Development of an Assay for Antimicrobial Susceptibility Testing of Coxiella burnetii

Abstract

Coxiella burnetii, a small Gram-negative bacterium, is the causative agent of Q fever, a zoonotic disease with initial flu-like symptoms that lead to both acute and chronic stages of infection. As with many obligate intracellular parasites, C. burnetii has been difficult to culture and therefore has been difficult to establish assays that examine a compound’s direct effect upon the bacterium. The development of an axenic medium for C. burnetii provided a method with which to culture the bacterium in liquid medium and thereby permitted our development of an assay for examining C. burnetii sensitivity to various anti-bacterial compounds. In order to establish the assay, the nonpathogenic C. burnetii Nine Mile Phase II Clone 4 (CBNMIIC4) strain was cultured (37°C) in acidified citrate cysteine medium (ACCM2) under hypoxic conditions. After 4 days in culture, the OD600 of the culture was adjusted to 0.10 (corresponding to approximately 6.8 x 108 bacteria per mL) and then added to wells in a 96-well plate containing dilutions of various anti-bacterial compounds. The plates were incubated (37°C) under hypobaric conditions for 5 days after which the OD600 was measured to determine whether the compounds affected CBMNIIC4 growth. Characterization of CBNMIIC4 growth was verified by measuring CBNMIIC4 genomic DNA and colony forming units in treatment cultures compared to the controls. The results of these studies provide a much-needed assay to assess the effects of anti-bacterial compounds upon C. burnetii growth.