In a system in which unphagocytosed bacteria were removed by differential centrifugation after a 30-min phagocytosis period, staphylococci associated with rabbit polymorphonuclear (PMN) leukocytes were completely protected from the effects of benzyl penicillin 1 μ/ml, but not completely protected from the effects of 5 μ/ml. When unphagocytosed bacteria were lysed with lysostaphin, effective protection could be observed over a range of penicillin concentrations from 0-25 to 200 μ/ml. C-benzyl penicillin failed to accumulate in rabbit PMN leukocytes, whether or not they had previously phagocytosed staphylococci, in conditions in which mouse peritoneal macrophages readily accumulated penicillin. Mixed granule extracts prepared from the PMN leukocytes interacted synergically with penicillin against staphylococci at physiological pH (7-2) but failed to show synergy at an intraphagolysosomal pH of 5.0 unless the bacteria were first sublethally treated with penicillin. Experiments in which the pH value of culture media was changed either from 7.2 to 5.0 or from 5.0 to 7.2 indicated that the partial nature of the protective effect of the intraphagolysosomal environment could be attributed to the growthlimiting effects of the low phagolysosomal pH, which prevents full expression of the synergic potential of granule contents and penicillin. The concentration-dependent nature of the protection and its incompleteness are explained by supposing that a proportion of the staphylococci not ingested during the 30-min phagocytosis period are modified by penicillin in a way that opsonises them and potentiates the intrinsic bactericidal mechanisms of the PMN leukocytes when the bacteria are subsequently ingested.


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