tsutsugamushistrain or isolate. as chloramphenicol, tetracyclines, macrolides, and rifampin have shown efficacious clinical outcomes [2]. In 1996, delayed clinical improvement was reported in patients who were infected by doxycycline-insensitiveO. tsutsugamushistrains in Chiang Rai, Thailand [3]. After treatment with antibiotics, mice infected with these strains showed higher mortality rates than those infected with the Karp strain, an antibiotic-susceptible strain [3,4].In vitroresistance to doxycycline has also been observed [3,5]. However, there has been no investigation into the mechanism responsible for the delayed defervescence in scrub typhus. We previously performed several experiments to characterize the antibiotic susceptibility ofO. tsutsugamushistrains. During these experiments, the bacterial counts in cultures of the AFSC-4 strain, a doxycycline-insensitive strain [5], were higher than those in cultures of the Boryong strain, an antibiotic-susceptible strain, regardless of whether these cultures contained antibiotics. We hypothesized that if the AFSC-4 strain was capable of growing more rapidly than other strains, a higher amount of bacteria would be present at the time of antibiotic susceptibility testing. Thus, the rapidly growing strain would appear to be resistant to antibiotics, even when the antibiotic susceptibilities of these strains might not be different from one another. To verify this hypothesis, we compared the infectivity and growth rates of variousO. tsutsugamushistrains. Twelve strains ofO. tsutsugamushiwere evaluated in this study: 6 strains were laboratory-adapted strains and the other 6 were clinical isolates. The AFSC-4 strain was kindly provided by Dr. Daniel Strickman, Naval Medical Research Institute, Bethesda, MD, USA. Other laboratory-adapted strains were Boryong, Gilliam (ATCC VR-312), Karp (ATCC VR-150), Kato (Niigata strain), and Kuroki. The 6 clinical isolates were passaged 4-6 times in ECV304 cells after isolation from patients and stored at -70 until use. Sequence analysis of the gene encoding the 56-kDa type-specific antigen ofO. tsutsugamushiidentified these isolates as the following genotypes: Boryong (Kuroki clade), Neimeng-65 (Gilliam clade), Yonchon (Japanese Gilliam clade), Je-cheon (Karp-related clade), and Young-worl (Saitama clade) [6]. Stock solutions of eachO. tsutsugamushistrain were inoculated onto monolayers of ECV304 cells seeded in culture flasks (75 cm2). When the infected ECV304 cells showed maximum cytopathic effects, the degree of infection of ECV304 cells as assessed by immunofluorescence (IF) staining was found to be approximately equal in each experiment. The cells were disrupted with glass beads and centrifuged at 350 g for 5 min. To avoid the death of ECV304 cells by overgrowth ofO. tsutsugamushiwithin 72 hr, the resultant supernatant was divided equally into 40 aliquots, of which 4 aliquots were used. The amount of each inoculum was one-tenth the amount that is usually used in antibiotic susceptibility tests in our laboratory. Each aliquot was inoculated onto monolayers of ECV304 cells grown in shell vials and incubated for 4 hr. At the end of Bay 41-4109 less active enantiomer the initial incubation period, the inocula were replaced with fresh medium and the cultures were maintained for 72 hr. Immunofluorescence staining was performed at 4, 24, 48, and 72 hr post-inoculation (hpi) withO. tsutsugamushi. ECV304 cells infected with each strain ofO. tsutsugamushion a cover slip of the shell vial were fixed with Bay 41-4109 less active enantiomer acetone. The infected cells were then stained with mouse polyclonal antibodies in phosphate-buffered saline (PBS) for 30 min at 37, washed briefly with PBS Tween-20 (PBST), treated with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (Jackson ImmunoResearch, West Grove, PA, USA.) for 30 min in a moist chamber, and finally washed 3 Bay 41-4109 less active enantiomer times with PBST. To clearly defineO. tsutsugamushi, the host cells were counter-stained with 0.003% Evans blue in PBS. After the slides had been washed, they were placed in mounting medium (Vector Laboratories, Burlingame, CA, USA) and examined at 400 magnification under a fluorescence microscope (Zeiss, Germany) with a confocal laser scanning system (Bio-Rad, Hercules, CA, USA). The IF-positive foci per field were enumerated. Although the ECV304 cells were inoculated with approximately equal amounts of eachO. tsutsugamushistrain, quantification of the bacterial number in each culture at 4 hpi revealed differences among the strains or isolates (Fig. 1). Interestingly, the bacterial count in a culture of the AFSC-4 strain Bay 41-4109 less active enantiomer was 2-5 Rac-1 times higher than that in cultures of other strains or isolates at 4 hpi. Additionally, the numbers of.