BACTERICIDAL AND BACTERIOLYTIC ACTIVITY OF USTERIA

Skalka, B.! Bactericidal and Bacteriolytic ActiTJity of Listeria. Acta vet. Bmo, 61, 1992: 23-28. Sirains of all known species of the genus Listeria exerted bactericidal and bacteriolytic effects on Micrococcus luteus and bacteriolytic effects on Micrococcus lylae. The bactericidal effect was tested on living cells and the bacteriolytic effect on heat-killed cells of micrococ:cal strains. The sample of 141 listeria strains examined comprised 80 L. monocytogenes strains, 10 L. ifJantJfJii strains, 45 L. innocua strains, 2 strains each of L. seeligeri and L. gray; and 1 strain each of L. welshimeri and L. murray;. No effect was observed on the other micrococcal species, namely M. roseus, M. kristinae, M. sedentarius, M. TJarians and M. nishinomiyaensis. The bactericidal and bacteriolytic activity observed iIi the study can be included in the characteristic properties of the genus Listeria. Listeria spp., Micrococcus spp., bactericidal actiTJity, bacteriolytic activity The genus Listeria now comprises seven species constituting two groups. The first of them comprises Listeria monocytogenes and Listeria ifJanovii, both of them pathogenic for man and animals, and Listeria innocua, Listeria seeligeri and Listeria welshimeri. The second group comprises Listeria grayi and Listeria murray; (Rocourt 1988). This recent classification has introduced a change in that L. gray; and L. murray; are no longer classified as a separate genus Murraya as suggested previously (Stuart and Welshimer 1974) and represents also a change as compared with the recent edition of Bergey's Manual of Systematic Bacteriology (Sneath et al. 1986) where the two species were classified within the ·genus Listeria only as "species incertae sedis" (Seeliger and Jon es 1986). Moreover, Listeria denitrijicans has been transferred to a new genus Jonesia as J. denitrijicons (Rocourt et al. 1987). The characteristics of listeria reportedly include the capacity of some strains to produce substances having the properties ofbacteriocins. These substances were named "monocins" (Sword and Pickett 1961) or "listeriocins" (Tubylewicz 1963) and exerted their effects primarily on L. monocytogenes strains. More recently however, these substances have been found to be defective bacteriophages of listeria (Bradley and Devar 1966; OrteI1989). Mollerach et al. (1988) obtained a L. innocua strain producing an exosubstance referred to as "linnocuicin 819" which showed both bactericidal and bacteriolytic activity. This exosubstance, however, exerted bactericidal and bacteriolytic effects only on one Listeria strain, namely L. ifJanovii 818. Moreover, it had a weak growth-inhibiting effect on Micrococcus luteus strain ATCC 9 341, one L. welshimsri strain 1Uld one L. denitrijicans strain. Another isolated observation was the production of lysozyme by some L. monocytogenes strains, virulent for white mice (Seeliger and Jones 1986). This enzyme is produced in varying intensity by some staphylococci (Varaldo et al. 1980; Llimmler 1989) and exerts its effects primarily on Micrococcus lureus strains (Kocur 1986). The present study is concerned in detail. with a phenomenon described in our previous studies (Skalka 1986, 1988). Materials and Methods Media Brain Iieart Infusion Agar CM 375 (Oxoid Ltd.) and Blood Agar Base No.4 (Imuna) were used.

The characteristics of listeria reportedly include the capacity of some strains to produce substances having the properties ofbacteriocins.These substances were named "monocins" (Sword and Pickett 1961) or "listeriocins" (Tubylewicz 1963) and exerted their effects primarily on L. monocytogenes strains.More recently however, these substances have been found to be defective bacteriophages of listeria (Bradley and Devar 1966;OrteI1989).Mollerach et al. (1988) obtained a L. innocua strain producing an exosubstance referred to as "linnocuicin 819" which showed both bactericidal and bacteriolytic activity.This exosubstance, however, exerted bactericidal and bacteriolytic effects only on one Listeria strain, namely L. ifJanovii 818.Moreover, it had a weak growth-inhibiting effect on Micrococcus luteus strain ATCC 9 341, one L. welshimsri strain 1Uld one L. denitrijicans strain.Another isolated observation was the production of lysozyme by some L. monocytogenes strains, virulent for white mice (Seeliger and Jones 1986).This enzyme is produced in varying intensity by some staphylococci (Varaldo et al. 1980;Llimmler 1989) and exerts its effects primarily on Micrococcus lureus strains (Kocur 1986).
The present study is concerned in detail.with a phenomenon described in our previous studies (Skalka 1986(Skalka , 1988)).

Strains Examined
The sam;>le of strains examined com?rised 141 listeria strains, nam'!ly 80 L. monocytogenes strains inch:ding CCM 5576, CC\i 5879, CCM 5577, CCM 5578, CCM5 .The CCM strains were obtained from the Czechoslovak Collection of Microorganisms, Bmo. and the Li, Mau and M strains.from the Czechoslovak National Colection of Type Cultures (CNCTC;, Prague.The remaining strains were isolated in the author's laboratory.

Demonstration of Bactericidal Activity
A suspension of each micrococcal strain was prepared in saline in a• density corresponding to the 1st degree of McFarland's turbidity standard.The suspensions were added in 0.1 ml volumes to 10 ml volumes of melted agar medium cooled to 48°C.After mixing.the media with the incorporated suspensions were poured into Petri dishes and allowed to solidify.Mterwards the listeria strains to be examined and the staphylococci used as controls were cultured on the surface ot the media usiQl~ the .,spot-on-Iawn'•technique.The results were evaluated alter 24-h incubation at 37°C.

Demonstration of Bacteriolytic Activity
A suspension of each micrococcal strain was prepared in saline in a density corresponding to the 4th degree of McFarland's turbidity standard.Prior to use the suspensions were autoclaved at 121°C for 20 minutes.The devitalized suspensions were added in 1 ml volumes to 9 ml volumes of melt~d agar medium.After mixing they were poured into Petri dishes.The subsequent procedure and evaluation of the results were as described above.

Results'
All the listeria strains examined exerted bactericidal effects on the strains of M. luteus.On the media into which suspensions of living M. luteus strains were incorporated an entirely transparent zone was observed round the listeria cultivation "spots".The width of the zone depended on the potency of the respective listeria strain rather than on its species.Nevertheless, this activity showed least • fluctuation and was almost equally intense in L. monocytogenes, L. ivanovii, L.
• grayi and L. murrayi strains and was least pronounced in L. seeligeri strains.A remarkably intense activity was shown by L. innocua strain bearing the laboratory designation Lin 23 which exerted bactericidal effects also on M. lylae, M. roseus, M. kristinae and M. varians.Of the 3 staphylococci used as controls S. hyicus had marked bactericidal effects on .all the micrococcal strains, whereas the other two staphylococci showed a negative activity in this respect (Fig. 1).
All the listeria strains showed bacteriolytic effects on the media with incorporated heat-killed suspensions of the cells of M. luteus and M. lylae strains and did not exert this effect on the remaining indicator micrococci.The manifesta-

Listeria
No.
-   tion of positive bacteriolysis was a zone round the cultivation "spots" generally less transparent than that observed upon the use of the indicator living cells.
The bacteriolytic zone in one and the same strain corresponded in size to the bactericidal zone in some cases and was larger or smaller than that in some others.Among the staphylococcal controls bacteriolytic effects on M. luteus and.M. lylae were exerted by S. aureus Mau 87/79 and S. hyicus M 51/88., the bacteriolytic zone shown by the latter being smaller in size than its bactericidal effect.S. simulans biovar staphylolyticus had no effects on any of the indicator micrococcal strains (Fig. 2).The results are summarized in Table 1.

Discussion
The effect of lysozyme, particularly of that produced by staphylococci., on • some micrococcal species, is a well-established fact (Satta et al. 1978;Kocur 1986).This enzyme., however., is not produced by all staphylococcal species (Uimmler 1989) and differences in its production may exist even within one and the same species (Kloos and Schleifer 1986).Therefore our choice of control staphylococcal strains was made with respect to the data reported by other investigators (Schindler and Schuhardt 1964;Satta et al. 1978., Varaldo et al. 1980and Uimmler 1989) as well as in the light of our previous experience (Skalka 1986(Skalka , 1988)).
The fact that• strains of all species of the genus Listeria exert bactericidal and bacteriolytic effects on both living and devitalized M. luteus strains and bacteriolytic effects on devitalized M. lylae strains has not been described before.
The differences observed in the intensity of the positive effects can be accounted for by both the individual capacity of the strains and by the fact that the inocula of the examined strain were not invariably of the same size.Although in L. seeligeri, L. welshimeri, L. gray;.and L. murrayi only one or two strains were tested, it seems reasonable to assume in view of the number of the other listeria species examined in our study that the bactericidal and bacteriolytic activity reported here can be generalized so as to cover the whole genus Listeria.Our results exclude the possibility that the activities observed may be in association with the pathogenity or virulence of listeria.The question whether the effects observed are caused by one or more substances awaits further investigation.
The present knowledge based on our further, hitherto unpublished, results allows us to assume that the antagonism observed is caused by a substance very similar to lysozyme or possibly to S. hyicus bacteriolysine (Lammler 1989).An. exception in this respect is L. innocua strain Lin 23 producing bactericin with a wide-spectrum.effect.This strain the activity of which surpasses that of the other listeria as well as that reported for "linnocuicin" (Mollerach et al. 1988) will be the subject of our further study.
The results reported here differ from the descriptions by other writers (Sword and Pickett 1961;Tubylewicz 1963;Mollerach et al. 1988).Most of these studies were conducted at the time that the present classification of listeria did not exist.
The control strains used in our study corresponded in their effects to the data published previously (Varaldo et al. 1980;Kocur 1986;Skalka 1986Skalka , 1988)).The observation of Lammler (1989) who reported the effects of S. hyicus on dead M. luteus cells was extended by our finding of the effects on dead M. lylae cells and on living cells of M. ltiteus and other micrococcal species.
In the light of the present results we suggest that the bacteriolytic and bactericidal activity of listeria described should be included in the characteristic properties of the genus Listeria.

Table 1
Effects of List ... ia spp.and control strains on Micrococcus spp.