Biphasic culture system for rapid Campylobacter cultivation

Rollins, David M.; Coolbaugh, James C.; Walker, Richard I.; Weiss, Erik D.

doi:10.1128/aem.45.1.284-289.1983

Cited by 43 publications

(14 citation statements)

References 18 publications

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“…Consequently, quantitative comparisons of actual numbers of adherent bacteria obtained from different experiments should be made with caution. In addition, the reversible expression of flagella (4) and the peculiar morphologic changes seen in C. jejuni strains (25) may have also contributed to interexperimental variation, although routine microscopic examination of the cultures for motile, spiral forms was done to reduce this possibility. The reproducibility of the assay, however, was very good in that it was never difficult to distinguish adherent C. jejuni strains bound to INT 407 cells or mucus from the BSA and polystyrene controls.…”

Section: Resultsmentioning

confidence: 99%

“…Blood agar plates were incubated at 42°C in polybags (Levin Brothers Paper Co., Chicago, Ill.) with an atmosphere of 85% N2-10% C02-5% 02. Growth from the blood agar plates was subsequently used to inoculate 25-cm2 tissue culture flasks containing the biphasic culture system described by Rollins et al (25).…”

Section: Methodsmentioning

confidence: 99%

“…Labeling of the bacteria. Campylobacter strains were grown in a biphasic brucella broth-brucella agar system (25) supplemented with 0.01 mCi of sodium [3H]acetate per ml (specific activity, 90 mCi/mmol; New England Corp. Boston, Mass.). The inoculated tissue culture flasks were incubated at 370 for 18 to 20 h. Following incubation the cells were harvested, washed twice in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Hanks (HH) buffer (pH 7.4) and suspended in the same buffer.…”

Section: Methodsmentioning

confidence: 99%

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Identification and characterization of two Campylobacter jejuni adhesins for cellular and mucous substrates

McSweegan¹,

Walker²

1986

Infect Immun

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232

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Campylobacterjejuni is able to colonize the human intestinal mucosa and cause disease. For this reason, it was important to investigate mechanisms by which C. jejuni adheres to epithelial cells and intestinal mucus gel. All strains of C. jejuni used were able to adhere to INT 407 epithelial cells and mucus, but high adherence to one substrate did not necessarily indicate comparable adherence to the other. The adherence of C. jejuni to cells was inhibited partially by treating the bacterial cells with proteases or glutaraldehyde or by adding a certain carbohydrate (fucose or mannose) to the medium. The flagellum of C. jejuni was identified as a potential adhesin by comparing adherence of flagellated and aflagellated variants. Shearing of the bacterial cells to remove the flagella reduced bacterial adhesion, whereas immobilization of the flagellum with KCN increased adhesion. Purified flagella showed specific, fucose-resistant binding to epithelial cells but not to intestinal mucus. The presence of a second, nonproteinaceous adhesin was suggested because no single treatment of the bacteria completely inhibited adhesion. Lipopolysaccharide (LPS) was identified as another C. jejuni adhesin. [3H]LPS specifically bound to epithelial cells, and this phenomenon was inhibited by periodate oxidation of the LPS or glutaraldehyde fixation of the epithelial cells. LPS, unlike flagella, was fucose sensitive and inhibited binding of whole bacterial cells to INT 407 cells. LPS was also able to bind to intestinal mucus gel. These data indicate that both flagella and LPS are important in adhesion to the mucosal surface. Campylobacter jejuni is a frequent agent of diarrhea in humans (5), yet the interplay of its virulence factors in pathogenesis is poorly understood. The mechanisms by which C. jejuni interacts with the intestinal mucosa and initiates diarrhea are also unclear. Adhesion of bacteria to mucosal surfaces is necessary for colonization and subsequent pathogenesis. This process is mediated by chemotactic factors (9) and bacterial appendages (adhesins) which can include pili, flagella, capsules, glycocalyces, lipopolysaccharides, and cell-associated lectins (1, 2, 7). Complete evaluation of adhesion mechanisms of many organisms, however, has only recently begun. Newell and Pearson (24) used scanning electron microscopy to demonstrate in vitro adherence of C. jejuni to intestinal epithelial cells and saw morphologic evidence that this adhesion could be mediated by flagella. Later, flagellated organisms were shown to adhere in greater numbers to cells than did an aflagellated variant (23). Dijs and De Graaf (8) showed that C. jejuni cells do not possess fimbriae or pili but are able to agglutinate a variety of mammalian erythrocytes. Later, Naess et al. (22) were able to show in vitro adherence of C. jejuni strains to porcine small intestinal brush border preparations. Cinco et al. (6) also studied Campylobacter adherence to intestinal epithelial cells. The adhesion was inhibited partially by L-fuCose and D-mannose. This sug...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Identification and characterization of two Campylobacter jejuni adhesins for cellular and mucous substrates

McSweegan¹,

Walker²

1986

Infect Immun

Self Cite

232

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“…Cells to be used in metabolic experiments were cultivated from frozen stock cultures. Following a passage on 5% sheep blood agar in an atmosphere of 5% 02-10% C02-85% N2, the cells were passaged twice at 37°C in biphasic medium (18). The incubation time was 12 h unless otherwise indicated.…”

Section: Methodsmentioning

confidence: 99%

Substrate utilization by Campylobacter jejuni and Campylobacter coli

Westfall¹,

Rollins²,

Weiss³

1986

Appl Environ Microbiol

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An attempt was made to elucidate in Campylobacter spp. some of the physiologic characteristics that are reflected in the kinetics of CO2 formation from four '4C-labeled substrates. Campylobacterjejuni and C. coli were grown in a biphasic medium, and highly motile spiral cells were harvested at 12 h. Of the media evaluated for use in the metabolic tests, minimal essential medium without glutamine, diluted with an equal volume of potassium sodium phosphate buffer (pH 7.2), provided the greatest stability and least competition with the substrates to be tested. The cells were incubated with 0.02 M glutamate, glutamine, a-ketoglutarate, or formate, or with concentrations of these substrates ranging from 0.0032 to 0.125 M. All four substrates were metabolized very rapidly by both species. A feature of many of these reactions, particularly obvious with a-ketoglutarate, was an immediate burst of CO2 production followed by CO2 evolution at a more moderate rate. These diphasic kinetics of substrate utilization were not seen in comparable experiments with Escherichia coli grown and tested under identical conditions. With C. jejuni, C02 production from formate proceeded rapidly for the entire period of incubation. The rate of metabolism of glutamate, glutamine, and aketoglutarate by both species was greatly enhanced by increased substrate concentration. The approach to the study of the metabolism of campylobacters here described may be useful in detecting subtle changes in the physiology of cells as they are maintained past their logarithmic growth phase.

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“…The Campylobacter culture was thawed rapidly, diluted 1:10 with brucella broth (BBL Microbiology Systems, Cockeysville, Md. ), and inoculated into a biphasic medium (16) in 25-cm2, plastic tissue culture flasks (Costar, Cambridge, Mass.). The medium consisted of 5 ml of brucella broth plus 1.5% agar and 7.5% sheep blood, overlaid with 5 ml of brucella broth supplemented with 2.5 mM cysteine hydrochloride (Calbiochem-Behring Corp., La Jolla, Calif.) and 24 mM serine (Calbiochem-Behring).…”

Section: Methodsmentioning

confidence: 99%

Analysis of fatty acids of the genus Rochalimaea by electron capture gas chromatography: detection of nonanoic acid

Westfall¹,

Edman²,

Weiss³

1984

J Clin Microbiol

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The fatty acid compositions of Rochalimaea quintana, strains Fuller and Guadalupe, and R. vinsonii, the Canadian vole agent, were determined in an effort to further characterize these bacteria. The cells were saponified with 5% NaOH in 50% methanol and acidified to pH 2. The methanolysates were extracted with chloroform, derivatized with 2,2,2-trichloroethanol, and analyzed using a Hewlett-Packard gas chromatograph equipped with a frequency pulse-modulated electron capture detector and a 3% OV-101 packed-glass column. The fatty acid profiles of the three Rochalimaea strains were similar, with octadecenoic acid (C18:1) the most abundant, followed by octadecanoic (C18:0) and hexadecanoic (C160) acids. Moderate to trace amounts of other acids were also present. Unexpectedly, well-defined peaks of nonanoic acid (C9) were found consistently. A portion of this acid, but not all, was extractable with chloroform. Since Cg is not reported as a usual component of bacteria and most analyses do not include a search for this fatty acid, this study was extended to three strains of Legionella and one of Campylobacter. Comparable results were

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Biphasic culture system for rapid Campylobacter cultivation

Cited by 43 publications

References 18 publications

Identification and characterization of two Campylobacter jejuni adhesins for cellular and mucous substrates

Identification and characterization of two Campylobacter jejuni adhesins for cellular and mucous substrates

Substrate utilization by Campylobacter jejuni and Campylobacter coli

Analysis of fatty acids of the genus Rochalimaea by electron capture gas chromatography: detection of nonanoic acid

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