Decontamination of mycoplasma-contaminated Orientia tsutsugamushi strains by repeating passages through cell cultures with antibiotics

Abstract

BackgroundMycoplasmas-contamination of Orientia tsutsugamushi, one of the obligated intracellular bacteria, is a very serious problem in in vitro studies using cell cultures because mycoplasmas have significant influence on the results of scientific studies. Only a recommended decontamination method is to passage the contaminated O. tsutsugamushi strains through mice to eliminate only mycoplasmas under influence of their immunity. However, this method sometimes does not work especially for low virulent strains of O. tsutsugamushi which are difficult to propagate in mice. In this study, we tried to eliminate mycoplasmas contaminants from both high virulent and low virulent strains of the contaminated O. tsutsugamushi by repeating passage through cell cultures with antibiotics in vitro.ResultsWe cultured a contaminated, high virulent strain of O. tsutsugamushi using a mouse lung fibroblasts cell line, L-929 cell in the culture medium containing lincomycin at various concentrations and repeated passages about every seven days. At the passage 5 only with 10 μg/ml of lincomycin, we did not detect mycoplasmas by two PCR based methods whereas O. tsutsugamushi continued good growth. During following four passages without lincomycin, mycoplasmas did not recover. These results suggested that mycoplasmas were completely eliminated from the high virulent strain of O. tsutsugamushi. Furthermore, by the same procedures with 10 μg/ml of lincomycin, we also eliminated mycoplasmas from a contaminated, low virulent strain of O. tsutsugamushi. Our additional assay showed that 50 μg/ml of lyncomycin did not inhibit the growth of O. tsutsugamushi, although MICs of many mycoplasmas contaminants were less than 6 μg/ml as shown previously.ConclusionOur results showed an alternative method to eliminate mycoplasmas from the contaminated O. tsutsugamushi strains in place of in vivo passage through mice. Especially this notable method works for the decontamination not only from the high virulent strain also from the low virulent strain of O. tsutsugamushi. For further elimination, lincomycin at the limit concentration, which does not inhibit the growth of O. tsutsugamushi, can possibly eliminate most mycoplasmas from contaminated O. tsutsugamushi strains.

Rickettsia amblyommatis sp. nov., a spotted fever group Rickettsia associated with multiple species of Amblyomma ticks in North, Central and South America

“…To obtain a Mycoplasma -free stock of strain WB-8-2 T for deposition and distribution, 50 μg ml −1 lincomycin was added to the medium and cell cultures were exposed to the antibiotic-supplemented medium for 6 weeks (Ogawa et al , 2013). Cultures were shown to be negative for infection with Mycoplasma species at 4, 5 and 6 weeks by using the PCR assay (data not shown), after which time the antibiotic was removed from the medium.…”

Anti-Mycoplasma Activity of Daptomycin and Its Use for Mycoplasma Elimination in Cell Cultures of Rickettsiae

“…Mycoplasma organisms (except M. pneumoniae ) are susceptible to lincosamides, including clindamycin [2,15,27,28], whereas Rickettsia and Orientia are high-level resistant to clindamycin (MIC > 32 mg/L, Table 1). Studies have reported that lincosamides can decontaminate cell cultures from mycoplasmas [16,29]. However, lincosamides are bacteriostatic, and the MBC/MIC ratio was reportedly higher than 32 [30].…”

“…hominis and M . orale ) were tested in that study [16]. Fluoroquinolones and aminoglycosides have the potential for mycoplasma decontamination in Orientia culture due to the intrinsic resistance of Orientia to these classes of antibiotics [17].…”

The intracellular pathogen Orientia tsutsugamushi responsible for scrub typhus induces lipid droplet formation in mouse fibroblasts

“…The mycoplasma-free, highly virulent Ikeda strain of O. tsutsugamushi [9] was used in this study. For the inoculation, the Ikeda strain was deposited on monolayers of L-929 cells for 1 h. The inoculated cells were washed twice with Dulbecco's phosphate buffered saline (PBS, SigmaeAldrich Japan Co.…”