Mechanisms of pathogenesis in Listeria monocytogenes infection. I. Influence of iron. J. Bacteriol. 92: 536-542. 1966.-The effects of ferric andferrous ironaswell asother cationson Listeria infection in mice were studied. Iron compounds caused a reduction in the LD5 dose of Listeria, and, when added to a synthetic medium, proved stimulatory for in vitro growth of the organism. Bacterial counts on spleen and liver tissue from irontreated mice showed that iron injections caused more rapid growth of bacteria and resulted in higher numbers of organisms in the tissue. The reticuloendothelial system did not appear to be impaired by this treatment. Immunized animals were not affected by iron treatment during challenge. Mice with experimentally induced hemolytic anemia showed increased susceptibility to listeriosis, whereas thosetreated with Desferal, a specific iron-chelating agent, appeared more resistant. Iron proved stimulatory for the avirulent strain, 9037-7, and resulted in an LD50 of 1.3 X 104 organisms in iron-treated animals. Growth of L. monocytogenes and mortality from experimental infection appeared to be correlated with availability of iron to the bacteria. The results suggest that host iron metabolism may play a part in the onset and progress of Listeria infections.

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The effects of Listeria monocytogenes hemolysin on lysosomes and phagocytic cells were investigated. Hemolysin caused release of /3-glucuronidase and acid phosphatase from suspensions of rabbit and rat lysosomes prepared from liver homogenates. The degree of lysis was proportional to the concentration of hemolysin added. There appeared to be no significant difference between the sensitivities of rat and rabbit lysosomes to disruption. Studies on the effect of pH and temperature on lytic activity suggested that hemolysin could function under conditions which might exist within phagocytic cells. Peritoneal exudates from rabbits and mice were exposed to hemolysin and observed by phase microscopy. Hemolysin possessed leucocidal activity and caused degranulation of both rabbit and mouse cells. Optimal activity against lysosomes and peritoneal exudate cells required activation of hemolysin with a reducing agent and could be prevented if hemolysin was previously incubated with cholesterol. ment, Jamaica Plains, Mass. L. monocytogenes strain A 4413, serotype 4b, was used for immunization. Cultivation. Stock cultures of L. monocytogenes were maintained on frozen tryptose agar (Difco) slants. Cultivation for production of hemolysin was in Brain Heart Infusion broth (BHI, Difco) at 37 C for 24 hr. Cultivation for immunization was in tryptose broth (Difco) at 37 C for 18 hr.

Listeria monocytogenes was examined for superoxide dismutase (SOD) activity. Two catalase-negative strains possessed at least twofold greater SOD activities than the catalase-positive L. monocytogenes strains examined. Growth conditions such as aeration and iron concentration influenced the specific activity of SOD obtained from cells cultured in defined media. L. monocytogenes SOD from crude extracts and after partial purification was analyzed by polyacrylamide gel electrophoresis. Iron was associated with the single band of SOD activity detected in the gels. SOD activity appeared to be primarily extracytoplasmic. Survival of organisms in a superoxide-generating medium was studied, with photoactivation of riboflavin used as the source of free radical formation. Virulent, catalase-positive L. monocytogenes strains were relatively resistant to killing in a pH 7 superoxidecontaining medium. An intact-cell assay for SOD was developed, which used the superoxide-generating system and employed the superoxide-dependent oxidation of sulfite, added to the medium, and inhibition of this oxidation by SOD. Maximal SOD activities of intact cells were observed when 100 to 400 ,g (dry weight) of viable Listeria cells per ml was added to the medium. A possible role for SOD in the pathogenesis of listeric infection is discussed. Facultative intracellular pathogens such as Listeria monocytogenes must possess means of overcoming the nonspecific immune responses mediated by phagocytic cells and various humoral factors. The sequence in phagocytic killing of bacteria includes the formation of a toxic superoxide (*02) free radical (5) which is later eliminated by the enzyme superoxide dismutase (SOD) (22). Indirect evidence also exists for a role of * 02 in leukocyte-mediated antimicrobial activity. Baehner et al. (6) showed that the nitroblue tetrazolium reduction by leukocytes is dependent on *°02 generated by phagocytic cell metabolism, and Curnutte et al. (11) observed that neutrophils obtained from patients with chronic granulomatous disease produced low or undetectable quantities *°2 SODs from Escherichia coli have been found to contain manganese (16) or iron (31). Yost and Fridovich (32) demonstrated that E. coli cells grown in iron-rich media are more resistant to killing by phagocytes than E. coli grown in irondeficient media. Increased resistance appeared to correlate with increased levels of iron-SOD rather than with catalase or other effects of iron.

SUMMARYThe bacteriophages of Listeria monocytogenes have been studied with respect to isolation techniques and their use as diagnostic tools and as aids in epidemiological investigations. The occurrence of lysogeny was investigated in 123 strains isolated from human and animal sources throughout the world. Conventional procedures for isolation of phage were unreliable with Listeria since lysogenic strains did not always, by spontaneous lysis, release a detectable amount of phage. However, after exposure to ultraviolet radiation, such strains were induced to produce up to lo7 plaque-forming particles/ml. Some strains which did not release phage produced substances after irradiation possibly analogous to colicines. The lytic spectrum of 11 phages against 149 strains of Listeria was studied and a system of classification, with five of these phages, was used to place 127 of these strains in 8 phage types. Nearly all of the untypable strains were rough, undergoing dissociation, or were lysogenic. Phage susceptibility appeared to be closely associated with the serological type of the strain, but showed no relation to the animal source or the geographical origin. These studies indicated that Listeria phages could be used as a means of generic identification and also as a substitute for or an adjunct to serological typing in epidemiological investigations.