Direct Microscopic Observation of Human Neutrophil-Staphylococcus aureus Interaction In Vitro Suggests a Potential Mechanism for Initiation of Biofilm Infection on an Implanted Medical Device

Abstract

The ability of human neutrophils to clear newly attached Staphylococcus aureus bacteria from a serum-coated glass surface was examined in vitro using time-lapse confocal scanning laser microscopy. Quantitative image analysis was used to measure the temporal change in bacterial biomass, neutrophil motility, and fraction of the surface area policed by neutrophils. In control experiments in which the surface was inoculated with bacteria but no neutrophils were added, prolific bacterial growth was observed. Neutrophils were able to control bacterial growth but only consistently when the neutrophil/bacterium number ratio exceeded approximately 1. When preattached bacteria were given a head start and allowed to grow for 3 h prior to neutrophil addition, neutrophils were unable to maintain control of the nascent biofilm. In these head-start experiments, aggregates of bacterial biofilm with areas of 50 μm2 or larger formed, and the growth of such aggregates continued even when multiple neutrophils attacked a cluster. These results suggest a model for the initiation of a biofilm infection in which a delay in neutrophil recruitment to an abiotic surface allows surface-attached bacteria time to grow and form aggregates that become protected from neutrophil clearance. Results from a computational model of the neutrophil-biofilm surface contest supported this conceptual model and highlighted the stochastic nature of the interaction. Additionally, we observed that both neutrophil motility and clearance of bacteria were impaired when oxygen tension was reduced to 0% or 2% O2.

Intravital Multiphoton Examination of Implant-Associated Staphylococcus aureus Biofilm Infection

“…Despite neutrophils appearing to respond to biofilm infection by increasing migration velocity and displacement, their movements were less directed toward the implant/infection than neutrophils in sterile tissues. These observation are consistent with a recent report that a delay in neutrophil recruitment to the implant surface may permit S. aureus time to grow and form biofilm (Ghimire et al, 2019 ). We anticipate that neutrophil deviation is a result of S. aureus biofilm products interfering with neutrophil cytokine/chemokine signaling and the production of multiple leukocyte inhibitors and toxins (Rooijakkers et al, 2006 ).…”

“…Neutrophils are the first line of cellular immune defense against invading bacterial pathogens; their coordinated recruitment and activity are essential to preventing and eliminating infection. Compared to macrophages, neutrophils have shown a much higher propensity for biofilm invasion and phagocytosis in vitro (Gunther et al, 2009 ; Thurlow et al, 2011 ; Ghimire et al, 2019 ). We observed significant differences in the cell migration behaviors of neutrophils in response to infected versus sterile implants.…”

Identification and Characterization of Planktonic Biofilm-Like Aggregates in Infected Synovial Fluids From Joint Infections

“…However, the antimicrobial susceptibility of all the tested bacteria drastically decreased when assayed in SF, which represents an important issue considering that infections are likely to start from one single cell ( Gerlini et al, 2014 ) and joint infections might arise from the contamination of sporadic cells. Furthermore, Ghimire et al recently showed that neutrophils are unable to efficiently phagocytize staphylococcal aggregates exceeding 10 μm in diameter and that they are supposed to be recruited rapidly and in sufficient numbers to scavenge all bacterial cells and prevent them to aggregate and grow to critical dimensions ( Ghimire et al, 2019 ).…”

Evading the host response: Staphylococcus “hiding” in cortical bone canalicular system causes increased bacterial burden

“…Supportive of this finding is the implant–inoculum interplay concept of implant infection developed by Ghimire et al 30 Their in vitro work demonstrated that both time before exposure to neutrophils and quantity of initial bacteria inoculation were critical determinants of successful Staphylococci implant infection. Novel hypotheses in neutrophil identification and phagocytosis of bacteria were explored, revealing that variation in the host–implant interface impacts the delicate balance between either bacteria persistence or eradication.…”

“…Finally, new evidence produced by Ghimire et al 30 demonstrates the importance of bioburden, as a ratio of bacteria compared to the number of available neutrophils, in overwhelming the host immune system to establish a biofilm-associated infection. Summarized simply, at key early time points of exposure, a high ratio of neutrophils to bacteria seemed to clear the implant of colonization; whereas high bacteria to neutrophil ratio overwhelmed the system and allowed a biofilm to form.…”