Intraphagocytic Protection of Staphylococci from Extracellular Penicillin

Abstract

SUMMARY. 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 pg/ml, but not completely protected from the effects of 5 pg/ml. When unphagocytosed bacteria were lysed with lysostaphin, effective protection could be observed over a range of penicillin concentrations from 0.25 to 200 pg/ml. 14C-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 p H 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.

Evaluation of antibacterial agents in a high-volume bovine polymorphonuclear neutrophil Staphylococcus aureus intracellular killing assay

“…Our results with aminocyclitol, novobiocin, vancomycin (5,8,14,17,18,22), penicillin, macrolide, and quinolone antibiotics (14,(16)(17)(18) are in agreement with those in the literature. Although erythromycin has been reported to penetrate phagocytes (12,20), the compound was ineffective in killing intracellular bacteria.…”

“…This intracellular location may afford the microbe protection from the effects of the antibiotic (5,8). Some of the reasons suggested for this protection are intracellular inactivation of the antibiotic (10,14,15), lack of penetration of the antibiotic into the phagocyte (10,20), and resistance of the ingested bacteria to the action of antibiotic (10).…”

Effect of low intraphagolysosomal pH on antimicrobial activity of antibiotics against ingested staphylococci

“…We have shown here, and elsewhere (Lam and Mathison, 1982) that pH has a critical role in the expression of antimicrobial activity in leukocytes; pH values inimical to bacterial growth impair the antimicrobial activities of accumulated antibiotics that kill bacteria within leukocytes at pH values permitting growth. We strongly advocate that bacteria that do not multiply intracellularly should not be used to assess the intracellular activity of antibiotics.…”

“…Since the report of Rous and Jones (1916) it has been observed repeatedly that many microbial pathogens are protected from antibiotics to which they are susceptible when inside mammalian cells (Magoffin and Spink, 1951;Shaffer, Kucera and Spink, 1953;Holmes et al, 1966;Solberg and Hellum, 1978;Easmon, 1979;Lam and Mathison, 1982). Little is known about how the intracellular environment protects ingested staphylococci from lethal extracellular antimicrobials.…”

“…In contrast, rifampicin does not kill Mycobacterium tuberculosis in stimulated rabbit peritoneal macrophages ( C h i and Grassi, 1970) or S. aureus in mouse resident peritoneal macrophages (Pesanti, 1980). The low intraphagolysosomal pH after ingestion of a particle by a PMN leukocyte inhibits the growth of staphylococci and the bactericidal activity of penicillin (Lam and Mathison, 1982). The following questions, therefore, arise: (i) are staphylococci in this dormant state more resistant to the action of other antibiotics such as aminoglycosides and rifampicin; and (ii) is there a relationship between the inhibition of staphylococcal growth by the low intracellularpH and sensitivity of the bacteria to intracellular antibiotics?…”