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

BMC Microbiology

Methodology Article

Motohiko Ogawa¹  Masaaki Satoh¹  Shuji Ando¹  Tsuneo Uchiyama² 

[1] Department of Virology 1, National Institute of Infectious Diseases/1-23-1, Toyama, Shinjuku-ku, 162-8640, Tokyo, Japan;Institute of Health Biosciences, The University of Tokushima Graduate School/3-18-15, Kuramoto-cho, 770-8503, Tokushima, Japan;

关键词: Orientia tsutsugamushi;    Intracellular bacteria;    Mycoplasma;    Contamination;    Elimination;    Cell culture;    Antibiotics;   

DOI  :  10.1186/1471-2180-13-32

 received in 2012-08-02, accepted in 2013-01-17,  发布年份 2013

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Ogawa et al.; licensee BioMed Central Ltd. 2013