Cell Extract-Containing Medium for Culture of Intracellular Fastidious Bacteria

Abstract

The culture of fastidious microorganisms is a critical step in infectious disease studies. As a proof-of-concept experiment, we evaluated an empirical medium containing eukaryotic cell extracts for its ability to support the growth of Coxiella burnetii. Here, we demonstrate the exponential growth of several bacterial strains, including the C. burnetii Nine Mile phase I and phase II strains, and C. burnetii isolates from humans and animals. Low-oxygen-tension conditions and the presence of small hydrophilic molecules and short peptides were critical for facilitating growth. Moreover, bacterial antigenicity was conserved, revealing the potential for this culture medium to be used in diagnostic tests and in the elaboration of vaccines against C. burnetii. We were also able to grow the majority of previously tested intracellular and fastidious bacterial species, including Tropheryma whipplei, Mycobacterium bovis, Leptospira spp., Borrelia spp., and most putative bioterrorism agents. However, we were unable to culture Rickettsia africae and Legionella spp. in this medium. The versatility of this medium should encourage its use as a replacement for the cell-based culture systems currently used for growing several facultative and putative intracellular bacterial species.

Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project's Most Wanted taxa

“…Although these microbes could be grown in their natural habitats (9), where effects such as cross-feeding (11) and microbe-host interactions (12,13) are present, some biological samples, such as clinical biopsies, are often limited in quantity. This makes it challenging to set up cultivation experiments in large scale with these native media, but creates opportunities for miniaturized methods.…”

Current and Past Strategies for Bacterial Culture in Clinical Microbiology

“…Singh et al developed an axenic liquid medium based on an extract of eukaryotic cells, allowing the growth of a broad spectrum of bacteria. The eukaryotic cell extract-based universal empirical medium, which was tested for culturing of C. burnetii as a proof of concept, supported the growth of other fastidious bacteria and putative bacterial bioter- rorism agents (120). Rapid growth (Ͻ72 h) was observed for T. whipplei, Yersinia pestis, Vibrio cholerae, Bacillus anthracis, Shigella dysenteriae, Brucella melitensis, Bordetella pertussis, and Bartonella henselae.…”

“…Moreover, colonies were observed on ACCM-2 agarose (372). More recently, Singh et al showed the exponential growth of C. burnetii in an empirical medium containing eukaryotic cell extracts (120). With the medium being refreshed every 2 days, low oxygen tension and the presence of small hydrophilic molecules and short peptides were critical for its growth.…”

“…Solid media in miniaturized formats adapted to automated scanners could become the standard for culture and first-line antibiotic susceptibility testing (M. Drancourt, unpublished data). Axenic culture of as-yet-uncultured pathogens is another exciting challenge, as is the discovery of new opportunistic pathogens using the extended spectrum of axenic cell extract-containing media, such as that used for Mycobacterium bovis (120), and cell-based systems, including protozoa (150), offered by culturomics (154).…”

“…Treponema pallidum subsp. pallidum can be propagated only in laboratory animals (rabbits) by intratesticular, intradermal, intravenous, or intracisternal inoculation (120). T. pallidum grows slowly, with a doubling time of 30 to 33 h (107), and a mean of 10 10 bacteria has been harvested from the testis of a rabbit.…”