Improved Antibiotic Susceptibility Test of Orientia tsutsugamushi by Flow Cytometry Using Monoclonal Antibody

Cell culture and O. tsutsugamushi

Cells of the human umbilical-vein-derived endothelial cell line ECV304 were maintained in M199 medium (Gibco BRL, Gaithersburg, MD, U.S.A.) supplemented with 10% (v/v) heated-inactivated fetal bovine serum (Gibco BRL) in a humidified atmosphere containing 5% CO₂ at 37℃. The Boryong serotype of O. tsutsugamushi (15) was cultivated in ECV304 cells, as described previously (16). The Thailand strains (AFSC-4, AFSC-7, TA686, TA678, TA716, TA763, and TH1817) were kindly provided by Dr. Daniel Strickman, Naval Medical Research Institute, U.S.A.. The Karp (ATCC VR-150) and Gilliam (ATCC-VR-312) strains were obtained from American Type Culture Collection; Kato strain was donated from Dr. Hiroshi Tanaka, the Institute of Medical Science, Tokyo University, Japan. Kuroki and Kawasaki strains were donated from Dr. Akira Tamura, Department of Microbiology, Niigata College of Pharmacy, Japan. All bacterial strains were cultivated in ECV304 cells. When infected ECV304 cells showed maximum cytopathic effects, the cells were disrupted with glass bead (diameter 1.0 mm) and centrifuged at 300×g for 5 min. The resulting supernatants were immediately used to infect further ECV304 cells.

Treatment with doxycycline

ECV304 cells grown in 6-well plates were incubated with O. tsutsugamushi for 3 hr, allowing time for O. tsutsugamushi to attach to and enter the host cells. At the end of the initial incubation period, the inoculums were replaced with fresh medium containing two-fold dilutions of doxycycline (Sigma, St. Louis, MO, U.S.A.) from 0.2 µg/mL to 0.00625 µg/mL, and the cells were then re-incubated in 5% CO₂ at 37℃ for three days. Doxycycline was prepared in aliquots of 0.5 mL at an active concentration of 5,000 µg/mL in sterile distilled water. These aliquots were stored frozen at -20℃ until required.

Monoclonal antibodies and selection of antibodies for the application in flow cytometry

MAb FS15 and FS10 react against a linear epitope on 56-kDa major outer membrane protein of O. tsutsugamushi (17). Other MAbs (Rb167, Rb134, M686-8, and Shim107) were obtained from cell fusion experiments of spleen cells of mice immunized with live O. tsutsugamushi, as described previously (6, 18). For its application to flow cytometry, MAb FS15 was purified on an AffinityPak Protein A Column (Pierce Biotechnology Rockford, IL, U.S.A.) and conjugated with fluorescein isothiocyanate (FITC) with EZ-label Fluorescein Protein Labeling Kit (Pierce Biotechnology), according to the manufacturer's instruction.

To select a suitable MAb for the application in flow cytometry, we tested the immunoreactivities of MAbs to various strains of O. tsutsugamushi using IFA. Among the tested MAbs, we selected the MAb that had broad reactivity to many strains and stained the bacteria brightly.

Immunofluorescent antibody staining

The reactivities of MAbs to various strains of O. tsutsugamushi were examined using IFA. ECV304 cells infected with each strain were fixed with acetone-methanol (1:1) or acetone and treated with dilutions of MAbs in phosphate-buffered saline (PBS) for 1 hr at 37℃. After the cells had been washed briefly with PBS, they were treated with FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, U.S.A.) for 1 hr in a moist chamber, and then washed three times with PBS. To clearly define the O. tsutsugamushi, the host cells were counter-stained with 0.003% Evans blue in PBS. After the slides had been washed, they were placed on mounting medium (Vector Laboratories, Burlingame, CA, U.S.A.) and examined under a fluorescence microscope (Nikon, Japan) with a confocal laser scanning system (Bio-Rad, Hercules, CA, U.S.A.).

Immunogold electron microscopy

ECV304 cells infected with O. tsutsugamushi were grown for seven days and were fixed with 0.5% glutaraldehyde, 4% paraformaldehyde and 3.5% sucrose and postfixed with 1% osmium tetroxide (OsO₄). After dehydration in an ethanol series, the pellets were embedded in Epon 812. After the collection of ultrathin sections on Formvar/carbon-coated nickel grids, the grids were floated on drops of 3% sodium metaperiodate for 30 min. The immunogold labeling procedure (19) was done using MAb FS15 and 15 nm gold-conjugated goat anti-mouse IgG. After immunogold labeling, the grids were stained with uranyl acetate and lead citrate, and was viewed with a Hitachi H7100 electron microscope.

Flow cytometry assay and data processing

To quantify O. tsutsugamushi in ECV304 cells, infected cells were trypsinized, washed with PBS, and centrifuged at 300×g for 5 min. The cells were fixed with 70% ethanol for 1 hr on ice and stored at -20℃ until analyzed. Before their application to flow cytometry, fixed cells were washed with PBS and the resulting cell pellets were resuspended in 500 µL of FITC-conjugated MAb FS15 diluted with PBS, and analyzed with a flow cytometer (Becton Dickinson, Mansfield, MA, U.S.A.) equipped with a 15 mW argon laser. Data were collected on 10,000 cells and analysis was performed using LYSIS II software (Becton Dickinson). To calculate an estimate of the total mass of bacteria in the cells, the percentage of positive cells was multiplied by the mean fluorescence intensity of these cells to obtain the growth index (GI). The MIC was defined as the concentration of antibiotics at which GI was reduced by 90% relative to that of control cells that were infected and incubated in the absence of doxycycline.

Reactivity of MAb selected for the application in flow cytometry

The tested MAbs could stain O. tsutsugamushi brightly in IFA (Fig. 1A). Although the MAbs showed similar morphologies of bacteria, their staining patterns differed slightly according to the target antigens. In addition, the bacterial particles stained by MAbs (FS10, FS15, and Rb167) directed against the 56-kDa scrub typhus antigen (Sta56) were brighter and bigger than those stained by MAbs (Rb134, M686-8, and Shim107) directed against other antigens. Therefore, we reasoned that MAbs against Sta56 are suitable as the primary antibodies for the use in flow cytometry. Among them, we selected FS15 because the target epitope of this MAb has been well characterized in a previous study (17). Furthermore, the antigenic epitope is located at the surface of the cell envelope as shown by immunogold electron microscopy (Fig. 2A). As Sta56 has strain-specific and group-specific epitopes, we tested the reactivity of FS15 to other strains of O. tsutsugamushi. As shown in Fig. 2B. MAb FS15 stained most of the O. tsutsugamushi strains (Boryong, Karp, Kato, AFSC-4, AFSC-7, TA686, TA763, TH1817, Kawasaki and Kuroki) originating from several Asian nations in IFA, except Gilliam, TA678, and TA716 strains.

Fig. 1
IFA staining of MAbs Boryong strains of O. tsutsugamushi. MAbs directed against a Sta56 show brighter and bigger bacterial particles than those seen by MAbs against other antigens (×80).

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Fig. 2
Reactivity of MAb FS15 selected for the application in flow cytometry. (A) Immunogold staining of O. tsutsugamushi with MAb FS15 shows that epitope of FS15 was located at the surface of the cell envelope (×25,000). (B) MAb FS15 reacts positively against 10 strains out of 13 strains from various nations (×20).

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Growth of O. tsutsugamushi measured by flow cytometry

To test the applicability of FITC-conjugated FS15 to the quantification of the total mass of O. tsutsugamushi by flow cytometry, we first used this MAb to determine the growth of O. tsutsugamushi. We infected the ECV304 cells with high (84.6% cells are infected 3-day postinfection) and low numbers (12.2% cells are infected 3-day postinfection) of O. tsutsugamushi and measured bacterial growth for seven days using flow cytometry. Under both conditions, flow cytometry demonstrated the significant growth of O. tsutsugamushi, which appeared at as early as one day after infection and progressed further until seven days after infection. This is evident in the right-hand shift of the entire peak of cells infected with high numbers of bacteria (Fig. 3A) and the appearance of a new peak in cells infected with low numbers of bacteria (Fig. 3B). Because three days incubation was sufficient to demonstrate bacterial growth by flow cytometric parameters, we selected this time point for subsequent work.

Fig. 3
Flow cytometric histograms of ECV304 cells infected with a high dose (A) or low dose (B) of Boryong strain at the indicated times. Control represented the result from uninfected cells. The percentages of positive cells for O. tsutsugamushi are marked in the histogram.

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Determination of antibiotic susceptibility of O. tsutsugamushi by flow cytometry

To test the applicability of flow cytometry to the determination of the doxycycline susceptibility of the Boryong strain, which is the most prevalent strain in Korea (15), infected ECV304 cells were treated with various concentrations of doxycycline for three days, and then bacterial growth was measured by flow cytometry. As shown in Fig. 4A, the fluorescence peak shifted to left as the concentration of doxycycline increased, as indicated by the percentage of positive cells in each histogram. The percentage of positive cells infected with Boryong after treatment with 0.1 µg/mL doxycycline was 1.7% (Fig. 4A). In the presence of 0.1 or 0.2 µg/mL doxycycline, no bacterial growth was observed and the percentage of positive cells decreased to that of the control (uninfected) cells. Thus, the MIC was determined to be 0.1 µg/mL.

Fig. 4
Comparison of susceptibility of the Boryong and AFSC-4 strains to doxycycline. Flow-cytometric histograms of ECV304 cells infected with the Boryong (A) or AFSC-4 strain (B) in the presence of increasing concentrations of doxycycline (0-0.2 µg/mL) were measured after three day incubation. Control represents the result from the uninfected cells. The histograms are representative of three independent experiments. The GI of each strain was calculated and plotted as a percentage of fluorescence intensity (C), where 100% represents the percentage of infected cells without doxycycline treatment. Bars represent means±SD (n=3).

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Next, we used our method to determine the doxycycline susceptibility of O. tsutsugamushi strain AFSC-4, which is reported to be resistant to doxycycline (5). The percentage of positive cells infected with AFSC-4 after treatment with 0.1 µg/mL doxycycline was 3.1% and the MIC was 0.1 µg/mL (Fig. 4B). When we compared the doxycycline susceptibility of the Boryong and AFSC-4 strains, both strains showed similar patterns of inhibition by doxycycline. The percentages of GI of Boryong and AFSC-4 strains after the treatment with 0.1 µg/mL doxycycline were 1.29% and 4.11% respectively, where 100% represented the percentage of infected cells without doxycycline treatment (Fig. 4C).