Comparative fingerprinting analysis of Campylobacter jejuni subsp. jejuni strains by amplified-fragment length polymorphism genotyping.
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Amplified-fragment length polymorphism (AFLP) analysis with the endonucleases BglII and MfeI was used to genotype 91 Campylobacter jejuni subsp. jejuni strains from outbreaks and sporadic cases. AFLP-generated fragments were labeled with fluorescent dye and separated by capillary electrophoresis. The software packages GeneScan and GelCompar II were used to calculate AFLP pattern similarities and to investigate phylogenetic relationships among the genotyped strains. The AFLP method was compared with two additional DNA-based typing methods, pulsed-field gel electrophoresis (PFGE) using SmaI and restriction fragment length polymorphism analysis on PCR products (PCR-RFLP) of the flaA and flaB genes. We found that AFLP analysis of C. jejuni strains is a rapid method that offers better discriminatory power than do both PFGE and PCR-RFLP. AFLP and, to a lesser extent, PCR-RFLP could differentiate strains within the same PFGE profiles, which also makes PCR-RFLP an alternative to PFGE. We were able to clearly distinguish 9 of 10 recognized outbreaks by AFLP and to identify similarities among outbreak and sporadic strains. Therefore, AFLP is suitable for epidemiological surveillance of C. jejuni and will be an excellent tool for source identification in outbreak situations. (+info)
Sequence polymorphism, predicted secondary structures, and surface-exposed conformational epitopes of Campylobacter major outer membrane protein.
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The major outer membrane protein (MOMP), a putative porin and a multifunction surface protein of Campylobacter jejuni, may play an important role in the adaptation of the organism to various host environments. To begin to dissect the biological functions and antigenic features of this protein, the gene (designated cmp) encoding MOMP was identified and characterized from 22 strains of C. jejuni and one strain of C. coli. It was shown that the single-copy cmp locus encoded a protein with characteristics of bacterial outer membrane proteins. Prediction from deduced amino acid sequences suggested that each MOMP subunit consisted of 18 beta-strands connected by short periplasmic turns and long irregular external loops. Alignment of the amino acid sequences of MOMP from different strains indicated that there were seven localized variable regions dispersed among highly conserved sequences. The variable regions were located in the putative external loop structures, while the predicted beta-strands were formed by conserved sequences. The sequence homology of cmp appeared to reflect the phylogenetic proximity of C. jejuni strains, since strains with identical cmp sequences had indistinguishable or closely related macrorestriction fragment patterns. Using recombinant MOMP and antibodies recognizing linear or conformational epitopes of the protein, it was demonstrated that the surface-exposed epitopes of MOMP were predominantly conformational in nature. These findings are instrumental in the design of MOMP-based diagnostic tools and vaccines. (+info)
Antibodies, directed towards Campylobacter jejuni antigens, in sera from poultry abattoir workers.
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Occupational exposure of susceptible humans to Campylobacter jejuni appears to result in resistance to disease. This is believed to be due to acquired protective immunity. To support this hypothesis the levels of C. jejuni-specific IgG and IgM antibodies were determined in sera from poultry abattoir workers. Such individuals are persistently exposed to C. jejuni, but apparently rarely acquire campylobacteriosis. Sera from 43 short-term workers (employed < or = 1 month), 78 long-term workers and 40 blood donors were investigated by ELISA. In 51 individuals a second serum sample, taken at least 1 month after the first, was also investigated. Eight workers had C. jejuni-positive faecal cultures and only one, a short-term worker, had symptoms of campylobacteriosis. There were significantly higher levels of specific IgG antibodies in long-term workers than in either of the other groups. There was no significant difference detectable in specific IgM antibody levels between any of the groups. The results provide supporting evidence that long-term exposure to C. jejuni induces circulating antibodies which reflect apparent reduced susceptibility to disease. Western blotting showed flagellin and polypeptides of 45, 40, 32 and 30 kD bound antibodies significantly more frequently by sera from long-term workers than short-term workers and blood donors. The most commonly detected antigens were the 40-kD (80%) and flagellin (55%). The results indicate that specific serum IgG responses induced by endemic exposure to C. jejuni might be directed towards a small number of protein antigens with apparently conserved epitopes. (+info)
Evaluation of phenotypic and genotypic methods for subtyping Campylobacter jejuni isolates from humans, poultry, and cattle.
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Six methods for subtyping of Campylobacter jejuni were compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (RiboPrinting); random amplified polymorphic DNA typing (RAPD); pulsed-field gel electrophoresis (PFGE); restriction fragment length polymorphisms of the flagellin gene, flaA (fla-RFLP); and denaturing gradient gel electrophoresis of flaA (fla-DGGE). The methods were evaluated and compared on the basis of their abilities to identify isolates from one outbreak and discriminate between unrelated isolates and the agreement between methods in identifying clonal lines. All methods identified the outbreak strain. For a collection of 80 supposedly unrelated isolates, RAPD and PFGE were the most discriminatory methods, followed by fla-RFLP and RiboPrinting. fla-DGGE and serotyping were the least discriminative. All isolates included in this study were found to be typeable by each of the methods. Thirteen groups of potentially related isolates could be identified using a criterion that at least four of the methods agreed on clustering of isolates. None of the subtypes could be related to only one source; rather, these groups represented isolates from different sources. Furthermore, in two cases isolates from cattle and human patients were found to be identical according to all six methods. (+info)
A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein.
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Many bacterial pathogens encode a multisubunit toxin, termed cytolethal distending toxin (CDT), that induces cell cycle arrest, cytoplasm distention, and, eventually, chromatin fragmentation and cell death. In one such pathogen, Campylobacter jejuni, one of the subunits of this toxin, CdtB, was shown to exhibit features of type I deoxyribonucleases. Transient expression of this subunit in cultured cells caused marked chromatin disruption. Microinjection of low amounts of CdtB induced cytoplasmic distention and cell cycle arrest. CdtB mutants with substitutions in residues equivalent to those required for catalysis or magnesium binding in type I deoxyribonucleases did not cause chromatin disruption. CDT holotoxin containing these mutant forms of CdtB did not induce morphological changes or cell cycle arrest. (+info)
Detection and characterization of autoagglutination activity by Campylobacter jejuni.
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In several gram-negative bacterial pathogens, autoagglutination (AAG) activity is a marker for interaction with host cells and virulence. Campylobacter jejuni strains also show AAG, but this property varies considerably among strains. To examine the characteristics of C. jejuni AAG, we developed a quantitative in vitro assay. For strain 81-176, which shows high AAG, activity was optimal for cells grown for < or = 24 h, was independent of growth temperature, and was best measured for cells suspended in phosphate-buffered saline at 25 degrees C for 24 h. AAG activity was heat labile and was abolished by pronase or acid-glycine (pH 2.2) treatment but not by lipase, DNase, or sodium metaperiodate. Strain 4182 has low AAG activity, but extraction with water increased AAG, suggesting the loss of an inhibitor. Strain 6960 has weak AAG with no effect due to water extraction. Our study with clinical isolates suggests that C. jejuni strains may be grouped into three AAG phenotypes. A variant derived from strain 81116 that is flagellate but immotile showed the strong AAG exhibited by the parent strain, suggesting that motility per se is not necessary for the AAG activity. AAG correlated with both bacterial hydrophobicity and adherence to INT407 cells. Mutants which lack flagella (flaA, flaB, and flbA) or common cell surface antigen (peb1A) were constructed in strain 81-176 by natural transformation-mediated allelic exchange. Both AAG activity and bacterial hydrophobicity were abolished in the aflagellate mutants but not the peb1A mutant. In total, these findings indicate that C. jejuni AAG is highly associated with flagellar expression. (+info)
Role of catalase in Campylobacter jejuni intracellular survival.
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The ability of Campylobacter jejuni to penetrate normally nonphagocytic host cells is believed to be a key virulence determinant. Recently, kinetics of C. jejuni intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies conducted by Pesci et al. indicate that superoxide dismutase contributes to intraepithelial cell survival, as isogenic sod mutants are 12-fold more sensitive to intracellular killing than wild-type strains. These findings suggest that bacterial factors that combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. Zymographic analysis indicated that C. jejuni expresses a single catalase enzyme. The gene encoding catalase (katA) was cloned via functional complementation, and an isogenic katA mutant strain was constructed. Kinetic studies indicate that catalase provides resistance to hydrogen peroxide in vitro but does not play a role in intraepithelial cell survival. Catalase does however contribute to intramacrophage survival. Kinetic studies of C. jejuni growth in murine and porcine peritoneal macrophages demonstrated extensive killing of both wild-type and katA mutant strains shortly following internalization. Long-term cultures (72 h postinfection) of infected phagocytes permitted recovery of viable wild-type C. jejuni; in contrast, no viable katA mutant bacteria were recovered. Accordingly, inhibition of macrophage nitric oxide synthase or NADPH oxidase permitted recovery of katA mutant C. jejuni. These observations indicate that catalase is essential for C. jejuni intramacrophage persistence and growth and suggest a novel mechanism of intracellular survival. (+info)
Epidemiologic application of pulsed-field gel electrophoresis to an outbreak of Campylobacter jejuni in an Austrian youth centre.
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We report the first documented Campylobacter jejuni outbreak in an Austrian youth centre. Sixty-four children were involved of which 38 showed classical signs of campylobacter gastroenteritis. Since unpasteurized milk distributed by a local dairy was suspected to be the source of infection, stool samples were collected from 20 cows providing the milk. Five of the cows tested positive for C. jejuni. These isolates together with 37 clinical samples were compared by pulsed-field-gel electrophoresis (PFGE). The PFGE patterns, using the restriction endonucleases SmaI and SalI, were identical for the human and bovine isolates. This finding confirmed that the outbreak was caused by the consumption of unpasteurized milk contaminated with C. jejuni. (+info)