Development of a Coxiella burnetii culture method for high-throughput assay to identify host-directed therapeutics

The intracellular Gram-negative bacterium, Coxiella burnetii, is a worldwide zoonotic pathogen and the causative agent of Q fever. The standard of care for C. burnetii infections involves extended periods of antibiotic treatment and the development of doxycycline-resistant strains stress the need for new treatment strategies. A previously developed axenic medium has facilitated in vitro growth of the organism. In this study, we have developed a simple culture method that is inexpensive, reliable and utilizes a modular hypoxic chamber system for either small or large scale production of bacteria without the need of a tri-gas incubator. This method provides consistent growth and yields sufficient viable bacteria within four days of culture and can be used for high-throughput screening. The viable bacteria were quantified by counting colony forming units and total bacteria were enumerated using a genomic equivalent method. The characterized bacterial inoculum was then used to optimize cell-based high-throughput immunofluorescence assays with a goal to quantify intracellular bacteria and then screen and identify compounds that inhibit early stages of C. burnetii infection in macrophages.