Effects of human serum on bacterial competition with neutrophils for molecular oxygen

Abstract

A dialyzable factor(s) in human serum is known to stimulate gonococcal oxygen consumption. Its effect on other human pathogens was investigated. A 10% serum solution increased peak O2 consumption for Escherichia coli and Staphylococcus aureus to 157% (P less than 0.05) and 199% (P less than 0.02), respectively, of their O2 consumption when suspended in Hanks balanced salt solution, compared with a 356% increase for Neisseria gonorrhoeae with serum. Dialyzed serum lacked stimulatory capacity. Bacteria, serum, and neutrophils are often incubated to evaluate neutrophil bactericidal activity. Samples of 10(8) N. gonorrhoeae, S. aureus, and E. coli turned resazurin colorless (anaerobic conditions, Eh less than -42 mV) after 7.4, 13.3, and 15.1 min, respectively. Because neutrophil formation of reactive oxygen intermediates requires ambient O2, the effect of live bacteria and serum on this process was explored. After 5 min of incubation of 10(8) N. gonorrhoeae or S. aureus in 10% normal or dialyzed serum, 10(5) neutrophils were added. Phorbol myristate acetate was then added to assure neutrophil stimulation, and luminol-dependent luminescence was measured. N. gonorrhoeae and S. aureus incubation in normal serum decreased peak LDL 91.7 and 88.6%, respectively, relative to incubation in dialyzed serum. A sample of 10(8) E. coli totally eliminated LDL. A sample of 10(8) E. coli incubated in Hanks balanced salt solution for 5 min also eliminated phorbol myristate acetate induced neutrophil H2O2 production. LDL inhibition increased in proportion to bacterial concentration and time of incubation and was prevented by inclusion of KCN. Increasing the concentration of neutrophils to 10(8) (1:1 particle-to-cell ratio) only partially reversed LDL inhibition. Re-aeration of the system allowed brief LDL which persisted only if KCN was added. Addition of KCN after bacterial incubation also permitted LDL, arguing against depletion of other factors from the media or accumulation of bacterially derived inhibitory substances. A dynamic competition for O2 occurs between bacteria and neutrophils. Serum stimulation of bacterial O2 utilization may contribute to virulence by increasing bacterial capacity to inhibit neutrophil function.