A simple technique for mass cultivation of Campylobacter fetus.
Studies using 86 media for maximum growth of Campylobacter fetus for antigen production showed that a diphasic medium (solid base with liquid overlay) was most suitable. The solid base was double strength cystine heart agar. The liquid overlay was thioglycollate medium of Brewer (135-C) without agar. This medium yielded maximum growth of C. fetus in six days with good motility, less clumping and less filament formation than all other media tried. (+info)
Detection of campylobacter in gastroenteritis: comparison of direct PCR assay of faecal samples with selective culture.
The prevalence of campylobacter gastroenteritis has been estimated by bacterial isolation using selective culture. However, there is evidence that certain species and strains are not recovered on selective agars. We have therefore compared direct PCR assays of faecal samples with campylobacter culture, and explored the potential of PCR for simultaneous detection and identification to the species level. Two hundred unselected faecal samples from cases of acute gastroenteritis were cultured on modified charcoal cefoperazone deoxycholate agar and subjected to DNA extraction and PCR assay. Culture on CCDA indicated that 16 of the 200 samples contained 'Campylobacter spp.'. By contrast, PCR assays detected campylobacters in 19 of the 200 samples, including 15 of the culture-positive samples, and further identified them as: C. jejuni (16), C. coli (2) and C. hyointestinalis (1). These results show that PCR offers a different perspective on the incidence and identity of campylobacters in human gastroenteritis. (+info)
Presence of Campylobacter and Salmonella in sand from bathing beaches.
The purpose of this study was to determine the presence of thermophilic Campylobacter spp. and Salmonella spp. in sand from non-EEC standard and EEC standard designated beaches in different locations in the UK and to assess if potentially pathogenic strains were present. Campylobacter spp. were detected in 82/182 (45%) of sand samples and Salmonella spp. in 10/182 (6%). Campylobacter spp. were isolated from 46/92 (50%) of samples from non-EEC standard beaches and 36/90 (40%) from EEC standard beaches. The prevalence of Campylobacter spp. was greater in wet sand from both types of beaches but, surprisingly, more than 30% of samples from dry sand also contained these organisms. The major pathogenic species C. jejuni and C. coli were more prevalent in sand from non-EEC standard beaches. In contrast, C. lari and urease positive thermophilic campylobacters, which are associated with seagulls and other migratory birds, were more prevalent in sand from EEC standard beaches. Campylobacter isolates were further characterized by biotyping and serotyping, which confirmed that strains known to be of types associated with human infections were frequently found in sand on bathing beaches. (+info)
Clonality of Campylobacter sputorum bv. paraureolyticus determined by macrorestriction profiling and biotyping, and evidence for long-term persistent infection in cattle.
Eighteen strains of Campylobacter sputorum bv. paraureolyticus (isolated over a 12-month period from seven dairy cows contained in a single herd) were examined by resistotyping, and macrorestriction profiling using pulsed field gel electrophoresis (PFGE). The resistotypes of these strains were identical, although repeat testing indicated resistance to metronidazole was not a reliable trait for typing purposes. Five SmaI-derived genotypes were identified among the 18 strains. In 5 of 7 cows, isolates obtained from the same animal, but from different time periods, were genotypically indistinguishable, indicating persistence of infection. Macrorestriction profiles of 5 strains representing the 5 SmaI genotypes and 8 other strains of C. sputorum from various sources, were prepared using 4 endonucleases (SmaI, SalI, BamHI and KpnI). The only other strain of C. sputorum bv. paraureolyticus examined (a Canadian isolate from human faeces), was found to have a SmaI macrorestriction profile identical with one of the five clones isolated from the cattle. Moreover, SalI and BamHI profiles of all bv. paraureolyticus strains were similar, while digestion with KpnI was not observed. By contrast, the seven strains of C. sputorum bv. sputorum yielded various macrorestriction profiles with all the enzymes used, and features distinguishing the two biovars studied could be identified. This study indicates that C. sputorum can persist in cattle for at least 12 months and exhibits a clonal population genetic structure. (+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)
Cloning and characterization of two bistructural S-layer-RTX proteins from Campylobacter rectus.
Campylobacter rectus is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes, csxA and csxB (for C. rectus S-layer and RTX protein) were cloned from C. rectus and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of C. fetus and Crs of C. rectus. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca2+. These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All C. rectus isolates studied contained copies of either the csxA or csxB gene or both; csx genes were absent from all other Campylobacter and Helicobacter species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots. (+info)
Different invasion phenotypes of Campylobacter isolates in Caco-2 cell monolayers.
The pathogenesis of campylobacter enteritis is not well understood, but invasion into and translocation across intestinal epithelial cells may be involved in the disease process, as demonstrated for a number of other enteric pathogens. However, the mechanisms involved in these processes are not clearly defined for campylobacters. In this study, isolates were compared quantitatively in established assays with the enterocyte-like cell line, Caco-2, to determine the extent to which intracellular invasion contributes to translocation across epithelial cell monolayers, and whether isolates vary in this respect. Ten fresh Campylobacter isolates were compared and shown to differ in invasiveness by a factor of 10-fold by following their recovery from gentamicin-treated Caco-2 cells grown on nonpermeable tissue-culture wells. Four of these isolates with contrasting invasive ability were also shown to vary in their ability to translocate across Caco-2 cells grown on semipermeable Transwell inserts by a factor >10. However, translocation did not quantitatively correlate with the intracellular invasiveness of these isolates. Isolate no. 9752 was poorly invasive but had modest translocation ability, isolate no. 10392 was very invasive but did not translocate significantly and remained within the monolayer, isolate no. 9519 both translocated and invaded well, whereas, isolate no. 235 translocated very efficiently but was poorly invasive. Isolate no. 9519 also uniquely caused a transitory flattening of the Caco-2 cells and a possible drop in trans-epithelial electrical resistance (TEER) of the Transwell monolayers, whereas isolate no. 235 did not show these effects. Together these data demonstrate that there are significantly different 'invasion' phenotypes among Campylobacter strains involving different degrees of intracellular invasion, and either different rates of transcellular trafficking or, alternatively, paracellular trafficking. (+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)