Enduracidin induced accumulation of cell wall precursors(UDP-GlcNAc-lactyl-L-Ala.-D-Glu.-L-Lys. -D-Ala.-D-Ala.) in the growing cells of Staphylococcus aureus 209 P. Studies on enduracidin with 14C-L-glutamic acid indicate that the primary effect of this antibiotic is the inhibition of cell wall synthesis with mucopeptide.
The unique staphylococci, the E46 and E97 strains described in the preceding papers, were examined at cellular level, for the mechanism of high virulence for mice. An in vitro test with cultured mammalian cells was also studied to determine whether it was available as a criterion for the grading of virulence of these strains in comparison with the typical strains of Staphylococcus aureus, E111 and B40, and of S. epidermidis, E241 and B217. The cystopathic effects of all these living organisms, their culture filtrates, and nuclease were investigated when these materials had been inoculated onto the monolayers of HeLa and FL cells cultured in the YLE medium supplemented with 5% calf serum. The results obtained were as follows. 1) In the case of living organisms, cultured cells were invaded most vigorously by the S. aureus strains, moderately by the unique strains, and very slightly by the S. epidermidis strains. The consequent appearance of cystopathic effect was in parallel with the above grading of invasivess among those strains. 2) The cytopathic effect of the culture filtrate was also the most conspicuous in the S. aureus strains. The unique staphylococci, however, came next with a small difference. It was noted for them to cause an obvious karyolysis in host cells. The S. epidermidis strains gave rise to the slightest effect. 3) The susceptibility of the cultured cells to cytopathic effect due to the culture filtrate was proved considerably stronger in HeLa cells than in FL cells. Such a difference, however, was not recognized in the invasiveness of organisms into their host cells. 4) Addition of the nuclease isolated from the B40 strain to HeLa cells resulted in a conspicuous karyolysis in these host cells. The results of the cell culture method mentioned above were not identical with those of the virulence test by intraperitoneal inoculation of mice reported in the preceding paper. Thus it may be concluded that such test in vitro by cell culture is not satisfactory for the determination of the grading of virulence of the unique staphylococci, but that it would be available, to some extent, for checking the cytopathic effect of the extracellular metabolites, mainly nuclease.
An attempt was made to obtain an aid for the identification of pathogenic staphylococci by using the routine agar plate method to check their lysozyme activity. This paper deals with basic conditions for the determination of the enzyme activity. The determination was made by the turbidity and the agar gel diffusion methods with the culture filtrate of the Smith strain of Staphylococcus aureus as enzyme solution and with acetone-dried cells of the ATCC 4698 strain of Micrococcus lysodeikticus as substrate. The following results were obtained. 1) In the turbidity method, it was demonstrated that the culture filtrate of the Smith strain after cultivation with shaking at 37°C for 24 hours possessed a lysozyme activity, and that the acetone-dried cells of the M. lysodeikticus strain were appropriate as substrate. The optimum pH was proved to be 6.5 in this system. 2) The enzyme activity was activated, to some extent, by sodium chloride in the range of 0.05-0.1M, but was completely inhibited at a molarity exceeding 0.3M. The enzymatic action was inactivated by heating at 100°C for 5 minutes or at 60-80°C for 10 minutes, so long as a culture filtrate of pH 7.8 was employed. 3) The enzyme activity was parallel to the grade of microbial growth, reaching its highest level at 18 hours of incubation. Thereafter it remained at the same level up to 72 hours of incubation. 4) Such lysozyme activity was also demonstrated by the agar gel diffusion method in a similar fashion.