This presentation will develop the hypothesis that early events in the innate immune response to a microbially contaminated implant determine whether a biofilm forms and a persistent infection occurs. A corollary of this hypothesis is that innate immunity can be locally guided or boosted to reduce infections on indwelling medical devices. To gain insight into the important phenomena in the interaction between a nascent biofilm and phagocytes, two systems were used to directly and dynamically image the activity of neutrophils against small aggregates of surface-attached Staphylococcus aureus. In vitro, human neutrophils were stained red and added to a serum-coated glass cover slip with pre-attached GFP-expressing S. aureus. In vivo, multiphoton confocal intravital imaging was accomplished in a mouse model in which a young S. aureus biofilm attached to a glass coverslip was interfaced to the bottom of the dermis in a mouse skin flap model. Bacteria were imaged via GFP, neutrophils by tdTomato (CatchupIVM-red mice), collagen by second harmonic generation, and vasculature by injected quantum dots. Neutrophil speed, migration, discovery of bacteria, and killing of bacteria were tracked and quantified through image analysis. Collectively these observations suggest that: 1) the timescale for arrival of neutrophils at the biofilm-affected material surface in this animal model is variable with a median longer than three hours, 2) bacteria continue to grow prior to neutrophil arrival both in vitro and in vivo, and 3) formation of bacterial aggregates makes it more difficult for neutrophils to clear the nascent biofilm.
Phil Stewart from Montana State University