Effect of probenecid on phagocytosis and intracellular killing of Staphylococcus aureus and Escherichia coli by human monocytes and granulocytes.

The present study concerns the effects of probenecid on the phagocytosis and intracellular killing of Staphylococcus aureus and Escherichia coli by human monocytes and granulocytes. In both monocytes and granulocytes the inhibitory effect on phagocytosis was very small. Inhibition of intracellular killing of S. aureus by monocytes and granulocytes by probenecid was concentration dependent, being half-maximal at about 2 mM probenecid, and near-maximal at about 5 mM probenecid. The intracellular killing could also be inhibited when probenecid was added when this process was already started. Probenecid also inhibited the intracellular killing of E. coli by granulocytes, but not by monocytes. In the concentration range used, probenecid had no toxic effect on phagocytes or bacteria during the 2 hr of the experiments.

The antibacterial activity of benzylpenicillin against Staphylococcus aureus ingested by granulocytes.

Granulocytes that had ingested pre-opsonized Staphylococcus aureus for 3 min at 37 degrees C and then had been washed twice, were incubated in the presence of benzylpenicillin. The antibiotic showed a concentration-dependent effect on cell-associated S. aureus between 0.005 and 0.1 mg/l benzylpenicillin. The antibacterial effect on cell-associated S. aureus was equal to that on opsonized S. aureus in suspension. Microscopic studies with lysostaphin showed that the number of extracellular bacteria was negligible and that the decrease in the number of cell-associated bacteria reflected the true rate of intracellular killing of S. aureus. Contrary to the generally accepted opinion that benzylpenicillin does not act intracellularly in granulocytes, we were able to show intracellular antibacterial activity of benzylpenicillin against S. aureus under the experimental conditions we used.

The influence of human monocytes on the antibacterial activity of vancomycin and teicoplanin against Staphylococcus aureus.

The influence of human monocytes on the antibacterial activity of vancomycin and teicoplanin against Staphylococcus aureus was investigated. Monocytes, which neither phagocytosed nor killed bacteria because no serum was present in the incubation medium, enhanced the effect of both antibiotics. When phagocytosis and intracellular killing were induced by the use of pre-opsonized S. aureus or the addition of serum during the incubation, the antibacterial effect of vancomycin and teicoplanin was diminished. Both antibiotics showed intracellular activity against S. aureus ingested by monocytes. In the first hour of incubation the rate of killing of ingested S. aureus was greater than that of non-phagocytosed S. aureus.

Influence of human monocytes on the antibacterial activity of kanamycin and gentamicin for Staphylococcus aureus.

The present study was performed to compare the antibacterial activities of kanamycin and gentamicin on Staphylococcus aureus phagocytosed by human monocytes and on nonphagocytosed S. aureus. The method used permitted the measurement of the effect of antibiotics on intracellular bacteria independent of phagocytosis and intracellular killing by the monocytes. A morphological assay with lysostaphin established the intracellular localization of about 70% of the cell-associated S. aureus in the monocyte-bacterium suspension. After 1 h of incubation, the antibacterial activity of both aminoglycosides was greater against intracellular than against nonphagocytosed S. aureus, but after 3 h, the reverse was true. The maximal effect on phagocytosed S. aureus, i.e., killing of about 98% of the bacteria, was reached in the first hour of incubation at kanamycin and gentamicin concentrations of 5 and 1 microgram/ml, respectively. A cell-free medium in which monocytes had been incubated increased the antibacterial activity of kanamycin, indicating that monocytes secrete a factor that enhances the antibacterial activity of aminoglycosides.

Comparison of the effect of phenoxymethylpenicillin, cloxacillin, and flucloxacillin on Staphylococcus aureus phagocytosed by human monocytes.

The antibacterial effect of phenoxymethylpenicillin, cloxacillin, and flucloxacillin on pre-opsonized Staphylococcus aureus ingested by human monocytes and on preopsonized S. aureus in suspension was compared. The antibiotics were 1.7-3 times more effective against intracellular S. aureus than against S. aureus in suspension. No influence of acid stability or lipid solubility of the drugs on the antibacterial effect against intracellular S. aureus was demonstrated.

Effect of penicillin G on Staphylococcus aureus phagocytosed by human monocytes.

The effect of penicillin G on Staphylococcus aureus after phagocytosis by human monocytes was compared with the effect on nonphagocytosed bacteria. Intracellular S. aureus was obtained by incubation of monocytes and preopsonized S. aureus for 3 min at 37 C; phagocytosis was stopped by cooling the test tubes in crushed ice. Nonphagocytosed bacteria were removed by differential centrifugation followed by two washes of the cells. Morphological studies with lysostaphin confirmed the intracellular localization of approximately 70% of the bacteria after this procedure. The antibacterial activity of penicillin G was assessed by incubation of monocytes containing ingested S. aureus with the drug at 37 C, and determination of the number of viable cell-associated bacteria was assessed microbiologically at various intervals. Inactivation of the intracellular killing mechanism of the monocytes by omission of serum during incubation allowed measurement of the effect of penicillin G apart from the bactericidal activity of the cells. The effect of penicillin G on nonphagocytosed bacteria was assessed after incubation of preopsonized S. aureus in medium with the drug. The results show that the effect of penicillin G on intracellular S. aureus is two to seven times stronger than the effect on nonphagocytosed S. aureus.

Interaction between human monocytes and penicillin G in relation to the antibacterial effect on Staphylococcus aureus.

The influence of the presence of monocytes on the effect of penicillin G on Staphylococcus aureus was studied. Conditions were varied in such a way that phagocytosis and intracellular killing of S. aureus by monocytes did not occur, was short lasting and limited, or long lasting and extensive. Synergism between penicillin G and monocytes was observed in the absence of and during limited phagocytosis and intracellular killing by the monocytes. When phagocytosis and intracellular killing were optimal, addition or antagonism between penicillin G and monocytes was observed. Enhancement of the antibacterial effect of penicillin G was also obtained in the presence of monocyte supernatant (the cell-free medium in which monocytes had been incubated for 3 hr). This indicates that monocytes secrete a factor that enhances the antibacterial activity of penicillin G. Serum inhibits the activity of this factor.

Adherence of lysostaphin to and penetration into human monocytes.

The effect of lysostaphin on Staphylococcus aureus phagocytosed by monocytes was investigated. The results showed that lysostaphin adheres to monocytes by a temperature-independent mechanism, is not adequately removed from monocytes by washing, and penetrates by means of a temperature-dependent mechanism. In in vitro assays of monocyte function, phagocytosed S. aureus can be killed by lysostaphin after penetration of the cells during incubation or by adhering lysostaphin when the monocytes are disrupted.

Interaction of povidone-iodine compounds, phagocytic cells, and microorganisms.

The interaction between povidone-iodine, phagocytic cells, and microorganisms was studied. Three preparations of povidone-iodine were investigated: commercially available povidone-iodine solution Betadine, pure high-molecular-weight povidone-iodine as used in Betadine, and a low-molecular-weight povidone-iodine. Low concentrations of povidone-iodine (approximately 0.005%) have considerable activity in vitro. The concentrations used clinically (0.1 to 20%) are toxic for granulocytes and monocytes. Leukocytes reduce the in vitro microbicidal activity of povidone-iodine. No differences of any importance were found between the three preparations of povidone-iodine.