Virulent strains of Pasteurella pestis frequently undergo rapid attenuation during growth at 37 C in liquid media (Wats and Puduval, 1940; Devignat and Schoetter, 1942; and Fukui et al., 1957). This change in the bacterial population was related to a characteristic nutritional difference between virulent and avirulent cell types and was prevented by the presence of adequate amounts of calcium ions in the growth medium (Higuchi, Kupferberg, and Smith, 1959). In contrast to virulent cell types, the few avirulent cells initially present or arising by mutation, proliferate at 37 C in calcium deficient media, and thus rapidly outgrow the original virulent population. It was observed that an unusually high mutation rate in P. pestis from virulence to avirulence was an important factor in these processes. The present work considers the development of a differential plating medium for the detection of avirulent cell types and its application for the estimation of the mutation rate (virulence -avirulence) in P. pestis. A preliminary report on a part of this work has been presented previously (Higuchi and Smith, 1959).

https://jb.asm.org/content/jb/81/4/605.full.pdf

Studies on the nutrition and physiology of Pasteurella pestis. VI. A differential plating medium for the estimation of the mutation rate to avirulence.

A highly successful modus operandi in research is to perform an experiment and then decide what problem was solved; the alternative procedure of devising experiments in order to answer preexisting questions may be more difficult. Unfortunately, the latter approach cannot be avoided by those concerned with infectious diseases because the question of why a given microorganism is virulent is automatically framed by its discoverer—in this case, the plague bacillus by Yersin (1894) and the causative agent of pseudotuberculosis by Malassez, and Vignal (1883). The delay of almost a century in obtaining satisfactory answers to these questions simply illustrates that the complexity of the host-parasite relationship is formidable and that experiments relevant to the expression of virulence have not been designed. At the same time, considerable progress towards this end has been made with other pathogens, especially in correlating nutritional requirements and metabolic patterns with the bacterium’s favored habitat in vivo (Moulder, 1962). Nevertheless, the diverse phenomena observed during infection with intracellular parasites cannot always be duplicated in simplified experimental systems. Furthermore, the infectivity of many microorganisms is low or erratic in laboratory animals and the response of the normal host to acute and chronic phases of disease may be quite distinct.

The genus Yersinia: biochemistry and genetics of virulence.

The cultivation of Pasteurella pestis in chemically defined media has been investigated by Rao (1939, 1940), Berkman (1942), Doudoroff (1943), Rockenmacher, et al. (1952), and Hills and Spurr (1952). These studies have shown that at a temperature of 27 C the minimal growth requirements of this species are satisfied by media containing relatively few amino acids and no added accessory growth factors. Although there have been some disagreements as to which amino acids were to be designated as essential, it has been established generally that phenylalanine, cysteine, and methionine are required for most strains of P. pestis. However, the requirement for cysteine could be satisfied by inorganic sulfur compounds such as sulfite, thiosulfate, etc., (Englesberg, 1952). The highest yields obtained in synthetic media, heretofore, have been of the order of 1 billion cells per ml. Because much higher yields (60 to 100 billion cells per ml) have been obtained in a complex medium (Higuchi and Carlin, 1957), it was believed that yields in synthetic media could be greatly improved. The present report deals with the development of a chemically defined medium which is capable of supporting growth of a number of strains of P. pestis to yield viable cell populations in the range of 40 to 60 billion cells per ml.

Studies on the nutrition and physiology of Pasteurella pestis. II. A defined medium for the growth of Pasteurella pestis.

A cryptococcal meningitis model in corticosteroid-treated rabbits was used to assess the requirement for the phosphoribosylaminoimidazole gene (ADE2) for virulence of Cryptococcus neoformans. A wild-type strain (H99), an ade2 auxotroph of H99 (M001), and a randomly selected prototrophic transformant of M001 (M001.1c) which had received the cloned ADE2 cDNA copy were inoculated intrathecally into immunosuppressed rabbits. While M001 was avirulent in the central nervous system model, virulence was completely restored to wild-type pathogenicity in the prototrophic transformant. This study identifies the pathogenic importance of an endogenous adenine pathway in this yeast and confirms that purine biosynthesis is a potential target for antifungal therapy. It also demonstrates that the virulence of C. neoformans can be molecularly changed and detected within a clinically relevant animal model.

https://iai.asm.org/content/iai/61/10/4446.full.pdf

The gene encoding phosphoribosylaminoimidazole carboxylase (ADE2) is essential for growth of Cryptococcus neoformans in cerebrospinal fluid.

The host as a growth medium.

The basis of virulence in Pasteurella pestis: attempts to induce mutation from avirulence to virulence.

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The basis of virulence in Pasteurella pestis: attempts to induce mutation from avirulence to virulence.