Cloning and expression of the dnaK gene of Campylobacter jejuni and antigenicity of heat shock protein 70.
Campylobacter jejuni is a leading cause of infectious diarrhea throughout the world. In addition, there is growing evidence that Guillain-Barre syndrome, an inflammatory demyelinating disease of the peripheral nervous system, is frequently preceded by C. jejuni infection. In the present study, the hrcA-grpE-dnaK gene cluster of C. jejuni was cloned and sequenced. The dnaK gene consists of an open reading frame of 1,869 bp and encodes a protein with a high degree of homology to other bacterial 70-kDa heat shock proteins (HSPs). The overall percentages of identity to the HSP70 proteins of Helicobacter pylori, Borrelia burgdorferi, Chlamydia trachomatis, and Bacillus subtilis were calculated to be 78.1, 60.5, 57.2, and 53. 8%, respectively. Regions similar to the Escherichia coli sigma70 promoter consensus sequence and to a cis-acting regulatory element (CIRCE) are located upstream of the hrcA gene. Following heat shock, a rapid increase of dnaK mRNA was detectable, which reached its maximum after 20 to 30 min. A 6-His-tagged recombinant DnaK protein (rCjDnaK-His) was generated in E. coli, after cloning of the dnaK coding region into pET-22b(+), and purified by affinity and gel filtration chromatography. Antibody responses to rCjDnaK-His were significantly elevated, compared to those of healthy individuals, in about one-third of the serum specimens obtained from C. jejuni enteritis patients. (+info)
Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells.
Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (10(7) cells ml-1) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride-4',6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 microliter mg of protein-1 for the culturable form and 10.96 microliter mg of protein-1 after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water. (+info)
Evaluation of accuracy and repeatability of identification of food-borne pathogens by automated bacterial identification systems.
The performances of five automated microbial identification systems, relative to that of a reference identification system, for their ability to accurately and repeatedly identify six common food-borne pathogens were assessed. The systems assessed were the MicroLog system (Biolog Inc., Hayward, Calif.), the Microbial Identification System (MIS; MIDI Inc., Newark, Del.), the VITEK system (bioMerieux Vitek, Hazelwood, Mo.), the MicroScan WalkAway 40 system (Dade-MicroScan International, West Sacramento, Calif.), and the Replianalyzer system (Oxoid Inc., Nepean, Ontario, Canada). The sensitivities and specificities of these systems for the identification of food-borne isolates of Bacillus cereus, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and verotoxigenic Escherichia coli were determined with 40 reference positive isolates and 40 reference negative isolates for each pathogen. The sensitivities of these systems for the identification of these pathogens ranged from 42.5 to 100%, and the specificities of these systems for the identification of these pathogens ranged from 32.5 to 100%. Some of the systems had difficulty correctly identifying the reference isolates when the results were compared to those from the reference identification tests. The sensitivity of MIS for the identification of S. aureus, B. cereus, E. coli, and C. jejuni, for example, ranged from 47.5 to 72. 5%. The sensitivity of the Microlog system for the identification of E. coli was 72.5%, and the sensitivity of the VITEK system for the identification of B. cereus was 42.5%. The specificities of four of the five systems for the identification of all of the species tested with the available databases were greater than or equal to 97.5%; the exception was MIS for the identification of C. jejuni, which displayed a specificity of 32.5% when it was tested with reference negative isolates including Campylobacter coli and other Campylobacter species. All systems had >80% sensitivities for the identification of Salmonella species and Listeria species at the genus level. The repeatability of these systems for the identification of test isolates ranged from 30 to 100%. Not all systems included all six pathogens in their databases; thus, some species could not be tested with all systems. The choice of automated microbial identification system for the identification of a food-borne pathogen would depend on the availability of identification libraries within the systems and the performance of the systems for the identification of the pathogen. (+info)
Cloning, mutation and distribution of a putative lipopolysaccharide biosynthesis locus in Campylobacter jejuni.
A region encoding ORFs with homology to known lipopolysaccharide (LPS) biosynthesis genes was isolated from two strains of Campylobacter jejuni. One of the strains produces LPS, but the second strain is reported to produce only lipooligosaccharide (LOS) and therefore lacks the O-chain. The two strains shared six predicted ORFs, but an additional ORF, orfE, of unknown function was identified in the LOS-producing strain. Mutation of the shared wbeE (rfbE) homologue (orfF) or deletion of five of the seven genes reduced core reactivity with specific antiserum without affecting O-chain production. Mutation of either the capD homologue (orfG) or the unique orfE had no detectable effect on LOS or LPS production. The presence or absence of orfE in 36 isolates of C. jejuni did not correlate with LOS/LPS phenotype or serotype. However, after insertion of orfE into a LPS-producing orfE-negative strain the O-chain ladder was no longer detectable on Western blots. We were not able to disrupt the wbaP (rfbP) homologue (orfC) in C jejuni. (+info)
Campylobacter jejuni is the most commonly reported bacterial cause of foodborne infection in the United States. Adding to the human and economic costs are chronic sequelae associated with C. jejuni infection--Guillian-Barre syndrome and reactive arthritis. In addition, an increasing proportion of human infections caused by C. jejuni are resistant to antimicrobial therapy. Mishandling of raw poultry and consumption of undercooked poultry are the major risk factors for human campylobacteriosis. Efforts to prevent human illness are needed throughout each link in the food chain. (+info)
The risk of Guillain-Barre syndrome following infection with Campylobacter jejuni.
To estimate the incidence of Guillain-Barre syndrome (GBS) following Campylobacter jejuni infection (CI) we studied three populations where outbreaks of CI had occurred involving an estimated 8000 cases. No case of GBS was detected in the 6 months following the outbreaks in the local populations. The point estimate for the risk of GBS following CI estimated in this study was 0 in 8000 (95% confidence interval 0-3). (+info)
Detection of cytolethal distending toxin activity and cdt genes in Campylobacter spp. isolated from chicken carcasses.
This study was designed to determine whether isolates from chicken carcasses, the primary source of Campylobacter jejuni and Campylobacter coli in human infections, commonly carry the cdt genes and also whether active cytolethal distending toxin (CDT) is produced by these isolates. Campylobacter spp. were isolated from all 91 fresh chicken carcasses purchased from local supermarkets. Campylobacter spp. were identified on the basis of both biochemical and PCR tests. Of the 105 isolates, 70 (67%) were identified as C. jejuni, and 35 (33%) were identified as C. coli. PCR tests amplified portions of the cdt genes from all 105 isolates. Restriction analysis of PCR products indicated that there appeared to be species-specific differences between the C. jejuni and C. coli cdt genes, but that the restriction patterns of the cdt genes within strains of the same species were almost invariant. Quantitation of active CDT levels produced by the isolates indicated that all C. jejuni strains except four (94%) had mean CDT titers greater than 100. Only one C. jejuni strain appeared to produce no active CDT. C. coli isolates produced little or no toxin. These results confirm the high rate of Campylobacter sp. contamination of fresh chicken carcasses and indicate that cdt genes may be universally present in C. jejuni and C. coli isolates from chicken carcasses. (+info)
Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay.
A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8, 700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as +info)