Formation and cell-medium partitioning of autoinhibitory free fatty acids and cyclodextrin's effect in the cultivation of Bordetella pertussis

Fröhlich, B.; d’Alarcao, Marc; Feldberg, Ross S.; Nicholson, Mary L.; Siber, George R.; Swartz, Randall W.

doi:10.1016/0168-1656(95)00155-7

Cited by 16 publications

(19 citation statements)

References 34 publications

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“…Cyclodextrins can also replace FBS in Helicobacter pylori culture medium (19) and similarly improves growth of Bordetella pertussis (17). Improved growth may result from M␤-CD sequestration of inhibitory metabolites, such as certain fatty acids (11).…”

Section: Discussionmentioning

confidence: 99%

Isolation from Animal Tissue and Genetic Transformation of Coxiella burnetii Are Facilitated by an Improved Axenic Growth Medium

Omsland¹,

Beare²,

Hill

et al. 2011

Appl Environ Microbiol

192

258

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We recently described acidified citrate cysteine medium (ACCM), which supports host cell-free (axenic) growth of Coxiella burnetii. After 6 days of incubation, greater than 3 logs of growth was achieved with the avirulent Nine Mile phase II (NMII) strain. Here, we describe modified ACCM and culture conditions that support improved growth of C. burnetii and their use in genetic transformation and pathogen isolation from tissue samples. ACCM was modified by replacing fetal bovine serum with methyl-␤-cyclodextrin to generate ACCM-2. Cultivation of NMII in ACCM-2 with moderate shaking and in 2.5% oxygen yielded 4 to 5 logs of growth over 7 days. Similar growth was achieved with the virulent Nine Mile phase I and G isolates of C. burnetii. Colonies that developed after 6 days of growth in ACCM-2 agarose were approximately 0.5 mm in diameter, roughly 5-fold larger than those formed in ACCM agarose. By electron microscopy, colonies consisted primarily of the C. burnetii small cell variant morphological form. NMII was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice. A completely axenic C. burnetii genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 1/2 weeks. Transformation experiments demonstrated clonal populations in colonies and a transformation frequency of approximately 5 ؋ 10 ؊5 . Cultivation in ACCM-2 will accelerate development of C. burnetii genetic tools and provide a sensitive means of primary isolation of the pathogen from Q fever patients.Coxiella burnetii is a wide-ranging zoonotic pathogen that causes a debilitating influenza-like illness in humans called Q fever (20). Following infection of a eukaryotic host cell, this intracellular bacterium replicates exclusively within a phagolysosome-like parasitophorous vacuole (PV) (16). C. burnetii directs development of the PV (15, 24) and manipulates other host cell functions such as apoptotic signaling (18,29). The mechanisms by which C. burnetii modifies the host cell and causes disease are largely unknown. Indeed, lipopolysaccharide is the only defined virulence factor of C. burnetii with the full-length molecule of phase I bacteria required for full virulence (21).The obligate intracellular nature of C. burnetii has severely impeded development of genetic tools for virulence factor discovery. Beare et al. (3) recently genetically transformed C. burnetii to chloramphenicol resistance and mCherry red fluorescent protein expression using the mariner-based Himar1 transposon. However, a significant limitation of the procedure is the 8 to 12 weeks required for expansion and clonal isolation of individual transposon mutants using cell culture-based propagation of C. burnetii. Moreover, the micromanipulation method used for harvesting clonal C. burnetii from an individual PV is technically challenging.The recent description of a method for axenic (host cellfree) propagation of C. burnetii is a significant...

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Section: Discussionmentioning

confidence: 99%

Isolation from Animal Tissue and Genetic Transformation of Coxiella burnetii Are Facilitated by an Improved Axenic Growth Medium

Omsland¹,

Beare²,

Hill

et al. 2011

Appl Environ Microbiol

192

258

View full text Add to dashboard Cite

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“…Still, investigators have yet to decipher the mechanism for the reduction in the concentration of Ptx per optical density unit ([Ptx]/OD) commonly observed in wild-type strains grown in shake flasks and fermentation containing standard and more complex media (15,16). In this study, we show that B. pertussis autoregulates Ptx production through the metabolic conversion of cysteine to SO 4 2Ϫ and pyruvic acid.…”

mentioning

confidence: 74%

“…Studies have shown that virulence factor expression is influenced by temperature, pH, glutamate levels, free fatty acids, and environmental compounds like nicotinic acid and SO 4 2Ϫ (15,16,23,25,26,35,37,56). Addition of heptakis-(2,6-Odimethyl)-␣-cyclodextrin to the medium increases Ptx yields significantly (15,16,58).…”

Section: Discussionmentioning

confidence: 99%

“…Addition of heptakis-(2,6-Odimethyl)-␣-cyclodextrin to the medium increases Ptx yields significantly (15,16,58). However, addition of this complex compound to the growth medium may pose adverse effects if it contaminates the formulated vaccine product.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the presence of free fatty acids in the media has been reported to be either inhibitory or stimulatory for growth (13). Addition of heptakis-(2,6-O-dimethyl)-␣-cyclodextrin to sequester free fatty acids has significantly increased Ptx production (15,16,58). Outer membrane vesicles produced in Stainer-Scholte cultures contain significant amounts of adenylate cyclase, lipopolysaccharide, and Ptx (18).…”

mentioning

confidence: 99%

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