El Consulado de Cádiz y el Reglamento de Comercio Libre en 1765

Tomo I ; pág. 79-9

Comparative genotypic and pathogenic examination of Campylobacter concisus isolates from diarrheic and non-diarrheic humans

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter concisus </it>is an emerging enteric pathogen, yet it is commonly isolated from feces and the oral cavities of healthy individuals. This genetically complex species is comprised of several distinct genomospecies which may vary in pathogenic potential.</p> <p>Results</p> <p>We compared pathogenic and genotypic properties of <it>C. concisus </it>fecal isolates from diarrheic and healthy humans residing in the same geographic region. Analysis of amplified fragment length polymorphism (AFLP) profiles delineated two main clusters. Isolates assigned to AFLP cluster 1 belonged to genomospecies A (based on genomospecies-specific differences in the 23S rRNA gene) and were predominantly isolated from healthy individuals. This cluster also contained a reference oral strain. Isolates assigned to this cluster induced greater expression of epithelial IL-8 mRNA and more frequently contained genes coding for the zonnula occludins toxin and the S-layer RTX. Furthermore, isolates from healthy individuals induced greater apoptotic DNA fragmentation and increased metabolic activity than those from diarrheic individuals, and isolates assigned to genomospecies A (of which the majority were from healthy individuals) exhibited higher haemolytic activity compared to genomospecies B isolates. In contrast, AFLP cluster 2 was predominated by isolates belonging to genomospecies B and those from diarrheic individuals. Isolates from this cluster displayed greater mean epithelial invasion and translocation than cluster 1 isolates.</p> <p>Conclusion</p> <p>Two main genetically distinct clusters (i.e., genomospecies) were identified among <it>C. concisus </it>fecal isolates from healthy and diarrheic individuals. Strains within these clusters differed with respect to clinical presentation and pathogenic properties, supporting the hypothesis that pathogenic potential varies between genomospecies. ALFP cluster 2 isolates were predominantly from diarrheic patients, and exhibited higher levels of epithelial invasion and translocation, consistent with known roles for these factors in diarrhoeal disease. Conversely, isolates from healthy humans and AFLP cluster 1 or genomospecies A (which were predominantly isolated from healthy humans) exhibited increased haemolytic ability, apoptotic DNA fragmentation, IL-8 induction, and/or carriage of toxin genes. Given that this cluster contains an oral reference strain, it is possible that some of the AFLP cluster 1 isolates are periodontal pathogens and may cause disease, albeit via a different mechanism than those from AFLP cluster 2.</p

Campylobacter jejuni induces transcellular translocation of commensal bacteria via lipid rafts

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter </it>enteritis represents a risk factor for the development of inflammatory bowel disease (IBD) via unknown mechanisms. As IBD patients exhibit inflammatory responses to their commensal intestinal microflora, factors that induce translocation of commensal bacteria across the intestinal epithelium may contribute to IBD pathogenesis. This study sought to determine whether <it>Campylobacter </it>induces translocation of non-invasive intestinal bacteria, and characterize underlying mechanisms.</p> <p>Methods</p> <p>Mice were infected with <it>C. jejuni </it>and translocation of intestinal bacteria was assessed by quantitative bacterial culture of mesenteric lymph nodes (MLNs), liver, and spleen. To examine mechanisms of <it>Campylobacter</it>-induced bacterial translocation, transwell-grown T84 monolayers were inoculated with non-invasive <it>Escherichia coli </it>HB101 ± wild-type <it>Campylobacter </it>or invasion-defective mutants, and bacterial internalization and translocation were measured. Epithelial permeability was assessed by measuring flux of a 3 kDa dextran probe. The role of lipid rafts was assessed by cholesterol depletion and caveolin co-localization.</p> <p>Results</p> <p><it>C. jejuni </it>81–176 induced translocation of commensal intestinal bacteria to the MLNs, liver, and spleen of infected mice. In T84 monolayers, <it>Campylobacter</it>-induced internalization and translocation of <it>E. coli </it>occurred via a transcellular pathway, without increasing epithelial permeability, and was blocked by depletion of epithelial plasma membrane cholesterol. Invasion-defective mutants and <it>Campylobacter</it>-conditioned cell culture medium also induced <it>E. coli </it>translocation, indicating that <it>C. jejuni </it>does not directly 'shuttle' bacteria into enterocytes. In <it>C. jejuni</it>-treated monolayers, translocating <it>E. coli </it>associated with lipid rafts, and this phenomenon was blocked by cholesterol depletion.</p> <p>Conclusion</p> <p><it>Campylobacter</it>, regardless of its own invasiveness, promotes the translocation of non-invasive bacteria across the intestinal epithelium via a lipid raft-mediated transcellular process.</p

Campylobacter jejuni induces transcytosis of commensal bacteria across the intestinal epithelium through M-like cells

<p>Abstract</p> <p>Background</p> <p>Recent epidemiological analyses have implicated acute <it>Campylobacter </it>enteritis as a factor that may incite or exacerbate inflammatory bowel disease (IBD) in susceptible individuals. We have demonstrated previously that <it>C. jejuni </it>disrupts the intestinal barrier function by rapidly inducing epithelial translocation of non-invasive commensal bacteria via a transcellular lipid raft-mediated mechanism ('transcytosis'). To further characterize this mechanism, the aim of this current study was to elucidate whether <it>C. jejuni </it>utilizes M cells to facilitate transcytosis of commensal intestinal bacteria.</p> <p>Results</p> <p><it>C. jejuni </it>induced translocation of non-invasive <it>E. coli </it>across confluent Caco-2 epithelial monolayers in the absence of disrupted transepithelial electrical resistance or increased permeability to a 3 kDa dextran probe. <it>C. jejuni</it>-infected monolayers displayed increased numbers of cells expressing the M cell-specific marker, galectin-9, reduced numbers of enterocytes that stained with the absorptive enterocyte marker, <it>Ulex europaeus </it>agglutinin-1, and reduced activities of enzymes typically associated with absorptive enterocytes (namely alkaline phosphatase, lactase, and sucrase). Furthermore, in <it>Campylobacter</it>-infected monolayers, <it>E. coli </it>were observed to be internalized specifically within epithelial cells displaying M-like cell characteristics.</p> <p>Conclusion</p> <p>These data indicate that <it>C. jejuni </it>may utilize M cells to promote transcytosis of non-invasive bacteria across the intact intestinal epithelial barrier. This mechanism may contribute to the inflammatory immune responses against commensal intestinal bacteria commonly observed in IBD patients.</p

Catheterization of Intestinal Loops in Ruminants

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (<24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (<6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period

Non-therapeutic administration of a model antimicrobial growth promoter modulates intestinal immune responses

<p>Abstract</p> <p>Background</p> <p>The development of efficacious alternatives to antimicrobial growth promoters (AGP) in livestock production is an urgent issue, but is hampered by a lack of knowledge regarding the mode of action of AGP. The belief that AGP modulate the intestinal microbiota has become prominent in the literature; however, there is a lack of experimental evidence to support this hypothesis. Using a chlortetracycline-murine-<it>Citrobacter rodentium </it>model, the ability of AGP to modulate the intestinal immune system in mammals was investigated.</p> <p>Results</p> <p><it>C. rodentium </it>was transformed with the tetracycline resistance gene, <it>tet</it>O, and continuous oral administration of a non-therapeutic dose of chlortetracycline to mice did not affect densities of <it>C. rodentium </it>CFU in feces throughout the experiment or associated with mucosal surfaces in the colon (i.e. at peak and late infection). However, chlortetracycline regulated transcription levels of Th1 and Th17 inflammatory cytokines in a temporal manner in <it>C. rodentium</it>-inoculated mice, and ameliorated weight loss associated with infection. In mice inoculated with <it>C. rodentium</it>, those that received chlortetracycline had less pathologic changes in the distal colon than mice not administered CTC (i.e. relative to untreated mice). Furthermore, chlortetracycline administration at a non-therapeutic dose did not impart either prominent or consistent effects on the colonic microbiota.</p> <p>Conclusion</p> <p>Data support the hypothesis that AGP function by modulating the intestinal immune system in mammals. This finding may facilitate the development of biorationale-based and efficacious alternatives to AGP.</p

Development of a comparative genomic fingerprinting assay for rapid and high resolution genotyping of Arcobacter butzleri

Sherpa Romeo green journal. Open access, distributed under the terms of the Creative Commons Attribution (CC-BY) License.Background Molecular typing methods are critical for epidemiological investigations, facilitating disease outbreak detection and source identification. Study of the epidemiology of the emerging human pathogen Arcobacter butzleri is currently hampered by the lack of a subtyping method that is easily deployable in the context of routine epidemiological surveillance. In this study we describe a comparative genomic fingerprinting (CGF) method for high-resolution and high-throughput subtyping of A. butzleri. Comparative analysis of the genome sequences of eleven A. butzleri strains, including eight strains newly sequenced as part of this project, was employed to identify accessory genes suitable for generating unique genetic fingerprints for high-resolution subtyping based on gene presence or absence within a strain. Results A set of eighty-three accessory genes was used to examine the population structure of a dataset comprised of isolates from various sources, including human and non-human animals, sewage, and river water (n=156). A streamlined assay (CGF40) based on a subset of 40 genes was subsequently developed through marker optimization. High levels of profile diversity (121 distinct profiles) were observed among the 156 isolates in the dataset, and a high Simpson’s Index of Diversity (ID) observed (ID > 0.969) indicate that the CGF40 assay possesses high discriminatory power. At the same time, our observation that 115 isolates in this dataset could be assigned to 29 clades with a profile similarity of 90% or greater indicates that the method can be used to identify clades comprised of genetically similar isolates. Conclusions The CGF40 assay described herein combines high resolution and repeatability with high throughput for the rapid characterization of A. butzleri strains. This assay will facilitate the study of the population structure and epidemiology of A. butzleri.Ye

Effect of antimicrobial growth promoter administration on the intestinal microbiota of beef cattle

Sherpa Romeo green journal. Open access journal. Creative Commons Attribution 2.0 Generic License (CC BY 2.0) appliesBackground: Antimicrobial growth promoters (AGPs) are antimicrobial agents administered to livestock in feed for prolonged periods to enhance feed efficiency. Beef cattle are primarily finished in confined feeding operations in Canada and the USA, and the administration of AGPs such as chlortetracycline and sulfamethazine (Aureo S-700 G) is the standard. The impacts of AGPs on the intestinal microbiota of beef cattle are currently uncertain; it is documented that AGPs administered to beef cattle pass through the rumen and enter the intestine. To ascertain the impacts of Aureo S-700 G on the small and large intestinal microbiota of beef cattle (mucosa-associated and within digesta), terminal restriction fragment length polymorphism (T-RFLP) analysis and quantitative PCR (qPCR) for total bacteria were applied. Beef cattle were maintained in an experimental feedlot (five replicate pens per treatment), and AGP treatment cattle were administered Aureo S-700 G in feed, whereas control cattle were administered no antimicrobials. As the intestinal microbiota of beef cattle has not been extensively examined, clone library analysis was applied to ascertain the primary bacterial constituents of the intestinal microbiota. Results: Comparative T-RFLP and qPCR analysis (n = 122 samples) revealed that bacterial community fingerprints and bacterial load within digesta differed from those associated with mucosa. However, the administration of Aureo S-700 G did not affect bacterial community fingerprints or bacterial load within the small and large intestine relative to control cattle. Analysis of >1500 near full length 16S rDNA clones revealed considerably greater bacterial diversity in the large relative to the small intestine of beef cattle. Mucosa-associated bacterial communities in the jejunum were dominated by Proteobacteria, and differed conspicuously from those in the ileum and large intestine. Although the ileum contained bacterial clones that were common to the jejunum as well as the cecum, Firmicutes clones associated with mucosa dominated in the ileum, cecum, and descending colon. In the descending colon, clone library analysis did not reveal a difference in the richness or diversity of bacterial communities within digesta relative to those associated with mucosa. However, T-RFLP analysis indicated a significant difference in T-RF relative abundance (i.e. difference in relative taxon abundance) between mucosa-associated and digesta communities attributed in part to the differential abundance of Bacteriodes, Alistipes, Oscillibacter, and unclassified Clostridiales. Conclusions: These data demonstrate that there was no significant difference in the composition of the predominant intestinal bacteria constituents within animals administered Aureo S-700 G and those not administered AGPs after a 28 day withdrawal period.Ye

Comparative variation within the genome of Campylobacter jejuni NCTC 11168 in human and murine hosts

Sherpa Romeo green journal. Open access, distributed under the terms of the Creative Commons Attibution License.Campylobacteriosis incited by C. jejuni is a significant enteric disease of human beings. A person working with two reference strains of C. jejuni National Collection of Type Cultures (NCTC) 11168 developed symptoms of severe enteritis including bloody diarrhea. The worker was determined to be infected by C. jejuni . In excess of 50 isolates were recovered from the worker’s stool. All of the recovered isolates and the two reference strains were indistinguishable from each other based on comparative genomic fingerprint subtyping. Whole genome sequence analysis indicated that the worker was infected with a C. jejuni NCTC 11168 obtained from the American Type Culture Collection; this strain (NCTC 11168-GSv) is the genome sequence reference. After passage through the human host, major genetic changes including indel mutations within twelve contingency loci conferring phase variations were detected in the genome of C. jejuni . Specific and robust single nucleotide polymorphism (SNP) changes in the human host were also observed in two loci (Cj0144c, Cj1564). In mice inoculated with an isolate of C. jejuni NCTC 11168-GSv from the infected person, the isolate underwent further genetic variation. At nine loci, mutations specific to inoculated mice including five SNP changes were observed. The two predominant SNPs observed in the human host reverted in mice. Genetic variations occurring in the genome of C. jejuni in mice corresponded to increased densities of C. jejuni cells associated with cecal mucosa. In conclusion, C. jejuni NCTC 11168-GSv was found to be highly virulent in a human being inciting severe enteritis. Host-specific mutations in the person with enteritis occurred/were selected for in the genome of C. jejuni , and many were not maintained in mice. Information obtained in the current study provides new information on host-specific genetic adaptation by C. jejuni .Ye