The effect of leukocyte hydrolases on bacteria VIII. The combined effect of leukocyte extracts, lysozyme, enzyme “cocktails,” and penicillin on the lysis ofStaphylococcus aureus and group a streptococci in vitro

Abstract

Cultures ofStaphylococcus aureus, which are harvested from the stationary phase of growth, are extremely resistant to lysis by extracts of human blood leukocytes. Such bacteria are, however, rendered susceptible to bacteriolysis when cultivated in the presence of subinhibitory concentrations of penicillin G, nafcillin, or cloxacillin (0.05μg/ml). The lytic effect of the leukocyte extracts on the penicillin-grown bacteria is further augmented by the addition of egg-white lysozyme. Staphylococci, which are harvested from the logarithmic phase of growth in ordinary media, are susceptible to lysis by leukocyte extracts, maximal lysis being achieved with about 100μg/ml of leukocyte extracts. On the other hand, penicillin-grown staphylococci are lysed by much smaller amounts of leukocyte extracts (20μg/ml), and much shorter periods of incubation are needed to achieve maximal lysis. Similar results are obtained when the leukocyte extracts are substituted by a cocktail of lytic agents which contain crude trypsin, lysolecithin, and lysozyme. Lysis of the staphylococci by the leukocyte extracts, fortified by lysozyme, is optimal at pH 5.0 and is accompanied by the solubilization of the bulk of glucosamine, known to be mostly concentrated in the peptidoglycan of the cell wall. Penicillin-grown staphylococci are also more susceptible than controls to lysis by a mixture of histone and lysozyme. The lysis, by leukocyte extracts and by the cocktail of both regular and penicillin-grown staphylococci, is strongly inhibited to the same extent by heparin, liquoid, histone, protamine sulfate, IgG, and human serum. On the other hand, no inhibition of lysis is achieved by chloramphenicol, streptomycin, erythromycin, KCN, HgCl₂, or by neutral polyelectrolytes. Group A streptococci, which are extremely resistant to degradation by leukocyte extracts or by the cocktail, when harvested from any phase of growth, also become susceptible to lysis by leukocyte extracts or by the cocktail when grown in the presence of small amounts of penicillin (0.004–0.008μ/ml). Bacteriolysis became even more pronounced when the reaction mixtures were incubated at 41 °C, a temperature likely to develop in patients with streptococcal infections. Electron-microscope examination of the staphylococci following treatment with leukocyte enzymes and penicillin revealed that both cell wall and cytoplasmic structures were severely damaged by the lytic agents. The mechanisms by which penicillin exposes the bacterial cell walls to cleavage by leukocyte extracts is discussed, and the phenomenon of enhanced susceptibility to lysis by leukocyte enzymes is related to the role played by undegraded bacterial constituents in the initiation of chronic inflammatory lesions.