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)
Cytolethal distending toxin genes in Campylobacter jejuni and Campylobacter coli isolates: detection and analysis by PCR.
Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). Knowledge of the prevalence and homogeneity of Campylobacter sp. cdt genes is incomplete. In this work, we identified four PCR primer pairs that collectively amplified cdt genes in all of the C. jejuni and Campylobacter coli strains tested. Restriction analyses of the cdt PCR products showed clear differences between the cdt genes of these two species, yet there were few heterogeneities noted between members of the same species. Consequently, it may be possible to speciate C. jejuni and C. coli isolates on the basis of restriction patterns within their cdt genes. (+info)
Differentiation of Campylobacter coli and C. jejuni by length and DNA sequence of the 16S-23S rRNA internal spacer region.
The internal spacer region (ISR) between the 16S and 23S rRNA genes of Campylobacter was investigated by PCR fragment length typing and DNA sequencing of clinical and chicken wild-type isolates. PCR fragment length typing showed one fragment of 859 nt in length for the 12 strains of Campylobacter coli investigated. Thirty-six of the Campylobacter jejuni subsp. jejuni strains possessed one fragment, which varied in size between 727 and 802 nt. Three strains showed two fragments between 501 and 923 nt. Strains of C. jejuni subsp. doylei, Campylobacter lari and Campylobacter upsaliensis possessed one or two fragments with lengths different from those of C. coli and C. jejuni subsp. jejuni. DNA sequences were obtained from 54 nt downstream of rrs up to rrl of four strains of C. coli, eight strains of C. jejuni subsp. jejuni, and one strain each of C. jejuni subsp. doylei and C. lari, selected to represent the different biotypes of Campylobacter. ISR lengths determined by PCR fragment length typing and DNA sequencing corresponded for 12 strains. For two strains of C. coli, PCR fragment length typing underestimated ISR lengths by 159 and 193 nt, probably related to incomplete resolution of the distal helical structures, which were not fully denatured during PAGE. For the 14 strains and the published C. jejuni subsp. jejuni sequence, the first 206-211 nt were conserved and included the two tRNA genes in the characteristic tRNA(Ala) to tRNA(Ile) order separated by a short 8-9 nt spacer region. Within the region downstream of tRNA(Ile) conserved regions were identified which allowed a separation of C. lari from C. coli and C. jejuni but not separation of C. coli from C. jejuni. The 69-282 nt longer variable regions in C. coli strains allowed separation of this species from C. jejuni, confirming results obtained by PCR typing. Certain nucleic acid positions in variable regions were related to the Lior biotypes. Sequence information from ISRs of more strains is needed to ascertain if separation of species and biotypes will be possible for diagnostic purposes. (+info)
Rapid identification of thermotolerant Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis from various geographic locations by a GTPase-based PCR-reverse hybridization assay.
Recently, a gene from Campylobacter jejuni encoding a putative GTPase was identified. Based on two semiconserved GTP-binding sites encoded within this gene, PCR primers were selected that allow amplification of a 153-bp fragment from C. jejuni, C. coli, C. lari, and C. upsaliensis. Sequence analysis of these PCR products revealed consistent interspecies variation, which allowed the definition of species-specific probes for each of the four thermotolerant Campylobacter species. Multiple probes were used to develop a line probe assay (LiPA) that permits analysis of PCR products by a single reverse hybridization step. A total of 320 reference strains and clinical isolates from various geographic origins were tested by the GTP-based PCR-LiPA. The PCR-LiPA is highly specific in comparison with conventional identification methods, including biochemical and whole-cell protein analyses. In conclusion, a simple method has been developed for rapid and highly specific identification of thermotolerant Campylobacter species. (+info)
High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting.
For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations. (+info)
Generation of a superoxide dismutase (SOD)-deficient mutant of Campylobacter coli: evidence for the significance of SOD in Campylobacter survival and colonization.
The microaerophilic nature of Campylobacter species implies an inherent sensitivity towards oxygen and its reduction products, particularly the superoxide anion. The deleterious effects of exposure to superoxide radicals are counteracted by the activity of superoxide dismutase (SOD). We have shown previously that Campylobacter coli possesses an iron cofactored SOD. The sodB gene of C. coli UA585 was insertionally inactivated by the site-specific insertion of a tetO cassette. Organisms harboring the inactivated gene failed to produce a biologically functional form of the enzyme. While the ability of this mutant to grow in aerobic conditions was unchanged relative to the parental strain, its survival was severely compromised when nongrowing cells were exposed to air. Accordingly, the SOD-deficient mutant was unable to survive for prolonged periods in model foods. Furthermore, inactivation of the sodB gene decreased the colonization potential in an experimental infection of 1-day-old chicks. In contrast, strain CK100, which is deficient in catalase activity, showed the same survival and colonization characteristics as the parental strain. These results indicate that SOD, but not catalase, is an important determinant in the ability of C. coli to survive aerobically and for optimal colonization within the chicken gut. (+info)
Comparative value of colonic biopsy and intraluminal fluid culture for diagnosis of bacterial acute colitis in immunocompetent patients. Infectious Colitis Study Group.
We compared the yield of intraluminal fluid culture to that of biopsy specimens obtained during colonoscopy for the diagnosis of bacterial colitis in 93 immunocompetent patients with a recent episode of diarrhea and macroscopic lesions of colitis. Stool culture findings were also available for 68 patients. At least one bacterial pathogen was isolated from the biopsy specimen, intraluminal fluid, or stool from 48 patients (51.6%). Salmonella species, Clostridium difficile, Klebsiella oxytoca, Shigella species, and Campylobacter species were recovered from 16 (17.2%), 15 (16.1%), 8 (8.6%), 7 (7.5%), and 4 (4.3%) of the patients, respectively. One Shigella species and one K. oxytoca strain were isolated from biopsy specimens but not from intraluminal fluid, and intraluminal fluid was the only positive specimen in 12 cases (yielding 1 Salmonella species, 2 Shigella species, 2 K. oxytoca, and 7 C. difficile isolates). In nine cases out of 10, toxin B was detected only in intraluminal fluid. A correlation of 91.2% was observed between stool and intraluminal fluid cultures for Salmonella, Shigella, and Campylobacter species isolations. Culture of biopsy specimens adds little to the diagnosis of infectious colitis, and stools and intraluminal fluids appear to have comparable value. (+info)