Neutrophil killing of Staphylococcus aureus In Sepsis
Neutrophils form the majority of circulating white blood cells and play a principle role in defending the body against invasions by microorganisms. Neutrophils engulf microorganisms into a phagosome where they kill them using reactive oxygen species and a range of antimicrobial proteins. Sepsis is described as a complicated clinical disorder that is generated from a dangerous host response to an infection. One of the main organisms responsible for causing infection in sepsis is Staphylococcus aureus. Pro-inflammatory cytokines are elevated in patients with sepsis. These cytokines are also known to prime neutrophils and as a result, superoxide production is elevated in neutrophils. This may help to destroy the pathogen more rapidly. The main aim of this study was to compare the ability of neutrophils to kill S. aureus in serum from healthy individuals to that in serum rich in inflammatory cytokines obtained from sepsis patients. To test the priming effect of sepsis serum, neutrophils were pre-incubated in either healthy or sepsis serum followed by stimulation with Formyl-methionyl-leucyl-phenylalanine. Cells incubated in sepsis serum followed by stimulation showed significantly increased superoxide production compared to cells incubated in healthy serum. S. aureus were pre-treated in either serum collected from healthy donors or serum from patients with sepsis and then incubated with neutrophils. A one-step neutrophil killing assay was used which gave the combined rate of phagocytosis and killing. I found that there was no significant differences in the rate of phagocytosis and killing of S. aureus by neutrophils in healthy and sepsis serum. Since neutrophils kill S. aureus mainly by an oxidative mechanism, the effect of diphenylene iodonium, an inhibitor of flavoproteins such as NADPH oxidase, was checked to see if there was any difference in the mechanism of killing in sepsis serum. I found that the contribution of NADPH oxidase derived oxidants to killing was similar in healthy or sepsis serum. S. aureus have adapted many protective mechanisms in order to avoid being destroyed by neutrophils. A two-step killing assay that separate rates of killing and phagocytosis, was used to assess whether S. aureus collected from patients after antibiotic treatment were more resistant to neutrophil killing. The result showed no difference in the rate of neutrophil phagocytosis and killing of clinical isolates compared to the lab strain of S. aureus. Hypochlorous acid is a powerful oxidant used by neutrophils to kill invading microorganisms. It is known that S. aureus can survive within neutrophil, despite exposure to hypochlorous acid potentially by upregulation of survival genes. Pretreatment of S. aureus with a sub-lethal dose of hypochlorous acid did not protect bacteria from a subsequent dose of hypochlous acid. In conclusion I have tested neutrophil killing of S. aureus in normal healthy serum and serum from sepsis patients. The results show that even though there was increased superoxide production by neutrophils pre-treated in sepsis serum compared to healthy serum, the rate of phagocytosis and killing were the same. This suggests that pre-treatment of neutrophils with pro-inflammaotry cytokines does not improve their ability to kill S. aureus.
Advisor: Winterbourn, Christine; Parker, Heather
Degree Name: Master of Science
Degree Discipline: Microbiology
Publisher: University of Otago
Keywords: Neutrophils; Sepsis
Research Type: Thesis