Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
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We describe rapid PCR-biprobe identification of Campylobacter spp. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of Campylobacter, Helicobacter, and Arcobacter spp. and 59 Penner serotype reference strains of Campylobacter jejuni and C. coli. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five taxa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR-enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following microaerobic culture. (+info)
Differentiation between Campylobacter hyoilei and Campylobater coli using genotypic and phenotypic analyses.
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Genotypic and phenotypic methods were applied to investigate differences between the closely related species Campylobacter hyoilei and Campylobacter coli. A unique DNA sequence from C. hyoilei was used to design a specific PCR assay that amplified a DNA product of 383 bp for all C. hyoilei strains, but not other Campylobacter species, including C. coli. The PCR assay could detect 100 fg pure C. hyoilei DNA, 2 x 10(2) c.f.u. ml(-1) using cultured cells and 8.3 x 10(3) c.f.u. 0.1 g(-1) in faeces. The C. hyoilei sequence utilized for specific detection and identification of this species showed similarities to sequences from bacteriophages Mu, P2 and 186, suggesting lysogination of the ancestral C. hyoilei genome. Activities of a set of 15 enzymes that participate in a variety of cellular functions, including biosynthesis, catabolism, energy generation, maintenance of redox balance and phosphate utilization, were tested using sets of strains of C. hyoilei and C. coli. Comparison of mean rates of enzyme activities revealed significant differences between species in the values determined for seven of these activities. Both the genetic and phenotypic data indicate that C. hyoilei is a unique Campylobacter species. (+info)
Genomic heterogeneity and O-antigenic diversity of Campylobacter upsaliensis and Campylobacter helveticus strains isolated from dogs and cats in Germany.
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A serotyping scheme based on heat-stable surface antigens was established for 101 Campylobacter upsaliensis and 10 Campylobacter helveticus strains isolated from 261 dogs and 46 cats of different ages originating from two geographically distinct regions in Germany. The prevalence of C. upsaliensis varied between 27.8% in juvenile dogs (<12 months of age) and 55.4% in adult dogs (P < 0.05). Of the cats, 19.6% harbored C. upsaliensis, whereas 21.7% carried C. helveticus. Of the C. upsaliensis isolates from both host species, 93.1% belonged to five different serogroups, two of them being prevalent at rates of 47.5 and 27.7%, with different frequencies in both regions. Six (54.6%) of the C. helveticus isolates also belonged to serotypes found among C. upsaliensis strains, whereas five (45.4%) possessed an O antigen unique for C. helveticus. In contrast, a considerable degree of genomic diversity of the isolates was assessed by macrorestriction analyses with the endonucleases SmaI and XhoI, using pulsed-field gel electrophoresis as well as enterobacterial repetitive intergenic consensus sequence PCR (ERIC PCR). Restriction with SmaI pointed towards the existence of clonal groups associated to some extent with serotypes, while restriction with XhoI disintegrated these groups to smaller noncoherent subgroups. Analysis of ERIC PCR profiles did not exhibit any associations with serotypes. In conclusion these data demonstrate the genomic heterogeneity among C. upsaliensis strains and indicate that the combination of SmaI restriction with serotyping is a useful tool to investigate the expansion of clonal groups of C. upsaliensis. (+info)
Basis of the superiority of cefoperazone amphotericin teicoplanin for isolating Campylobacter upsaliensis from stools.
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The optimum method for isolating Campylobacter upsaliensis from stools has not been clearly defined. In a preliminary study, cefoperazone amphotericin teicoplanin (CAT) selective medium isolated six C. upsaliensis strains which were not detected using modified cefoperazone charcoal deoxycholate (mCCDA). In order to identify the factors that underlie the superiority of CAT over mCCDA for isolating C. upsaliensis, we examined the effect of incubation time and antibiotic content of culture media on the growth of C. upsaliensis isolates using semiquantitative methods. The recovery of a subgroup of C. upsaliensis isolates from seeded stool specimens was also evaluated. Differences in growth of C. upsaliensis on CAT and mCCDA were modest and were not explained by the antibiotic profiles of the two media. Recovery of C. upsaliensis from spiked human feces on CAT was superior to that on mCCDA at lower concentrations of organisms (10(3) CFU/ml). We conclude that although CAT is more suitable than mCCDA for the isolation of C. upsaliensis from stools, the superiority of CAT for detecting this organism is not accounted for by the antibiotic composition of the medium. (+info)
Antimicrobial resistance of thermophilic Campylobacter.
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Campylobacter has become the leading cause of zoonotic enteric infections in developed and developing countries world-wide. Antimicrobial resistance has emerged among Campylobacter mainly as a consequence of the use of antimicrobial agents in food animal production. Resistance to drugs of choice for the treatment of infections, macrolides and fluoroquinolones has emerged as a clinical problem and interventions to reduce this are recommended. Resistance to fluoroquinolones and macrolides is mediated by chromosomal mutations. Resistance to other relevant antimicrobial agents, mediated by acquired resistance genes, has not become widespread so far. However, resistance genes originating from both Gram-positive and Gram-negative bacterial species have been found, showing the potential for acquired resistance to emerge in Campylobacter. (+info)
Pathogenicity and convalescent excretion of Campylobacter in rural Egyptian children.
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Campylobacter infection in developing countries has not received much public health attention because of the observation that infections are not associated with disease beyond the first 6 months of life. A cohort of 397 Egyptian children aged less than 3 years, who were observed twice weekly during 1995--1998, experienced an incidence of 0.6 episodes of Campylobacter diarrhea per child-year. A total of 13% of the Campylobacter diarrheal episodes were characterized by severe dehydration. Age-specific incidence rates (episodes per year) were 0.9 in infants aged less than 6 months, 1.5 in those 6--12 months, and 0.4 and 0.2 in the second and third years of life, respectively. Convalescent excretion of Campylobacter after a diarrheal episode might be enhancing transmission and contributing to this high incidence. Observed risk factors for Campylobacter diarrhea were poor hygienic conditions and the presence of animals in the house. Regardless of the child's age, a first infection by Campylobacter was associated with diarrhea (odds ratio = 2.45; 95% confidence interval: 1.61, 3.71); however, subsequent infections were associated with diarrhea only in children aged less than 6 months. This observation that natural infection did not confer protection during the first 6 months of life poses a challenge to vaccine development. (+info)
A comparison of the isolation rates of Salmonella and thermophilic Campylobacter species after direct inoculation of media with a dilute faecal suspension and undiluted faecal material.
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Regardless of media used, dilution of faecal samples before direct plating may improve isolation rates and reduce subcultures by freeing organisms from the faecal mass and diminishing competing flora. Despite the routine use of dilution in many laboratories, it has never been established properly whether direct or dilute inocula should be used in primary plating of faeces. A total of 3764 faecal samples was examined in four laboratories with a standardised methodology. The isolation rates, competing flora and confirmatory work performed for Salmonella spp. and Campylobacter spp. from primary plating media with a dilute faecal inoculum were compared with those after direct inoculation of faecal material. Inoculum effects on the isolation of Shigella spp. could not be assessed as only one isolate occurred during the study period. The overall isolation rates of both major enteric pathogens were unaffected by the inoculum. However, significantly fewer wasted subcultures were recorded with a dilute inoculum for Campylobacter spp., and competing florawas reduced in all cases without diluting out small numbers of the pathogen. (+info)
Antimicrobial susceptibilities of Campylobacter strains isolated from food animals in Belgium.
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Campylobacter spp. are a frequent cause of diarrhoea in man, originating mostly from poultry. It has been suggested that the veterinary use of antibiotics is largely responsible for resistance in human isolates, particularly to quinolones. During a 6 month period from June to December 1998, 677 Campylobacter isolates were obtained from healthy poultry and pigs. Samples were taken at Belgian slaughterhouses. Species identification was performed by biochemical tests, multiplex PCR and SDS-PAGE of whole-cell proteins. The in vitro susceptibility to six antimicrobial drugs was determined by the agar dilution method. Campylobacter jejuni was found more often in poultry than Campylobacter coli (79% C. jejuni versus 21% C. coli). In pigs the situation was reversed (6 versus 94%). Erythromycin resistance was significantly higher (P < 0.05) in C. coli, particularly in C. coli isolated from pigs (67.2%). Alarmingly high rates of resistance to ciprofloxacin were also noted, particularly for C. coli from broilers (62.1%). The latter indicates that resistance of Campylobacter in humans could derive from animals. (+info)