Identification of possible virulence marker from Campylobacter jejuni isolates.
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ABSTRACT:
SUBMITTER: Harrison JW
PROVIDER: S-EPMC4036754 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Recently the Type VI secretion system (T6SS), which can play a significant role in bacterial survival and pathogenesis, was reported in Campylobacter spp., having the hcp gene as a key component. METHODS:Campylobacteriosis is associated with the consumption of infected chicken meat. Our study aimed to explore the presence of T6SS in C. jejuni (n = 59) and C. coli (n = 57) isolates, from retail raw chicken and to investigate their pathogenic potential. The hcp gene was used as an indicator for the T6SS presence. RESULTS:Using multiplex PCR we have identified a significantly higher prevalence of hcp in C. coli isolates (56.1%) than in C. jejuni (28.8%) and AFLP analysis of the isolates showed a high degree of genetic similarity between the isolates carrying the hcp gene. Genome sequencing data showed that 84.3% of the C. coli and 93.7% of the C. jejuni isolates had all 13 T6SS open reading frames. Moreover, the virulence characteristics of hcp + isolates, including motility and the ability to invade human intestinal epithelial cells in vitro, were significantly greater than in the control strain C. jejuni 12502; a human isolate which is hcp positive. CONCLUSION:Overall, it was discovered that hcp (+) C. coli and C. jejuni isolated from retail chicken isolates posses genetic and phenotypic properties associated with enhanced virulence. However, since human infections with C. coli are significantly less frequent than those of C. jejuni, the relationship between virulence factors and pathogenesis requires further study.
| S-EPMC4511981 | biostudies-literature
Project description:Comparison of the entire coding sequence of flaA (1,764 nucleotides) from 15 isolates of Campylobacter jejuni showed two regions of high variability, one region approximately from base positions 700 to 1,450 and a short variable region (SVR) from base positions 450 to 600. Parsimony analysis of the SVR sequences yielded a dendrogram similar to that which was derived by analysis of the entire gene. PCR was used to generate templates, and the SVR was sequenced with primers constructed to hybridize to conserved flanking sequences. The SVRs of 22 isolates of C. jejuni from four outbreaks that have been well characterized and a larger panel of isolates from three additional outbreaks were sequenced. Analysis of the nucleotide sequences produced results that grouped the isolates very similarly to other subtyping techniques. Sequence data were also generated for isolates from three additional outbreaks. Categorizing the isolates by fla SVR DNA sequence placed them in epidemiologically relevant groups. Sequence analysis of the C. jejuni flaA SVR may be a useful tool for epidemiologic investigations and could complement or replace serotyping and other subtyping methods.
| S-EPMC230067 | biostudies-other
Project description:Campylobacter jejuni and Campylobacter coli are common causes of gastroenteritis in humans. Infection with C. jejuni or C. coli is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for C. jejuni and C. coli isolates based on the cadF virulence gene and its product. The cadF gene from C. jejuni and C. coli encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates with primers designed to amplify an internal fragment of the cadF gene. PCR was then used to amplify Campylobacter DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-C. jejuni 37-kDa serum. In addition, methanol-fixed samples of whole-cell C. jejuni and C. coli were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the cadF gene and its product are conserved among C. jejuni and C. coli isolates and that a PCR assay based on the cadF gene may be useful for the detection of Campylobacter organisms in food products.
| S-EPMC84446 | biostudies-literature
Project description:BackgroundThe aim of this study was to determine the prevalence of virulence-associated genes and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) analysis of Campylobacter spp. isolated from children with diarrhea in Iran.MethodsA total of 200 stool specimens were obtained from children under 5 years during July 2012 to July 2013. Detection of C. jejuni and C. coli was performed by standard biochemical and molecular methods. The presence of virulence-associated genes and genetic diversity of isolates was examined using PCR and ERIC-PCR analyses.ResultsA total of 12 (6%) Campylobacter spp. were isolated from patients including 10 (4.5%) C. jejuni and 2 (1.5%) C.coli. The flaA, cadF and ciaB genes were present in 100% of isolates, while no plasmid of virB11 gene was present in their genome. The prevalence of invasion-associated marker was 100% among C. coli and was not detected in C. jejuni isolates. The distribution of both pldA and the genes associated with cytolethal distending toxin (CDT) was 58.3% in C. jejuni isolates. Seven distinct ERIC-PCR profiles were distinguished in three clusters using ERIC-PCR analysis. Genotyping analysis showed a relative correlation with geographic location of patients and virulence gene content of isolates.ConclusionTo our knowledge, this is the first molecular survey of Campylobacter spp. in Iran concerning genotyping and virulence gene content of both C. jejuni and C. coli. ERIC-PCR revealed appropriate discriminatory power for clustering C. jejuni isolates with identical virulence gene content. However, more studies are needed to clearly understand the pathogenesis properties of specific genotypes.
| S-EPMC4048480 | biostudies-literature
Project description:Campylobacter jejuni is one of the most common causes of bacterial diarrhea worldwide and is the primary bacterial cause of food-borne illness. Adherence to and invasion of epithelial cells are the most important pathogenic mechanisms of Campylobacter diarrhea. Molecular characterization of invasive and noninvasive Campylobacter isolates from children with diarrhea and symptom-free children was performed by random amplified polymorphic DNA techniques (RAPD). A distinct RAPD profile with a DNA band of 1.6 kb was observed significantly more frequently among invasive (63%) than among noninvasive (16%) Campylobacter isolates (P = 0.000005). The 1.6-kb band was named the invasion-associated marker (IAM). Using specifically designed primers, a fragment of 518 bp of the iam locus was amplified in 85% of invasive and 20% of noninvasive strains (P = 0.0000000). Molecular typing with a PCR-restriction fragment length polymorphism assay which amplified the entire iam locus showed a HindIII restriction fragment polymorphism pattern associated mainly with invasive strains. Although cluster analysis of the RAPD fingerprinting showed genetic diversity among strains, two main clusters were identified. Cluster I comprised significantly more pathogenic and invasive isolates, while cluster II grouped the majority of nonpathogenic, noninvasive isolates. These data indicate that most of the invasive Campylobacter strains could be differentiated from noninvasive isolates by RAPD analysis and PCR using specific primers that amplify a fragment of the iam locus.
| S-EPMC87939 | biostudies-literature
Project description:A promoterless lacZ shuttle vector, which allowed screening of promoters by beta-galactosidase activity in Campylobacter jejuni and Escherichia coli, was developed. Chromosomal DNA fragments from C. jejuni were cloned into this vector; 125 of 1,824 clones displayed promoter activity in C. jejuni. Eleven clones with strong promoter activity in C. jejuni were further characterized. Their nucleotide sequences were determined, and the transcriptional start sites of the putative promoters in C. jejuni were determined by primer extension. Only 6 of these 11 promoters were functional in E. coli. The 11 newly characterized and 10 previously characterized C. jejuni promoters were used to establish a consensus sequence for C. jejuni promoters. The 21 promoters were found to be very similar. They contain three conserved regions, located approximately 10, 16, and 35 bp upstream of the transcriptional start point. The -10 region resembles that of a typical sigma70 E. coli promoter, but the -35 region is completely different. In addition a -16 region typical for gram-positive bacteria was identified.
| S-EPMC106926 | biostudies-literature
Project description:Campylobacter jejuni is the major cause of human bacterial diarrhea worldwide. Its pathogenic mechanism remains poorly understood. cj0371 is a novel gene that was uncovered using immunoscreening. There have been no previous reports regarding its function. In this study, we constructed an insertion mutant and complement of this gene in C. jejuni and examined changes in virulence. We observed that the cj0371 mutant showed significantly increased invasion and colonization ability. We also investigated the role of cj0371 in motility, chemotaxis, and growth kinetics to further study its function. We found that the cj0371 mutant displays hypermotility, enhanced chemotaxis, and enhanced growth kinetics. In addition, we localized the Cj0371 protein at the poles of C. jejuni by fluorescence microscopy. We present data that collectively significantly proves our hypothesis that cj0371 is a new virulence-associated gene and through the influence of chemotaxis plays a negative role in C. jejuni pathogenicity.
| S-EPMC4944492 | biostudies-literature
Project description:Analysis of nucleic acid polymorphism in the flagellin genes of Campylobacter jejuni was used to investigate genetic diversity among Campylobacter spp. in a commercial broiler flock. Three hundred single colonies of C. jejuni were isolated from fecal samples collected weekly for 3 weeks immediately before slaughter. Both the flaA and flaB genes were amplified by PCR, and the PCR product was digested with the restriction enzyme AluI. The fragments generated were then analyzed by agarose gel electrophoresis. Among the 300 recovered isolates, five different restriction fragment length polymorphism profiles were observed. Three of these profiles were dominant during the course of the study, and the other two profiles were detected at low frequency. Analysis of genetic variation in C. jejuni over the course of an experimental infection lasting 7 weeks indicated that there was no obvious drift in the flagellin gene type. These findings demonstrate that a range of bacterial genotypes can constitute the bacterial population within a commercial poultry flock, with the most likely sources of these types being multiple environmental exposure and/or genetic drift within the population. This degree of diversity must be considered in epidemiological analyses which utilize genetic typing methods that investigate Campylobacter contamination of any food source, including poultry, to ensure that the total gene pool for C. jejuni is evaluated.
| S-EPMC1389157 | biostudies-other
Project description:There are still major gaps in our understanding of the bacterial factors that influence the outcomes of human Campylobacter jejuni infection. The aim of this study was to compare the virulence-associated features of 192 human C. jejuni strains isolated from hospitalized patients with diarrhoea (150/192, 78.1%), bloody diarrhoea (23/192, 11.9%), gastroenteritis (3/192, 1.6%), ulcerative colitis (3/192, 1.5%), and stomach ache (2/192, 1.0%). Traits were analysed with genotypic and phenotypic methods, including PCR and extracellular matrix protein (ECMP) binding, adhesion, and invasion capacities. Results were studied alongside patient symptoms, but no distinct links with them could be determined. Since the capacity of C. jejuni to invade host epithelial cells is one of its most enigmatic attributes, a high throughput transcriptomic analysis was performed in the third hour of internalization with a C. jejuni strain originally isolated from bloody diarrhoea. Characteristic groups of genes were significantly upregulated, outlining a survival strategy of internalized C. jejuni comprising genes related (1) to oxidative stress; (2) to a protective sheath formed by the capsule, LOS, N-, and O- glycosylation systems; (3) to dynamic metabolic activity supported by different translocases and the membrane-integrated component of the flagellar apparatus; and (4) to hitherto unknown genes.
| S-EPMC7232156 | biostudies-literature
Project description:Campylobacter jejuni is the major cause of bacterial gastroenteritis in man, while it is generally regarded as a commensal of the avian gut. Consumption and handling of contaminated poultry meat products are major risk factors for human infection. The body temperature in man (37 °C) and chickens (42 °C) differ markedly, and differential gene regulation and protein expression at different temperatures may in part explain the behaviour in the two hosts. We performed proteomics analyses with C. jejuni cells grown at 37 °C and 42 °C. Time-of-flight mass spectrometry (Q-Tof) analysis was carried out after samples were digested with the Filter-Aided Sample Preparation (FASP) method and peptides were fractionated by strong anion exchanges. Differentially regulated proteins were identified by Mascot and Scaffold analyses. Triple quadrupole (QQQ) mass spectrometer analysis confirmed that a total of 33 proteins were differentially regulated between 37 °C and 42 °C. Several upregulated proteins were selected for their corresponding gene knock-out mutants to be tested for their virulence in the Galleria mellonella model. To correlate with other tissue/animal models, the GADH mutant was selected for its reduced ability to colonize chickens. At 37 °C, the mutants of outer membrane protein Omp50 and Chaperone GroEL significantly increased virulence; while at 42 °C, the mutants of YceI, Omp50, and GADH reduced virulence against Galleria mellonella compared with the wild type strains. The results of current and previous studies indicate that GADH is a virulent factor in G. mellonella and a colonization factor in chickens. The workflow of this study may prove a new way to identify stress related virulent factors. The implications of these findings are discussed for pathogenesis in the model and other hosts.
| S-EPMC6604965 | biostudies-literature