Effect of erythritol on the in vitro growth and respiration of Listeria monocytogenes.

In ruminants, such as cattle, sheep, and goats, common forms of listeriosis are abortion, stillbirth, or postnatal death of the young infected animal (M. L. Gray et al., Am. J. Vet. Res. 17: 510, 1956; J. W. Osebold et al., J. Am. Vet. Med. Assoc. 137:221, 1960). By experimental oral exposure of pregnant sheep and goats, M. L. Gray et al. have shown the reproductive system of these animals, especially the gravid uterus and its contents, to be highly susceptible to invasion by Listeria monocytogenes. In terms of tissue affinity, L. monocytogenes thus resembles those Brucella species which cause contagious abortion in cattle, sheep, and goats. Erythritol is localized in those ruminant tissues (placenta, chorion, fetal fluids) which are most susceptible to the Brucella, and the course of brucellosis in these animals may be explained by the presence of erythritol in the susceptible tissues (H. Smith et al., Nature 193:47, 1962; J. Keppie et al., Brit. J. Exptl. Pathol. 46:104, 1965). N. B. McCullough and G. A. Beal (J. Infect. Diseases 89:266, 1951) found that erythritol was the most effective of nine carbohydrates tested as sole carbon sources for B. abortus, B. suis, and B. melitensis in a basal medium. Recently, it has been demonstrated that B. abortus preferentially metabolizes erythritol as a carbon and energy source even in the presence of high glucose concentrations (J. D. Anderson and H. Smith, J. Gen. Microbiol. 38:109, 1965). The tendency for both Brucella and Listeria to localize in the placenta and associated tissues of pregnant ruminants, and the correlation between the presence of erythritol and the localization of the Brucella in these tissues, led us to determine whether L. monocytogenes attacks erythritol in vitro. Seven strains of L. monocytogenes were used in all experiments. The designations of these strains, with their serotypes in parentheses, and their sources are as follows: strains 7973 (1), 5348 (2), 5105 (3), 5214 (4a), and 1071 (4b) from J. H. Schubert, Communicable Disease Center, Atlanta, Ga.; strains A4413 (4b) and 9037-7 (serotype unknown) from M. E. Friedman, Fort Detrick, Frederick, Md. Stock cultures were maintained on tryptose agar slants at 4 C and were transferred every 4 months. Three types of experiments were carried out. These tested the ability of L. monocytogenes to (i) grow with erythritol as the sole carbohydrate, (ii) grow with erythritol and glucose as carbohydrates, and (iii) consume oxygen in the presence of erythritol. All seven strains of L. monocytogenes tested grew well in Trypticase broth only if it was supplemented with glucose or other utilizable carbohydrate. An observable turbidity increase in this broth in response to added erythritol would, therefore, suggest the utilization of this substance. Experiments were performed with autoclaved phosphate-buffered Trypticase (2%) broth at pH 7.2 as the basic medium. Solutions of glucose and erythritol were rendered bacteria-free by passage through a membrane ifiter (0.45 ,u; Millipore Filter Corp., Bedford, Mass.). Bacteria were grown in, and the turbidity was measured from, 13-mm cuvettes which contained a total volume of 3.0 ml after inoculation. Eight cuvettes were used for each strain: a control (Trypticase only), a glucose control (Trypticase plus 28 mm glucose), and six cuvettes containing Trypticase plus erythritol in concentrations of 0.2, 0.8, 3.1, 12.5, 50, and 200 mm. Each of the eight cuvettes was inoculated with approximately 107 cells (0.1 ml), grown in 2% Trypticase plus 28 mm glucose, and washed once by centrifugation in 2% Trypticase. Incubation at 37 C was stationary, except for mixing on a Vortex mixer just before each turbidity reading at 620 m,u. The growth response of strain A4413 is shown in Fig. 1; the other six strains gave similar responses. None of the seven strains showed a detectable increase in turbidity in Trypticase plus erythritol. All of these seven strains, however, demonstrated a marked turbidity increase in Trypticase plus glucose, reaching a maximum in 8 to 12 hr, at which time the pH was 6.0 to 6.1. A marked decrease in turbidity occurred during the next 12 hr, which might have been the result of death and lysis of the Listeria in the acid medium; H. Seeliger and G. Linzenmeier (Z. Hyg. Infektionskrankh. 136:335, 1953) noted that these