Standardized <em>In vitro</em> Assays to Visualize and Quantify Interactions between Human Neutrophils and <em>Staphylococcus aureus</em> Biofilms

Abstract

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years.Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms.These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Dioxane-Linked Novel Bacterial Topoisomerase Inhibitors Exhibit Bactericidal Activity against Planktonic and Biofilm Staphylococcus aureus In Vitro

“…Given the importance of staphylococcal biofilms in clinical settings ( 21 , 22 ), we evaluated the ability of 0147 and 0186 to prevent biofilm formation. To more readily detect S. aureus biofilm growth, 96-well plates were pretreated with poly- l -lysine, and the Luria broth (LB) growth medium was supplemented with 2% glucose and 2% NaCl ( 23 ). Approximately 10 7 CFU/mL of S. aureus was incubated with the test compound for 24 h at 37°C.…”

“…Crystal violet was then extracted in 33% glacial acetic acid and incubated at room temperature for 25 min. The absorbance was measured using the Molecular Devices SpectraMax i3x at OD 590 ( 23 ). The results represent an average of three or four independent experiments performed in triplicate and were analyzed by one-way analysis of variance (ANOVA) (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001;****, P < 0.0001) using GraphPad Prism version 9.4.1.…”