J Korean Med Sci.  2007 Feb;22(1):1-6. 10.3346/jkms.2007.22.1.1.

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

• Kim MJ
• Kim MK
• Kang JS

• KMID: 1713220
• DOI: http://doi.org/10.3346/jkms.2007.22.1.1

Abstract

Orientia tsutsugamushi causes scrub typhus, which is endemic in many countries in the Asia-Pacific region including Korea. Recent emergence of doxycycline-resistant strains from Thailand has underlined the importance of the susceptibility tests of O. tsutsugamushi to antibiotics. To improve the flow cytometric technique for the susceptibility test, we applied a monoclonal antibody (MAb) in the quantification of O. tsutsugamushi. With using MAb FS15, we determined the doxycycline susceptibility of two strains, Boryong and AFSC-4 strain which is reported to be doxycycline-sensitive and resistant, respectively. The growth of both strains was inhibited to below 10% of the control in the presence of 0.1 microgram/mL or higher concentrations of doxycycline. We suggest that our approach is more quantitative and reproducible than the conventional microscopic methods.

Keyword

Orientia tsutsugamushi; Doxycycline; Antibodies, Monoclonal; Flow Cytometry; Antibiotic Susceptibility; Microbial Sensitivity Tests

MeSH Terms

Orientia tsutsugamushi/*drug effects/growth & development
Microbial Sensitivity Tests/*methods
Flow Cytometry/*methods
Antibodies, Monoclonal/*immunology

Figure

• 

  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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  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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  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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