Characteristics of the internalization and intracellular survival of Campylobacter jejuni in human epithelial cell cultures.

The characteristics associated with the internalization and intracellular behavior of Campylobacter jejuni during short-term and long-term cultivation with INT 407 cells were examined. The internalization of C. jejuni by INT 407 cells was inhibited by cytochalasin dansylcadaverine, chemicals that disrupt microfilament formation and inhibit receptor cycling, respectively. Ammonium chloride and methylamine, two chemicals that inhibit endosomal acidification, did not affect C. jejuni internalization. Once internalized, C. jejuni were found exclusively with membrane-bound vacuoles. With regard to intracellular survival, a decline in the number of viable intracellular bacteria, as determined by protection from gentamicin, occurred during the initial phase of infection and when a low level of the antibiotic was maintained in the culture medium. However, the number of intracellular C. jejuni increased markedly after the removal of the antibiotic. In the absence of antibiotic, the infection led to the deterioration of the cell monolayers, indicating that C. jejuni is able to survive within epithelial cells and elicit a cytotoxic effect. The ability of C. jejuni to enter and exert deleterious effects on cells may reflect a pathogenic mechanism associated with enteritis caused by this organism.     

Adherence, enterotoxigenicity, invasiveness and serogroups in Campylobacter jejuni and Campylobacter coli strains from adult humans with acute enterocolitis

Two hundred Campylobacter jejuni and Campylobacter coli strains from the same number of adult Swedish patients with acute enterocolitis were tested regarding adherence to and invasiveness in HEp-2 cells and for enterotoxigenicity by the CHO-cell assay. The serogroup characteristics, heat-stable and heat-labile, for each strain were also investigated. Eighty-four percent of the strains were classified as C. jejuni and 16 percent as C. coli. All of the strains were adherent to HEp-2 cells, 39% were invasive and 31.5% enterotoxigenic. We found significantly more invasive strains in the non-enterotoxigenic group than in the enterotoxigenic one. There would seem to be no correlation between enterotoxigenicity or invasiveness and serogroup. The results of this study suggest the existence of multiple mechanisms for C. jejuni- and C. coli-induced diarrhoea and that the mechanisms may differ from one strain to another.     

Comparison of rectal swabs and stool cultures in detecting Campylobacter fetus subsp. jejuni.

Rectal swabs and stool specimens were compared for the detection of Campylobacter fetus subsp. jejuni in marmosets. Rectal swabs were superior to stool specimens for detection of Campylobacter fetus subsp. jejuni (P = 0.016). Preliminary human data are also presented.     

Mononuclear cell response in the liver of mice infected with hepatotoxigenic Campylobacter jejuni.

Intragastric inoculation with hepatotoxigenic strains of Campylobacter jejuni led to the death of mice during the late phase of infection. Histological study disclosed a massive infiltration of mononuclear cells in the liver, mimicking intrahepatic hypersensitivity. Neither enterotoxigenic nor enteroinvasive Escherichia coli induced such a lesion. However, the same histopathological change was induced by injecting the hepatotoxic factor of hepatotoxigenic C. jejuni intravenously on two occasions separated by 14 days. Neither a single injection of an increased dose of the hepatotoxic factor nor two injections, the second of which was heat-inactivated, induced this change. Pre-treatment with rabbit antibody to the hepatotoxic factor inhibited the development of the hepatic lesion. These results suggest that C. jejuni-induced hepatic lesions in mice may be caused, at least in part, by the active moiety of the hepatotoxic factor. The possible mechanisms by which the toxic factor induces hepatitis as a consequence of hypersensitivity are discussed in relation to Guillain-Barré syndrome and Reiter's syndrome associated with C. jejuni enteritis.     

Long-term infections with Campylobacter fetus subsp. jejuni.

Seventy-three apparently healthy, rural South African schoolchildren 6 to 8 or 13 to 16 years of age were examined five times over a 16-month period for fecal pathogens. Nine were positive for Campylobacter fetus subsp. jejuni. The organism was isolated intermittently from six children for at least 9 months and from three children for more than 1 year. Five of the long-term infections occurred among the 46 children aged 6 to 8 years (10.9%) versus one long-term infection among the 27 children aged 13 to 16 years (3.7%). It is not possible with present microbiological techniques to make a clear-cut distinction between reinfected subjects and chronic carriers.     

Fatal Campylobacter jejuni bacteraemia in patients with AIDS.

Two fatal cases of Campylobacter jejuni septicaemia in patients with AIDS were characterised by severe HIV-related immunodeficiency, negative stool cultures and presentation during hospitalisation, developing a clinical picture of fulminant septic shock despite therapy with appropriate antibiotics. Campylobacter spp. are important opportunist pathogens in HIV disease and may cause a septicaemic illness in the absence of enteric disease.     

Campylobacter jejuni Cocultured with Epithelial Cells Reduces Surface Capsular Polysaccharide Expression

The host cell environment can alter bacterial pathogenicity. We employed a combination of cellular and molecular techniques to study the expression of Campylobacter jejuni polysaccharides cocultured with HCT-8 epithelial cells. After two passages, the amount of membrane-bound high-molecular-weight polysaccharide was considerably reduced. Microarray profiling confirmed significant downregulation of capsular polysaccharide (CPS) locus genes. Experiments using conditioned media showed that sugar depletion occurred only when the bacterial and epithelial cells were cocultured. CPS depletion occurred when C. jejuni organisms were exposed to conditioned media from a different C. jejuni strain but not when exposed to conditioned media from other bacterial species. Proteinase K or heat treatment of conditioned media under coculture conditions abrogated the effect on the sugars, as did formaldehyde fixation and cycloheximide treatment of host cells or chloramphenicol treatment of the bacteria. However, sugar depletion was not affected in flagellar export (fliQ) and quorum-sensing (luxS) gene mutants. Passaged C. jejuni showed reduced invasiveness and increased serum sensitivity in vitro. C. jejuni alters its surface polysaccharides when cocultured with epithelial cells, suggesting the existence of a cross talk mechanism that modulates CPS expression during infection.The importance of the host cell environment in bacterial pathogenicity is an emerging paradigm in the study of bacterial infection. For example, human colonization creates a hyperinfectious state in Vibrio cholerae that appears to contribute to the epidemic spread of this intestinal pathogen (29). Earlier work with Campylobacter jejuni demonstrated that coculture conditions altered the protein synthesis and virulence of this organism (23). Stintzi et al. demonstrated major effects in gene expression among C. jejuni organisms exposed to pathogenic conditions (36). However, how the host cell environment alters Campylobacter pathogenesis remains largely unknown. C. jejuni is now known to produce capsular polysaccharide (CPS) (4, 21, 31). Although relatively little is known about the contribution of CPS to the pathogenicity of the organism during bacterial interaction with mucosal surfaces, existing data suggest that CPS in general plays a complex and dynamic role in the infection process (2, 33).In a study that was carried out before CPS was identified in C. jejuni, Babakhani and Joens reported that C. jejuni cocultured with primary swine intestinal cells showed increased mucoidy when regrown on solid medium (1). In addition, passaged C. jejuni showed enhanced invasiveness in vitro. We hypothesized that the change in mucoidy observed in these experiments reflected altered CPS expression by bacteria exposed to pathogenic conditions in vitro. We further speculated that altered CPS under these conditions might have relevance to pathogenic behavior among these organisms during infection. In this study, we show that coculture of C. jejuni with human intestinal epithelial cells causes a reproducible reduction in CPS staining. The presence of viable host cells and active protein synthesis by both bacteria and host cells is necessary for the effect on CPS. The alteration in CPS appears to be dependent on a soluble factor that is both heat labile and proteinase K sensitive. Following serial passage with HCT-8 cells, C. jejuni also showed other phenotypic changes, including reduced internalization into HCT8 epithelial cells and increased serum sensitivity.     

Antigenicity of Campylobacter jejuni flagella.

We studied the antigenicity of a wild-type flagellate and motile (F+M+) Campylobacter jejuni strain (81116) and two daughter mutants, one flagellate and immotile (F+M-) and one aflagellate and immotile (F-M-). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis of acid-extracted surface proteins, a 63-kilodalton (kDa) band identified from sheared flagella as the flagellar protein was present in the F+M+ and F+M- strains but not in the F-M- strain. No other differences in protein profile among the three strains were noted. By Western blotting, serum from rabbits immunized with either the F+M+ or F-M- strain detected a 63-kDa protein in the F+M+ and F+M- strains but not in the F-M- strain. That the F-M- antiserum recognized the 63-kDa band suggests that small amounts of this protein or a cross-reacting antigen is present on the F-M- strain. By counterimmunoelectrophoresis of the acid-extracted preparations with immune sera, all three strains were found to share three major antigens, but a fourth antigen with a net positive charge was present only in the F+M+ and F+M- strains. Antisera to five C. jejuni and two Campylobacter fetus strains recognized the 63-kDa protein of purified F+M+ flagella in Western blots, demonstrating a common antigen is present, but enzyme-linked immunosorbent assay results suggest that the sharing of this antigen among Campylobacter strains is variable.     

Occurrence of Campylobacter jejuni in pets living with human patients infected with C. jejuni

Campylobacter jejuni was recovered from four dogs (11%) and four cats (33%) living with Danish human patients infected with C. jejuni. Pulsed-field gel electrophoresis (PFGE) analysis revealed the occurrence of the same quinolone-resistant strain in a girl and her dog. C. jejuni isolates with closely related (>95% similarity) PFGE profiles occurred in humans and pets from different Danish counties.     

Chemotactic behavior of Campylobacter jejuni.

The chemotactic behavior of Campylobacter jejuni was determined in the presence of different amino acids, carbohydrates, organic acids, and preparations and constituents of mucin and bile. L-Fucose was the only carbohydrate and L-aspartate, L-cysteine, L-glutamate, and L-serine were the only amino acids producing a chemotactic (positive) response. Several salts of organic acids, including pyruvate, succinate, fumarate, citrate, malate, and alpha-ketoglutarate, were also chemoattractants, as were bile (beef, chicken, and oxgall) and mucin (bovine gallbladder and hog gastric). Most constituents of bile tested individually were chemorepellents, but the mucin component was chemoattractant. The chemotactic behavior of C. jejuni toward L-fucose, a constituent of both bile and mucin, may be an important factor in the affinity of the organism for the gallbladder and intestinal tract.     

Electron microscopy of Campylobacter jejuni.

Stools from 56 patients with gastroenteritis were cultured for Campylobacter jejuni. The five strains isolated were examined by electron microscopy. The campylobacter cells were pleomorphic and most displayed appearances similar to those of V. fetus. Morphological changes were observed in cultures subjected to prolonged incubation.     

Campylobacter jejuni antibodies in Nigerian children.

Titers of complement-fixing (CF) antibody to Campylobacter jejuni were demonstrated in 87 (36.7%) of 237 infants 6 to 15 months old in Jos, Nigeria. Of the total number of children examined, 81 had acute diarrhea and 27 of them (33.3%) were found to have CF antibodies in their serum. The remaining 156 children were asymptomatic, and 60 (38.4%) of them had CF antibodies. In the diarrheal group, 27 of 75 children 6 to 8 months old were CF antibody positive. There was no significant difference in the incidence of CF C. jejuni antibodies in the diarrheal and nondiarrheal infants (P greater than 0.05). Also, infants 9 to 15 months old had a higher incidence of CF antibodies (46.5%) than those 6 to 8 months of age (25%). The data suggest that the infants whose sera were CF antibody positive had had an exposure to C. jejuni. All 33 infants 6 to 8 months of age who had no diarrhea were CF antibody negative.     

Naturally occurring auxotrophs of Campylobacter jejuni and Campylobacter coli.

The nutritional requirements for 439 Campylobacter jejuni isolates and 46 Campylobacter coli isolates were determined by using a previously described chemically defined medium, campylobacter defined medium. With this medium, 45% of both human and nonhuman C. jejuni isolates demonstrated auxotrophic requirements. None of the 46 C. coli isolates studied demonstrated requirements for amino acids on campylobacter defined medium. The most common auxotrophic requirement among C. jejuni isolates was for methionine, which was present as a single requirement or in combination with other markers in 21% of human and 28% of nonhuman isolates. There was no correlation between plasmid carriage and auxotype, and a comparison of the Lior serotypes of 472 of the strains showed a correlation only between proline auxotrophs and Lior serotype 11 for strains isolated in the Seattle-King County region.     

Enriched brucella medium for storage and transport of cultures of Campylobacter fetus subsp. jejuni.

A semisolid brucella medium with 10% sheep blood was used for storage and transport of Campylobacter fetus subsp. jejuni and subsp. intestinalis and kept isolated alive about 3 weeks or longer at 25 degrees C or when sent through the regular mail.     

Colonization of mice by Campylobacter jejuni.

Both streptomycin-treated and untreated Swiss white mice were irregularly colonized when challenged orogastrically with between 1 and 10(11) viable organisms of either of two strains of Campylobacter jejuni. The organisms were occasionally recovered from portions of the intestinal tracts of these animals in numbers ranging from 10(1) to 10(3)/g when the challenge doses were 10(10) or more. When germfree mice were challenged with 10(8) organisms of either strain, the entire intestinal tracts of all the animals were colonized with C. jejuni in numbers ranging from 10(4) to 10(9)/g. The ceca were most heavily colonized. Both strains of C. jejuni multiplied anaerobically in brucella broth, except when the broth contained 60.80 mu eq of total volatile fatty acids (VFA) per ml at pH 6.75, simulating conditions in the ceca of untreated mice, or when it contained 21.63 mu eq/ml at pH 7.04, simulating conditions in the ceca of streptomycin-treated mice. Active multiplication occurred, however, in brucella broth without VFA at pH 7.02 that was incubated microaerobically, simulating conditions in the ceca of germfree mice. The results suggest that VFA operating under anaerobic conditions present in the intestinal tract of both streptomycin-treated and untreated conventional mice interfere with the multiplication of C. jejuni. The organisms actively multiply, on the other hand, in the absence of VFA at the higher oxidation-reduction potential of the intestinal tract of germfree mice.     

Serotyping of Campylobacter jejuni/coli.

Antisera were prepared from strains of Campylobacter jejuni/coli isolated from patients in six outbreaks of enteritis. Bactericidal antibodies, and agglutinating antibodies to heat-labile and heat-stable antigens, were demonstrated. These reactions were used to type a number of strains isolated from patients in each outbreak, and to distinguish 'epidemic' from 'non-epidemic' strains.     

Outer membrane characteristics of Campylobacter jejuni.

Outer membranes were isolated from type strains and wild-type isolates of Campylobacter jejuni and Campylobacter coli by sodium lauryl sarcosinate extraction, and the polypeptide complement and lipopolysaccharide (LPS) content were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles exhibited by membranes from both species were quite similar, but could be distinguished from the type strain of the genus, C. fetus subsp. fetus CIP5396. The sodium dodecyl sulfate electrophoretograms of C. jejuni and C. coli were dominated by a major polypeptide band. In the reference strain C. jejuni VC74, this polypeptide had an apparent molecular weight of 45,000, was heat modifiable, and was shown to be transmembrane by virtue of its peptidoglycan association and surface exposure. Two other proteins with approximate molecular weights of 37,000 and 73,000 were also surface exposed on C. jejuni VC74 and represented potential surface antigens. The LPS of C. jejuni and C. coli was of low molecular weight, suggesting that serotypic differences due to LPS were based on different carbohydrate compositions of core LPS. In contrast, the LPS of C. fetus CIP5396 exhibited O antigen polysaccharide chains of intermediate chain length. Fragments of outer membranes released during growth of C. jejuni VC74 displayed a polypeptide profile which differed from that of sarcosinate-extracted outer membranes. Radiolabeling demonstrated that the proteins exposed on the surface of this released membrane differed from those exposed on the cell surface and would likely contribute to the antigenic complexity of C. jejuni.