; M. phlei, 5 min; M. lepraemurium, 10 min) and then 1 min at 98 C with vacuum to dry the samples. Heat or chloroform alone may suffice for some microbes and release total ATP from plant and animal cells.

Studies towards the standardization of lepromin. Progress and prospects.

Because of the wide range of concentrations of Mycobacterium leprae in existing lepromins the authors studied methods of producing a standardizable lepromin containing 160 million bacilli/ml. The effects of using different dilutions of lepromin on the incidence of false-positive reactions were also studied.Progress reported includes a convenient method for preparing large batches of non-sedimenting lepromin, which is directly suitable for microscopic counting of Myco. leprae cells; and a validation of current methods for microscopic enumeration of Myco. leprae. Skin tests with diluted lepromins have demonstrated that dilutions up to 1:16 increase progressively the ability to distinguish between lepromatous and tuberculoid leprosy. This work has provided further evidence that 20 million bacilli/ml (a 1:8 dilution of the initial lepromin) should produce adequate Mitsuda reactions in general populations, provided that 3-mm reactions are taken as the criterion for 1+ positivity. The net effect of these findings is equivalent to expanding the world supply of lepromin by 8 times.Recommendations for further research are proposed.