Hard-tick-borne relapsing fever (HTBRF) is an emerging infectious disease throughout the temperate zone caused by the relapsing-fever spirochete Borrelia miyamotoi. Antibiotic treatment of HTBRF is empirically based on the treatment of Lyme borreliosis; however, the antibiotic susceptibility of B. miyamotoi has not been studied to date. Thus, we set out to determine the in vitro antimicrobial susceptibility of B. miyamotoi. A microdilution method with 96-well microtiter plates was used to determine the antibiotic susceptibilities of two B. miyamotoi strains isolated on two different continents (Asia and North America), two Borrelia burgdorferi sensu lato strains, and one Borrelia hermsii isolate for purposes of comparison. The MIC and minimal bactericidal concentration (MBC) were determined by both microscopy and colorimetric assays. We were able to show that relative to the B. burgdorferi sensu lato isolates, both B. miyamotoi strains and B. hermsii demonstrated greater susceptibility to doxycycline and azithromycin, equal susceptibility to ceftriaxone, and resistance to amoxicillin in vitro. The MIC and MBC of amoxicillin for B. miyamotoi evaluated by microscopy were 16 to 32 mg/liter and 32 to 128 mg/liter, respectively. Since B. miyamotoi is susceptible to doxycycline, azithromycin, and ceftriaxone in vitro, our data suggest that these antibiotics can be used for the treatment of HTBRF. Oral amoxicillin is currently used as an alternative for the treatment of HTBRF; however, since we found that the B. miyamotoi strains tested were resistant to amoxicillin in vitro, this issue warrants further study. KEYWORDS hard-tick-borne relapsing fever, relapsing-fever borrelia, Borrelia miyamotoi, Borrelia miyamotoi disease, antibiotic susceptibility, antimicrobials T he relapsing-fever spirochete Borrelia miyamotoi was first described in Japan in 1995 (1). While it is phylogenetically closely related to other relapsing-fever (RF) spirochetes, such as Borrelia hermsii, Borrelia turicatae (transmitted by soft ticks), and Borrelia recurrentis (transmitted by body lice), it is transmitted by hard-bodied Ixodes ticks across the temperate zone (2). Ixodes ticks concomitantly transmit spirochetes belonging to the Borrelia burgdorferi sensu lato group, which are known to cause Lyme borreliosis (3). The incidence of B. miyamotoi in Ixodes ticks is lower than that of B. burgdorferi sensu lato, with infection rates ranging from 0 to 15.4% in the United States and as high as 4% in Europe and Japan (2, 4, 5). RF Borrelia spirochetes cause a variety of diseases, which are characterized by episodes of high fever separated by periods of relative well-being. The clinical presentation of disease caused by B. miyamotoi, however, appears to differ from that of classical RF, since relapsing-fever episodes have been observed only in 10% of patients infected with B. miyamotoi, and their levels of spirochetemia are calculated to be low (6, 7). Since most patients with B. miyamotoi infection are usually treated with antibiotics, this ...