Selective Diagnostic Medium for Pathogenic Listeria spp.

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1983, P. 1432-1433 0095-1137/83/121432-02$02.00/0 Copyright © 1983, American Society for Microbiology

Vol. 18, No.6

Selective Diagnostic Medium for Pathogenic Listeria spp. BORIS SKALKA* AND JIRI SMOLA Department of Epizootiology and Microbiology, School of Veterinary Medicine, 612 42 Brno, Czechoslovakia

Received 27 July 1983/Accepted 12 September 1983

Pathogenic Listeria serovars produced complete hemolysis on agars containing 5% rabbit erythrocytes and 10 ,ug of acriflavin, 40 ,ug of nalidixic acid, and 7.5 activity units of equi factor per ml. Apathogenic Listeria innocua was nonhemolytic on this medium. The pathogenicity of Listeria spp. is closely CM331, brain heart infusion CM225, and brain associated with hemolytic activity, but most heart infusion agar CM375 (Oxoid Ltd.) were Listeria monocytogenes strains display a very used. All media and saline were prepared with weak, if any, hemolytic effect on sheep erythro- 2.5 mg of MgSO4 per ml. The hemolytic effects of strains of Listeria cytes (6, 8). A typical hemolysis is produced by strains of serovar 5, proposed as a new species, spp. observed on solid media containing 5% Listeria ivanovii (5). Based upon direct hemoly- (vol/vol) washed rabbit erythrocytes corresis on sheep blood agar and synergistic hemolyt- sponded to earlier descriptions (10). When rabbit blood agars were supplemented ic action with equi factor from Corynebacterium (Rhodococcus) equi, strains of Listeria spp. can with 10 ,ug of acriflavin and 40 jg of nalidixic be divided in four hemolytic groups (8). Subse- acid per ml, both L. innocua and many colonies quent assays have illustrated the hemolytic ac- of L. monocytogenes hemolytic group m1 were tion of Listeria spp. and the synergism with equi nonhemolytic. The direct hemolytic effect of the factor in media with man, horse, and rabbit m2 strains was evidently reduced, and the hemoerythrocytes (7, 9, 10). Rabbit erythrocytes are lytic zone of L. ivanovii strains was also smaller the most sensitive to the hemolytic activity of (Fig. 1). The addition of equi factor to the medium Listeria; nevertheless, the phenomenon of hemolysis is observed with the apathogenic spe- containing washed rabbit erythrocytes and baccies Listeria innocua (10), which has been de- terial inhibitors resulted in a positive hemolytic scribed as nonhemolytic (3, 4). Unlike the positive reaction with all strains of pathogenic Listeria spp., the hemolytic effect of L. innocua on rabbit blood is not enhanced by the equi factor (7-10). Sheep blood agars supplemented with equi factor are useful for in vitro differentiation of the pathogenic Listeria spp.; however, the addition of substances recommended for selective cultivation of Listeria spp. (2) suppress the hemolytic synergism, especially of L. monocytogenes strains (8). The aim of our study was to prepare a selective diagnostic medium for pathogenic Listeria. The trials were carried out with 69 strains of L. innocua, 136 strains of L. monocytogenes belonging to different serovars with properties of either the m1 or m2 hemolytic group, and 6 strains of L. ivanovii (or serovar 5). A detailed description of the strains has been presented (710). Equi factor was prepared from C. equi NCTC 1621. The techniques for preparing and assaying FIG. 1. R-I, Agar with washed rabbit erythrocytes, the activity were those described previously (7- acriflavin, and nalidixic acid. Growing on quadrants 9). are colonies of: i, L. innocua; ml and M2, hemolytic Nutrient agar CM3, Columbia agar base groups of L. monocytogenes, and 5, L. ivanovii. 1432

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reactions on sheep, human, equine, and particularly rabbit blood agars (8-10). The synergism of Listeria hemolysin(s) and equi factor on agar containing rabbit erythrocytes and antimicrobial agents offers a selective diagnostic medium for pathogenic species of the genus Listeria.

FIG. 2. REI, The same medium as that shown in Fig. 1., but supplemented with equi factor. Cultivated strains are identified as described in the legend to Fig. 1.

effect by pathogenic Listeria spp. The apathogenic species, L. innocua, remained nonhemolytic (Fig. 2). The optimum amount of equi factor was 7.5 activity units per ml of medium, although the described results could be observed with amounts ranging from 3 to 50 activity units per ml. There are divergent opinions on the hemolytic synergism of pathogenic Listeria species and C. equi. Originally, it was recommended for differentiation between L. monocytogenes and Erysipelothrix rhusiopathiae (1). Previous results (8, 9) call attention to its availability for differentiating within the genus Listeria. A team of authors stated that the C. equi exosubstance exerted synergism merely with the hemolysin of L. ivanovii (5), and they suggested use of this synergism to discern this new species from L. monocytogenes. Our experience indicates that in the presence of equi factor, both pathogenic species of Listeria exhibit hemolytic

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