ANTIBACTERIAL EFFECTS OF STAPHYLOCOCCUS HYICUS AND STAPHYLOCOCCUS CHROMOGENES

Skalka B.: Antibacterial Effects of Staphylococcus hyicus and Staphylococcus chromogenes. Acta vet. Bmo, 62, 1993: 39-47. Antibacterial effects of Staphylococcus hyicus (40 strains) and Staphylococcus chromo genes (20 strains) were tested on living and heat-devitalized cells of micrococcal, staphylococcal, streptococcal and corynebacterial indicator strains. All S. hyicus and S. chromo genes strains inhibited the growth of Micrococcus sedentarius and lysed devitalized cells of Micrococcus lylae but did not affect dead cells of M. sedentarius and only few of them inhibited the growth of M. lylae. Only a small number of S. hyicus strains produced intense effects on living indicators and had only very weak effects on their dead cells. The activity of some S. chromogenes strains, observed invariably on living indicators, did not reach the intensity recorded for active S. hyicus strains. Most S. hyicus and S. chromo genes strains were inactive towards the indicators. It is concluded that the antibacterial activity observed in the study should be ascribed to the characteristics of the individual strains and not to the general characteristics of S. hyicus and S. chromo genes species. Staphylococcus hyicus, Staphylococcus chromo genes, antibacterial effects, staphylococcal, micrococcal, streptococcal and corynebacterial indicator strains Staphylococcal exosubstances showing antibacterial activity are represented both by lytic enzymes, lysozyme (Welsch 1959) affecting somemicrococci,lysostaphin (Schindler and Schuhardt 1964) affecting staphylococci, and by substances having bactericidal or bacteriostatic effects, staphylococcins (Fredericq 1946), bacteriocin-like substances (Ivanovics 1962), micrococcins (Su 1948) and antibiotics of low molecular weight (Halbert et a1. 1953). Recently added to the afore-mentioned substances have been exosubstances of the species Staphylococcus hyicus nad Staphylococcus chromo genes. The first report of the antibacterial activity of S. hyicus and S. chromo genes (Balusek and Hajek 1985) described bactericidal and bacteriolytic activity observed in some strains of these two species. The lytic effects in particular then stimulated the interest of further investigators. Miillerand Blobel (1987) reported that a S. hyicus strain produced a bacteriolytic enzyme whose highly purified fractions, LE IlIa and LE IIlb, exerted intense effects on devitalized cells of some streptococci, staphylococci and micrococci. Subsequent studies not only confirm'rd the lytic effects of LE IlIb but also recommended the utilization of this lytic enzyme for extraction of both group and type specific streptococcal antigens (Frede et a1. 1989; Lammler and Wibawan 1990). It was suggested that the production of the lytic enzyme should be regarded as a constant trait of S. hyicus suitable for routine identification of this species (Lammler 1989, 1991a). The production was also described in S. chromogenes and its antigenic identity with the lytic enzyme of S. hyicus was demonstrated (Lammler 1991b). The present study was designed to test the antibacterial effects of S. hyicus and S. chrom()genes on micrococcal, staphylococcal, streptococcal and corynebacterial species with reference to possible practical utilization of these effects. Materials and Methods Culture Media , Brain'Heart Infusion Agar CM 375 (Oxoid Ltd.) and Blood Agar Base No.4 (Imuna) were used. '

Staphylococcal exosubstances showing antibacterial activity are represented both by lytic enzymes, lysozyme (Welsch 1959) affecting somemicrococci,lysostaphin (Schindler and Schuhardt 1964) affecting staphylococci, and by substances having bactericidal or bacteriostatic effects, staphylococcins (Fredericq 1946), bacteriocin-like substances (Ivanovics 1962), micrococcins (Su 1948) and antibiotics of low molecular weight (Halbert et a1. 1953).Recently added to the afore-mentioned substances have been exosubstances of the species Staphylococcus hyicus nad Staphylococcus chromo genes.The first report of the antibacterial activity of S. hyicus and S. chromo genes (Balusek and Hajek 1985) described bactericidal and bacteriolytic activity observed in some strains of these two species.The lytic effects in particular then stimulated the interest of further investigators.Miillerand Blobel (1987) reported that a S. hyicus strain produced a bacteriolytic enzyme whose highly purified fractions, LE IlIa and LE IIlb, exerted intense effects on devitalized cells of some streptococci, staphylococci and micrococci.Subsequent studies not only confirm'rd the lytic effects of LE IlIb but also recommended the utilization of this lytic enzyme for extraction of both group and type specific streptococcal antigens (Frede et a1. 1989;Lammler and Wibawan 1990).It was suggested that the production of the lytic enzyme should be regarded as a constant trait of S. hyicus suitable for routine identification of this species (Lammler 1989(Lammler , 1991a)).The production was also described in S. chromogenes and its antigenic identity with the lytic enzyme of S. hyicus was demonstrated (Lammler 1991b).
The present study was designed to test the antibacterial effects of S. hyicus and S. chrom()genes on micrococcal, staphylococcal, streptococcal and corynebacterial species with reference to possible practical utilization of these effects.(Sourek 1990).Strain designation given in the brackets refers to the respective working codes.

Demonstration of Antibacterial Activity
For demonstration of antibacterial activity the simultaneus technique of cultivation of indicator strains and strains examined or control strains was used.The indicator strains were incorporated into melted agar medium.For demonstration bacteriocin effects living cells of indicator strains were used, for demonstration of bacteriolytic activity use was made of heat-devitalized cells.The technique was described in detail previously (Skalka et aI. 1983;Skalka 1992).

Results
The active strains showed different intensity of effects on the indicator strains.An inhibition zone greater than 5 mm in diameter (observed in S. hyicus and in some control active strains) was scored as very intense.An inhibition zone of 2 to 5 mm in diameter was scored as intense and that smaller than 2 mm in diameter was recorded as weak.Where no clearly defined zone was observed, the effect was scored as negative.This scoring system was used in evaluating the effects observed on both living and devitalized cells of the indicator strains.The inhibition zones were generally defined more clearly on living than on dead cells of the indicators as can be seen in Fig. 1    results obtained in this series are summarized in Table 1 which, however, does ,not include the differences between the individual active and indicator strains •observed within the individual groups of intensity of the effects.The activity •of S. hyicus strains on living and devitalized M. luteus strains was the same; .only 35% of the strains were inactive.S. chromo genes strains showed less intense •effects on M.luteus: 45% and 55% of the strains produced no effects on M. luteus .livingand devitalized cells, respectively.All S. hyicus and S. chromo genes strains ,produced effects on M. sedentarius living cells but were entirely inactive towards devitalized cells of this species.An opposite effect was recorded for M. lylae: ,all S. hyicus and S. chromo genes strains affected its dead cells, whereas only 15% •of S. hyicus strains and 20% of S. chromo genes strains exerted their effects on living M. lylae cells.Both S. hyicus and S. chromo genes had very weak effects on M. roseus, M. varians, M. kristinae and M. nishinomiyaensis; nevertheless, the effects •<on living cells of these micrococcal species were invariably slighdy higher.
On the basis of the results obtained in the first series of our experiments the strains included in the second series were as follows: 7 S. hyicus strains, 3 S. chromogenes strains and 1 strain each of the micrococcal species.The active set was extended by including strains UT0007, KPJ III and K-6-WI that are known as 'producers of antibacterial exosubstances.The set of indicator strains was extended by including 5 S. aureus strains, 2 S. intermedius strains, 1 S. haemolyticus strain and 4 strains each of S. agalactiae, S. uberis, S. equi, C. pseudotuberculosis, A. pyo-,.genes,R. equi and 2 C. renale strains.The sensitivity of the indicators was tested using their living and devitalized cells.S. hyicus strain 664/86 produced effects on living cells of all staphylococcal, streptococcal and corynebacterial indicators, but in tests with their devitalized cells it was inactive except a weak effect on streptococci.A similar pattern of activity with less intense effects was recorded for strain 174/87.An antibacterial .activity on S. aureus strains of a higher intensity of effects than that shown by strain 664/86 was recorded for S. hyicus strain 695/87 which, as the only one of the S. hyicus and S. chromo genes strains, affected also S. aureus devitalized strains.
However, this strain was inactive on streptococcal and corynebacterial indicators.
Of the other S. hyicus strains only strain 173/87 exerted weak effects on living -cells of some staphylococcal flD.d corynebacterial indicators.The remaining S. hyicus strains were entirely inactive on staphylococcal, streptococcal and corynebacterial indicators.Of S. chromo genes strains only strain 709/87 affected living cells of some staphylococcal, streptococcal and corynebacterial indicators.
The other two strains of this species were inactive towards these indicators.
Among the active strains used as controls strain KPJ III showed the widest "Spectrum and a high intensity of effects, but only on living cells.Strain UTOOO7 affected only living cells of S. aureus, C. pseudotuberculosis, C. renale and A. pyogenes.Strain K-6-WI exerted intense effects on both living and devitalized cells of staphylococci but was inactive on the remaining indicators.
The results of the second series of experiments are presented in Table 2.

Discussion
Apart from the report by Balusek and Hajek (1985) the attention of the relevant studies was centred mainly on lytic effects of S. hyicus.Therefore our •study covered also those effects of S. hyicus and S, chrtimogenes that have received little attention so far, namely their effects on living indicator cells in continuation of our previous observatios (Skalka 1988), together with investigation of their effects on devitalized cells of indicator strains.The spectrum of indicators included in our study was wider than that described by other writers dealing with antibacterial effects of the two species.For the sake of comparison we also used produc-•ers of known staphylococcal exosubstances, namely bacteriocin BacRv staphylococcin having a wide-spectrum effect, and lysostaphin. .In the first series of our experiments conducted with micrococci it became clear that the activity of the two species under study as well as the sensitivity of the indicators varied from strain to strain and this observation was then confirmed in the second series of our experiments.The differences in effects were both quantitative (expressed in terms of their intensity) and qualitative (expressed as either positive or negative effect).In comparing our data with.thosereported by •other writers consideration should be given to the fact that we tested different strains and used different indicator strains, the only exception being M. luteus .strainCCM 169 which was used by Frede et al. (1989) and Lammler (1989Lammler ( , 1991aLammler ( , 1991 b) b).Some influence on the differences of the results may also be ascribed to the culture medium; the possible role of culture medium in this respect was pointed out by Varaldo et al. (1978aVaraldo et al. ( , 1978b) ) in their studies oflyso-  zyme activity of staphylococci.In our experiments the effects were substantially less pronounced on Blood Agar Base No.4 medium than on Brain Heart Infusion Agar which we then used consistently and which was also used by Balusek and Hajek (1985).Other writers preferred the use of Tryptone Soya Agar.

• •
In the first series of our experiments unequivocal results were obtained only • for M. sedentarius strains the living cells of which were affected by all S. hyicus and S. chromo genes strains which, however, were inactive on devitalized cells of this species.A:t;I.opposite, though not so unequivocal, effect was recorded for M. lylae the dead cells of which were lysed by all S. hyicus and S. chromo genes strains, whereas its living cells were affected by only a few strains of the two species.Differences in activity were found on both living and dead cells of M. luteus and not even strain CCM 169 was affected by all S. hyicus and S. chromo genes strains.This fact represents the greatest difference of our results from those reported by Lammler (1989Lammler ( , 1991 a) a).
The differences in antibacterial activity between the individual S. hyicus and S. chromo genes strains were even more pronounced in the second series of our experiments.It became apparent that within each of the two species there exist strains that can affect particularly living cells of micrococci, staphylococci, streptococci and corynebacteria along with strains lacking this characteristic or strains markedly active towards both living and dead cells of S. aureus.These observations  confirmed and extended the data reported by Balusek and Hajek (1985).The differences between our results with devitalized cells of streptococci and the descriptions reported by other writers (Mi.iller and Blobel 1987;Frede et a!. 1989;Lammler and Wibawan 1990) can be accounted for by the different form of active exosubstance.The afore-metioned writers used the purified fraction LE IIIb, which is a procedure more demanding in terms of technique and cost than the use of the substance produced by the growing strain "in situ" in our study.The effects of strains UT0007 and K-6-WI did not differ from those described previously (Schindler and Schuhardt 1964;Skalka et a1. 1983).Strain KPJ III showed the widest spectrum and the highest intensity of effects but affected only living cells of the indicator strains.Its antibacterial effects on living cells is comparable to those of strains 664/86 and 174/87 but differs from them by its inactivity on devitalized cells.
Our finding of the lack of uniform action of S. hyicus and S. chromo genes strains on living and dead cells of M. luteus prevents us from agreeing with the recommendation advanced by Uimmler (1989Uimmler ( , 1991 a) a).On the other hand, the uniform antibacterial effects of all S. hyicus and S. chromo genes strains on living M. sedentarius and dead M.lylae cells could be used in routine diagnostic work.However, tests less demanding in terms of technique and cost are available by which S. hyi-cus and S. chromo genes can be diagnosed (Skalka 1991a~ 1991b.Nevertheless, • studies of the antibacterial effects are of practical value for detailed characteristics of isolated S. hyicus and S. chromogenes strains~ particularly for epizootiological purposes.

Fig. 2 .
Fig. 2. Effects of the same strains as in Fig. 1 on devitalized cells of indicator M. luteus strain.• MIu (D) suspended in agar medium.
The activity of S. hyicus and S. chromo genes strains corresponded to the afore-.goingobservations.Of the active controls, UT0007 produced effects on living -cells of M .. luteus, M. roseus and M. sedentarius and on devitalized cells of M.luteus .311dM. lylae; in the remaining tests with micrococci it was inactive.KPJ III on living cells of M. luteus, M. lylae, M. roseus and M. kristinae but had no effect on living cells of the remaining three micrococcal "Species and on dead cells of the micrococcal indicators.K-6-WI had only weak effects on living cells of M . .sedentarius and on devitalized cells of M. luteus and M. lylae; in the remaining tests with micrococci it was inactive.
designation of the strains in the author's laboratory collection; L = living cells of indicator strains; D = dead cells of indicator strains.
Culture Media , Brain'Heart Infusion Agar CM 375 (Oxoid Ltd.) and Blood Agar Base No.4 (Imuna) were used.' Bacterial Strains • Strains Tested for Antibacterial Activity The set of strains examined comprised 40 strains of Staphylococcus hyicus and 20 strains of