Isolation from animal tissue and genetic transformation of Coxiella burnetii are facilitated by an improved axenic growth medium

Abstract

We recently described acidified citrate cysteine medium (ACCM), which supports host cell-free (axenic) growth of Coxiella burnetii. After 6 days of incubation, greater than 3 logs of growth was achieved with the avirulent Nine Mile phase II (NMII) strain. Here, we describe modified ACCM and culture conditions that support improved growth of C. burnetii and their use in genetic transformation and pathogen isolation from tissue samples. ACCM was modified by replacing fetal bovine serum with methyl-β-cyclodextrin to generate ACCM-2. Cultivation of NMII in ACCM-2 with moderate shaking and in 2.5% oxygen yielded 4 to 5 logs of growth over 7 days. Similar growth was achieved with the virulent Nine Mile phase I and G isolates of C. burnetii. Colonies that developed after 6 days of growth in ACCM-2 agarose were approximately 0.5 mm in diameter, roughly 5-fold larger than those formed in ACCM agarose. By electron microscopy, colonies consisted primarily of the C. burnetii small cell variant morphological form. NMII was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice. A completely axenic C. burnetii genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 1/2 weeks. Transformation experiments demonstrated clonal populations in colonies and a transformation frequency of approximately 5 × 10(-5). Cultivation in ACCM-2 will accelerate development of C. burnetii genetic tools and provide a sensitive means of primary isolation of the pathogen from Q fever patients.

Fig. 1.

Composite map of pJB-CAT and pJB-KAN. Plasmids contain a RSF1010 origin of replication and a chloramphenicol or kanamycin resistance gene under the control of the C. burnetii CBU1169 promoter.

Fig. 2.

Cultivation of C. burnetii in ACCM-2. (A) Growth of NMII in ACCM-2 with shaking (solid line) and ACCM without shaking (dotted line). (B) Growth of the virulent C. burnetii NMI (dotted line) and G (solid line) isolates in ACCM-2 with shaking. Growth was measured by enumerating GE, and results are from three separate cultures ± the standard error of the mean.

Fig. 3.

Colony formation by C. burnetii in ACCM and ACCM-2 agarose. (A) Representative ACCM plate showing NMII colonies that average approximately 0.1 mm in diameter. Scale bar, 10 mm. (B) Representative ACCM-2 plate showing NMII colonies that average approximately 0.5 mm in diameter. Scale bar, 10 mm. (C) Electron microscopy of colony-grown C. burnetii showing primarily the SCV morphological form. Scale bar, 0.5 μm.

Fig. 4.

NMII colonies in ACCM-2 agarose are clonal. Equal numbers of NMII and NMII-MC1, which contains a Himar1 transposon insertion between CBU0316 and CBU0317, were plated in ACCM-2 agarose. Fourteen random colonies were picked after 6 days of growth and genotyped as described in Materials and Methods. Each colony contained either NMII (1.6-kb band) or NMII-MC1 (4.8-kb band) but not both, showing the clonality of C. burnetii colonies in ACCM-2 agarose.

Fig. 5.

ACCM-2 supports culture of C. burnetii from infected animal tissue. ACCM-2 was inoculated with a 1:100 dilution of total lung, liver, or spleen homogenates derived from NMII-infected SCID mice infected by intraperitoneal or intratracheal routes as described in Materials and Methods. Closed symbols indicate the starting numbers of GE/ml. Open symbols indicate the numbers of GE/ml after a 7-day incubation. Depicted data are the means of three ACCM-2 cultures individually started with tissue homogenates from three infected mice.