Evaluation of Coxiella burnetii antibiotic susceptibilities by real-time PCR assay

Abstract

Coxiella burnetii is an obligate intracellular bacterium. The inability to cultivate this organism on axenic medium has made calculation of infectious units challenging and prevents the use of conventional antibiotic susceptibility assays. A rapid and reliable real-time PCR assay was developed to quantify C. burnetii cells from J774.16 mouse macrophage cells and was applied to antibiotic susceptibility testing of C. burnetii Nine Mile, phase I. For calculation of bacterial replication, real-time PCR performed equally as well as immunofluorescent-antibody (IFA) assay when J774.16 cells were infected with 10-fold serial dilutions of C. burnetii and was significantly (P < 0.05) more repeatable than IFA when 2-fold dilutions were used. Newly infected murine macrophage-like J774.16 cells were treated with 8 microg of chloramphenicol per ml, 4 microg of tetracycline per ml, 4 microg of rifampin per ml, 4 microg of ampicillin per ml, or 1 microg of ciprofloxacin per ml. After 6 days of treatment, tetracycline, rifampin, and ampicillin significantly (P < 0.01) inhibited the replication of C. burnetii, while chloramphenicol and ciprofloxacin did not. In general, these results are consistent with those from prior reports on the efficacy of these antibiotics against C. burnetii Nine Mile, phase I, and indicate that a real-time PCR-based assay is an appropriate alternative to the present methodology for evaluation of the antibiotic susceptibilities of C. burnetii.

FIG. 1.

Establishment of the standard curve for C. burnetii quantification. (A) Amplification plots of C. burnetii standards. Genome concentrations are (from left to right) 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², and 10¹. For each dilution the normalized fluorescence signal (ΔRn) is plotted against the PCR cycle number. (B) Standard curve generated from the C_t values of the amplification plots with ABI Sequence Detection software. This curve represents the standard curve only; no unknowns are represented in this graph.

FIG. 2.

Determination of PCR specificity. (A) Melting curve analysis performed with ABI Dissociation Curve software revealed that the com1-specific primer pair amplified a single predominant product with a melting temperature of 81.3°C. (B) Agarose gel electrophoresis of the amplified product with ethidium bromide staining shows the presence of a single band, confirming the specificity of the PCR assay. Lane 1, 100-bp molecular mass marker; lane 2, amplified product from a C. burnetii DNA standard dilution of 10⁷; lane 3, blank; lane 4, amplified product from a 1:200 dilution of the C. burnetii inoculum.

FIG. 3.

Validation of real-time PCR by comparison to IFA. The results are represented as the means and standard errors of three replicates. (A) Tenfold dilution series; (B) twofold dilution series. Circles, real-time PCR data; diamonds, IFA data.

FIG. 4.

Effects of chloramphenicol, tetracycline, rifampin, ampicillin, and ciprofloxacin on C. burnetii replication in J774.16 cells. Antibiotic-treated cells were assayed on day 7. Sham-treated cells were assayed on days 1, 2, 4, and 7 postinoculation and served as a control for bacterial replication. Open bar, day 1; black bar, day 2; stippled bar, day 4; striped bars, day 7. Results are reported as the means and standard errors for three replicates.