J Korean Med Sci.  2014 Feb;29(2):183-189. 10.3346/jkms.2014.29.2.183.

In Vitro Bacteriostatic Effects of Rifampin on Orientia tsutsugamushi

• Im JH
• Baek JH
• Lee JS
• Chung MH
• Lee SM
• Kang JS

• KMID: 1789973
• DOI: http://doi.org/10.3346/jkms.2014.29.2.183

Abstract

We performed an in vitro cell culture experiment to ascertain whether rifampin exhibits bactericidal effects against Orientia tsutsugamushi, the causative agent of scrub typhus. ECV304 cells were infected with the Boryong or AFSC-4 strain of O. tsutsugamushi and then, the cultures were maintained in media with increasing concentrations of rifampin, azithromycin, doxycycline, or chloramphenicol for 4 days. On day 5, the media were replaced with fresh antibiotic-free medium and the cultures were maintained until day 28. On days 5, 13, and 28, immunofluorescence (IF) staining of O. tsutsugamushi was performed. IF staining on days 13 and 28 revealed increasing numbers of IF-positive foci in all cultures, even in cultures initially exposed to the highest concentration of rifampin (80 microg/mL), azithromycin (80 microg/mL), doxycycline (20 microg/mL), or chloramphenicol (100 microg/mL). The present study reveals that rifampin has no bactericidal effect against O. tsutsugamushi as observed for azithromycin, doxycycline, and chloramphenicol. A subpopulation of the bacteria that are not killed by high concentrations of the antibiotics may explain the persistence of O. tsutsugamushi in humans even after complete recovery from scrub typhus with antibiotic therapy.

Keyword

Antibiotic Susceptibility Test; Azithromycin; Drug Resistance, Bacterial; Orientia tsutsugamushi; Rifampin; Scrub Typhus

MeSH Terms

Antibiotics, Antitubercular/*pharmacology
Cell Line, Tumor
Drug Resistance, Bacterial
Fluorescent Antibody Technique
Humans
Orientia tsutsugamushi/*drug effects/growth & development/metabolism
Rifampin/*pharmacology
Antibiotics, Antitubercular
Rifampin

Figure

• 

  Fig. 1 Serial photographs of immunofluorescence staining of ECV304 cells infected with Orientia tsutsugamushi (×200). Infected ECV304 cells were incubated in medium containing rifampin of increasing concentrations for 4 days. Then, the inocula were replaced with antibiotic-free medium and the culture was continued until day 28. The highest concentration of rifampin at which IF-positive foci are observed is 80 µg/mL on days 5, 13, and 28 in cultures infected by the Boryong or AFSC-4 strain. Cultures in medium not containing rifampin showed cell death due to overgrowth of O. tsutsugamushi before day 13.

• 

  Fig. 2 Serial photographs of infected ECV304 cells in medium containing azithromycin (see Fig. 1 for details) (×200). In the Boryong strain-infected cells, the highest concentrations of azithromycin at which IF-positive foci are observed are 10, 80, and 80 µg/mL on days 5, 13, and 28, respectively. In the AFSC-4 strain-infected cells, the highest concentration is 80 µg/mL on days 5, 13, and 28.

• 

  Fig. 3 Serial photographs of infected ECV304 cells in medium containing doxycycline (see Fig. 1 for details) (×200). Doxycycline at 40 and 80 µg/mL was toxic, resulting in death of ECV304 cells. In the Boryong strain-infected cells, the highest doxycycline concentrations at which IF-positive foci are observed are 10, 20, and 20 µg/mL on days 5, 13, and 28, respectively. In the AFSC-4 strain-infected cells, the highest concentration is 20 µg/mL on days 5, 13, and 28. Cultures infected with the AFSC-4 strain in medium containing 0.1 µg/mL doxycycline showed cell death because of overgrowth of O. tsutsugamushi on day 20.

• 

  Fig. 4 Serial photographs of infected ECV304 cells in medium containing chloramphenicol (see Fig. 1 for details) (×200). In the Boryong strain-infected cells, the highest concentrations of chloramphenicol at which IF-positive foci are observed are 40, 100, and 100 µg/mL on days 5, 13, and 28, respectively. In the AFSC-4 strain-infected cells, the highest concentration is 100 µg/mL on days 5, 13, and 28.

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