The relapsing fever spirochete Borrelia miyamotoi is cultivable in a modified Kelly-Pettenkofer medium, and is resistant to human complement

Abstract

BackgroundBorrelia miyamotoi is a relapsing fever spirochete found in Ixodes ticks in North America, Europe, and Asia, and has recently been found to be invasive in humans. Cultivation of this spirochete has not yet been described, but is important for patient diagnostics and scientific purposes. Host specificity of Borrelia species is dependent on resistance to host complement (serum resistance), and since B. miyamotoi has been identified as a human pathogen we were interested whether B. miyamotoi is resistant to human complement.MethodsWe inoculated B. miyamotoi strains LB-2001 and HT31 in modified-Kelly-Pettenkofer medium with 10% fetal calf serum (MKP-F), and used standard non-laborious Borrelia culture methods to culture the spirochetes. Next, we assessed serum sensitivity by a direct killing assay and a growth inhibition assay.ResultsWe were able to passage B. miyamotoi over 10 times using a standard culture method in MKP-F medium, and found B. miyamotoi to be resistant to human complement. In contrast to B. miyamotoi, Borrelia anserina - a relapsing fever spirochete unrelated to human infection- was serum sensitive.ConclusionsUsing a variation on MKP medium we were able to culture B. miyamotoi, opening the door to in vitro research into this spirochete. In addition, we describe that B. miyamotoi is resistant to human complement, which might play an important role in pathogenesis. We have also found B. anserina to be sensitive to human complement, which might explain why it is not related to human infection. Summarizing, we describe a novel culture method for B. miyamotoi and show it is resistant to human complement.

In Vitro Susceptibility of the Relapsing-Fever Spirochete Borrelia miyamotoi to Antimicrobial Agents

“…Spirochetes were enumerated using a Petroff-Hausser counting chamber and dark-field microscopy. B. burgdorferi sensu stricto B31 and B. afzelii PKo were cultured using modified Kelly-Pettenkofer (MKP) medium, and B. miyamotoi HT31, B. miyamotoi LB-2001, and B. hermsii HS1 were cultured using modified KellyPettenkofer medium with the addition of 10% heat-inactivated fetal calf serum (MKP-F medium), as described previously (14).…”

“…MIC measurement by dark-field microscopy. Spirochetes were counted in all wells after 72 h by dark-field microscopy, as described previously (14). Briefly, 5 l from each well was put on a microscopy slide and covered by a coverslip.…”

“…The antimicrobial susceptibility of B. miyamotoi has not yet been elucidated, due to difficulties with the cultivation of B. miyamotoi spirochetes in vitro, which has been reported only recently (13)(14)(15). In contrast, the in vitro susceptibilities of the causative agents of Lyme borreliosis and several RF Borrelia spirochetes have been extensively studied (16)(17)(18)(19).…”

The ecology of Lyme borreliosis risk : interactions between lxodes ricinus, rodents and Borrelia burgdorferi sensu lato

“…In experiment 2, mouse spleens and ears collected at the end of the experiment were cultured in MKP-F medium as described in detail previously (Wagemakers et al 2014). In short: a 4 mm tissue biopsy was disinfected and placed in 7 ml Modified Kelly-Pettenkofer medium containing rifampicin, fosfomycin and amphotericin B as antibiotics.…”

Borrelia miyamotoi: egy újabb, humán patogén, kullancs által terjesztett, visszatérő lázat okozó baktérium

“…A B. miyamotoi 2014 óta sikeresen tenyészthető mó-dosított Kelly-Pettenkofer-médiumban [43]. Ez valószí-nűleg segíteni fog abban, hogy közelebbről megért-hessük a kórokozó fertőzésdinamikáját, gazdában és vektorban való túlélésének, valamint az emberben kialakított kórképének a kulcsfaktorait.…”