Occasionally, combinations of antibiotics are used for the treatment of scrub typhus. However, the effectiveness of such combined therapies has rarely been evaluated. To date, no experimental studies have been performed; only 1 clinical study has assessed the efficacy of combined doxycycline and rifampin therapy. To elucidate the efficacies of other antibiotic combinations, we performed an experiment to evaluate the in vitro efficacy of antibiotic combinations against Orientia tsutsugamushi.

O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were cultured in antibiotic-containing media for 3-5 days and stained with FS15, a monoclonal antibody reacting against the linear epitope on the 56-kDa major outer membrane protein of O. tsutsugamushi. Thereafter, antimicrobial susceptibility was measured by flow cytometry and expressed as a growth index (total mass of Orientia). The growth indices of doxycycline (0.1 µg/mL), azithromycin (0.1 µg/mL), rifampin (0.0125 µg/mL), cefotaxime (2 and 20 µg/mL), and their various combinations (doxycycline+cefotaxime, doxycycline+rifampin, azithromycin+cefotaxime, and rifampin+cefotaxime) were measured. The above mentioned antibiotic concentrations, except for that of cefotaxime, represent the minimal inhibitory concentrations of each antibiotic.

The growth indices of doxycycline (4.67% and 0.52%), rifampin (2.35% and 0.26%), and azithromycin (7.54%) were within the range of full suppression of O. tsutsugamushi; in contrast, cefotaxime (87.60%) was in effective. The growth indices of doxycycline+rifampin were 0.10% and 0.10%, which were similar to those obtained with doxycycline or rifampin alone. The growth indices of doxycycline+cefotaxime were 3.99% and 3.65% in low-dose cefotaxime (2 µg/mL), and 3.69% and 4.40% in high-dose cefotaxime (20 µg/mL). The growth indices of rifampin+cefotaxime (2.19% and 2.19% at 2 µg/mL; 1.84% and 2.04% at 20 µg/mL cefotaxime) were similar to those obtained with rifampin alone (2.35% and 0.26%). Azithromycin+cefotaxime (11.06-14.63%) showed higher growth indices than azithromycin alone; this suggests that this combination may be antagonistic.

The anti-Orientia efficacies of doxycycline+rifampin, doxycycline+cefotaxime, and rifampin+cefotaxime were not antagonistic. The efficacy of the azithromycin+cefotaxime combination needs to be confirmed by more sensitive methods to exclude the possibility of antagonistic interactions between the antibiotics.