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Bacteria may play a role in esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), although evidence is limited to cross-sectional studies. In this study, we examined the relationship of oral microbiota with EAC and ESCC risk in a prospective study nested in two cohorts. Oral bacteria were assessed using 16S rRNA gene sequencing in pre-diagnostic mouthwash samples from n=81/160 EAC and n=25/50 ESCC cases/matched controls. Findings were largely consistent across both cohorts. Metagenome content was predicted using PiCRUST. We examined associations between centered log-ratio transformed taxon or functional pathway abundances and risk using conditional logistic regression adjusting for BMI, smoking, and alcohol. We found the periodontal pathogen Tannerella forsythia to be associated with higher risk of EAC. Further, we found that depletion of the commensal genus Neisseria and the species Streptococcus pneumoniae were associated with lower EAC risk. Bacterial biosynthesis of carotenoids was also associated with protection against EAC. Lastly, the abundance of the periodontal pathogen Porphyromonas gingivalis trended with higher risk of ESCC. Overall, our findings have potential implications for the early detection and prevention of EAC and ESCC.

The concentration of oxygen in the atmosphere is a common environmental factor which can also be a source of stress for microorganisms. Comparative analyses of the responses of the epsilon-proteobacteria Campylobacter jejuni, Helicobacter pylori and Wolinella succinogenes to elevated oxygen concentrations were carried out using transcriptomics. Microarray data were analysed to determine genes differentially expressed under elevated oxygen concentrations. The results indicated 158, 58 and 82 genes were upregulated and 46, 40 and 65 were downregulated in C. jejuni, H. pylori and W. succinogenes, respectively. The gene encoding the enzyme alkyl hydroperoxide reductase was the only one upregulated at higher oxygen tensions in all three bacterial species. No genes were found to be downregulated in all three species. Functional classification analyses were performed on the genes whose expression was modulated in order to identify common pathways and functional categories which were differentially expressed in the three organisms. Processes upregulated at higher oxygen tensions included translation, oxidative phosphorylation, antioxidation, and nucleic acid metabolism. ABC and ion-coupled transport proteins were generally downregulated at higher oxygen tensions. Finally, insights into the preferred environment were gained from the analyses of the bacterial responses, specifically motility and chemotaxis proteins. W. succinogenes preferred anaerobic conditions as opposed to C. jejuni and H. pylori preference for microaerobic conditions. These comparative studies provide a better understanding of bacterial adaptation to and interaction with their environment.

Cadmium ions are a potent carcinogen in animals, and cadmium is a toxic metal of significant environmental importance for humans. Response curves were used to investigate the effects of cadmium chloride on the growth of Camplyobacter jejuni. In vitro, the bacterium showed reduced growth in the presence of 0.1 mm cadmium chloride, and the metal ions were lethal at 1 mm concentration. Two-dimensional gel electrophoresis combined with tandem mass spectrometry analysis enabled identification of 67 proteins differentially expressed in cells grown without and with 0.1 mm cadmium chloride. Cellular processes and pathways regulated under cadmium stress included fatty acid biosynthesis, protein biosynthesis, chemotaxis and mobility, the tricarboxylic acid cycle, protein modification, redox processes and the heat-shock response. Disulfide reductases and their substrates play many roles in cellular processes, including protection against reactive oxygen species and detoxification of xenobiotics, such as cadmium. The effects of cadmium on thioredoxin reductase and disulfide reductases using glutathione as a substrate were studied in bacterial lysates by spectrophotometry and nuclear magnetic resonance spectroscopy, respectively. The presence of 0.1 mm cadmium ions modulated the activities of both enzymes. The interactions of cadmium ions with oxidized glutathione and reduced glutathione were investigated using nuclear magnetic resonance spectroscopy. The data suggested that, unlike other organisms, C. jejuni downregulates thioredoxin reductase and upregulates other disulfide reductases involved in metal detoxification in the presence of cadmium.

cope Overconsumption of energy-rich food is a major contributor to the obesity epidemic. The eating habits of many people are characterized by the cycling between overconsumption of energy-rich foods and dieting, the effects of which on the microbiota are currently unknown. Methods and results We compared the fecal microbiota of rats either continuously fed chow or palatable cafeteria diet to a “cycled” group switched between the two diets (chow for 4, cafeteria for 3 days/wk, n = 12/group) over 16 wk. Enriched bacterial metabolic pathways were predicted, and a range of metabolic parameters was correlated to microbial taxa and pathways. Cycled rats showed large excursions in food intake on each diet switch. When switched from chow to cafeteria, they overconsumed, and when switched back to chow they underconsumed relative to those maintained on the two diets. Metabolic parameters of cycled rats were intermediate between those of the other diet groups (p < 0.05). The microbiota of cycled rats was nearly indistinguishable from rats under constant cafeteria diet, and both groups were significantly different to the chow group. Correlation analyses identified microbial metabolic pathways associated with an obese phenotype. Conclusion These data suggest that continuous or intermittent exposure to palatable foods have similar effects on the gut microbiota.

The B2 phylogenetic group of Escherichia coli contains important pathogens such as extraintestinal pathogenic, adherent-invasive, and uropathogenic strains. In this study, we used comparative genomics and statistical methods to identify genetic variations that define a subset of pathogenic strains belonging to the B2 phylogenetic group. An initial proof of concept analysis indicated that five of the 62 E. coli strains available in the Kyoto Encyclopedia of Genes and Genomes database showed close association with B2 adherent-invasive E. coli, forming a subgroup within the B2 phylogenetic group. The tool, kSNP which uses a k-mer approach, and the statistical phenotype prediction tool PPFS2 were then employed to identify 29 high-resolution SNPs, which reaffirmed this grouping. PPFS2 analysis also provided indications that the clustering of this subgroup was highly consistent, and thus, could have a strong phenotypic basis rather than being only evolutionary. Protein homology analyses identified three proteins to be conserved across this subgrouping, two CRISPR-Cas proteins and a hypothetical protein. Functional analyses of these genetic and protein variations may provide insights into the phenotype of these strains.

Limited information is available on the systemic immunoglobulin response in patients infected with the emerging pathogen Campylobacter concisus. The aim of the present study was to detect anti-C. concisus antibodies in serum of 88 patients with C. concisus gastroenteritis. Specific IgG antibodies to C. concisus were measured in serum using an in-house enzyme-linked immunosorbent assay, and pooled donor serum was used as a control. The mean optical density was 0.135 (SEM: 0.020) for the 88 adult patients and 0.100 (SEM: 0.011) in controls. When using an optical density value equal to the mean +3 SEM for the control serum, 22 (25%) C. concisus-positive patients had increased IgG antibodies. Patients with high IgG levels more often reported headache, and they had a trend toward more mucus in stools, whereas IgG levels were unrelated to age, duration of diarrhea, number of stools per day, and weight loss.

Background Increasing demand for fecal microbiota transplantation (FMT) has created a need for stool banks sourced from long-term healthy donors. Here, we describe our experience in recruiting and screening fecal donors. Methods Mailbox, newspaper, and online advertisements were used. Potential donors were required to satisfy a prescreen telephone conversation, pass blood and stool investigations, then undertake a screening interview including medical history, physical examination, and evaluation of donor selection criteria. Results One hundred sixteen potential donors were prescreened of whom 74 failed-47 declined based on study donation requirements (primarily related to frequency and duration of donations), 13 had medical comorbidities, 6 variant Creutzfeldt-Jakob disease risk factors, 8 for other reasons. Thirty-eight completed stool and blood testing-1 failed blood testing (indeterminate hepatitis C serology), whereas 15 failed stool investigations (5 Dientamoeba fragilis, 5 Blastocystis hominis, 1 B. hominis and D. fragilis, 1 Giardia intestinalis plus D. fragilis, 1 Norovirus plus Clostridium difficile toxin positive, and 2 leucocytes or erythrocytes on stool microscopy). Of the 18 potential donors proceeding to screening interview, 6 were excluded (3 body mass index >30, 1 illicit drug use, 1 uncontrolled anxiety and concerns regarding compliance, 1 irregular bowel movements after new medication commencement). In total, only 12 of 116 (10%) potential donors were enrolled as study donors. Conclusions Recruitment of fecal donors for FMT is challenging with only a small percentage ultimately serving as donors. Many were unable or unwilling to meet the donor commitment requirements. A surprisingly large proportion of healthy asymptomatic donors failed stool testing, primarily due to gastrointestinal parasites.

© 2015 Baishideng Publishing Group Inc. All rights reserved. AIM: To explore gastroenterologist perceptions towards and experience with faecal microbiota transplantation (FMT). METHODS: A questionnaire survey consisting of 17 questions was created to assess gastroenterologists' attitude towards and experience with FMT. This was anonymously distributed in hard copy format amongst attendees at gastroenterology meetings in Australia between October 2013 and April 2014. Basic descriptive statistical analyses were performed. RESULTS: Fifty-two clinicians participated. Twenty one percent had previously referred patients for FMT, 8% more than once. Ninety percent would refer patients with Clostridium difficile infection (CDI) for FMT if easily available, 37% for ulcerative colitis, 13% for Crohn's disease and 6% for irritable bowel syndrome. Six percent would not refer any indication, including recurrent CDI. Eighty-six percent would enroll patients in FMT clinical trials. Thirty-seven percent considered the optimal mode of FMT administration transcolonoscopic, 17% nasoduodenal, 13% enema and 8% oral capsule. The greatest concerns regarding FMT were: 42% lack of evidence, 12% infection risk, 10% non infectious adverse effects/lack of safety data, 10% aesthetic, 10% lack of efficacy, 4% disease exacerbation, and 2% inappropriate use; 6% had no concerns. Seventy seven percent believed there is a lack of accessibility while 52% had an interest in learning how to provide FMT. Only 6% offered FMT at their institution. CONCLUSION: Despite general enthusiasm, most gastroenterologists have limited experience with, or access to, FMT. The greatest concerns were lack of supportive evidence and safety issues. However a significant proportion would refer indications other than CDI for FMT despite insufficient evidence. These data provide guidance on where education and training are required.

Campylobacter species are Gram-negative bacteria, their morphology varying from spiral to rod or curved in shape depending on the species. Some species are aflagellate whilst others have a single polar flagellum or bipolar flagella. The genus Campylobacter consists of 25 species, two provisional species and eight subspecies (as of August 2013) [1]. Campylobacter species have a widespread ecological distribution and are found in humans and domesticated or wild animals, including cattle, birds, reptiles and shellfish. C. jejuni is a leading cause of gastroenteritis worldwide. Other members of the Campylobacter species, including C. coli and C. fetus, are known pathogens in humans and animals. Several other species, including C. concisus, C. ureolyticus and C. lari, are known as ‘emerging Campylobacter species’, a term used to recognize their increasingly important role in human and animal diseases. In this chapter, we describe the epidemiology, genomic characteristics, mechanisms of attachment and invasion, toxin production, glycosylation, capsular polysaccharide production, biofilm formation and antibiotic resistance profile of members of the Campylobacter genus. We also summarize the role of host immune responses and the use of animal models in the understanding of the pathogenesis of these species.

Background: There is no general consensus about the specific oxygen and carbon dioxide requirements of the human pathogen Helicobacter pylori. This bacterium is considered a microaerophile and consequently, it is grown under atmospheres at oxygen tensions 5–19% and carbon dioxide tensions 5–10%, both for clinical and basic and applied research purposes. The current study compared the growth of H. pylori in vitro, under various gas atmospheres, and determined some specific changes in the physiology of bacteria grown under different oxygen partial pressures. Methods: Measurements of bacterial growth under various conditions were carried out employing classical solid and liquid culture techniques. Enzymatic activities were measured using spectrophotometric assays. Results: H. pylori and all the other Helicobacter spp. tested had an absolute requirement for elevated carbon dioxide concentrations in the growth atmosphere. In contrast with other Helicobacter spp., H. pylori can tolerate elevated oxygen tensions when grown at high bacterial concentrations. Under 5% CO2, the bacterium showed similar growth in liquid cultures under oxygen tensions from microaerobic (

Pathogenic species within the genus Campylobacter are responsible for a considerable burden on global health. Campylobacter concisus is an emergent pathogen that plays a role in acute and chronic gastrointestinal disease. Despite ongoing research on Campylobacter virulence mechanisms, little is known regarding the immunological profile of the host response to Campylobacter infection. In this study, we describe a comprehensive global profile of innate immune responses to C. concisus infection in differentiated THP-1 macrophages infected with an adherent and invasive strain of C. concisus . Using RNA sequencing (RNA-seq), quantitative PCR (qPCR), mass spectrometry, and confocal microscopy, we observed differential expression of pattern recognition receptors and robust upregulation of DNA- and RNA-sensing molecules. In particular, we observed IFI16 inflammasome assembly in C. concisus -infected macrophages. Global profiling of the transcriptome revealed the significant regulation of a total of 8,343 transcripts upon infection with C. concisus , which included the activation of key inflammatory pathways involving CREB1, NF-κB, STAT, and interferon regulatory factor signaling. Thirteen microRNAs and 333 noncoding RNAs were significantly regulated upon infection, including MIR221, which has been associated with colorectal carcinogenesis. This study represents a major advance in our understanding of host recognition and innate immune responses to infection by C. concisus .

Chronic inflammation has been associated with an increased risk of several human malignancies, a classic example being gastric adenocarcinoma (GC). Development of GC is known to result from infection of the gastric mucosa by Helicobacter pylori, which initially induces acute inflammation and, in a subset of patients, progresses over time to chronic inflammation, gastric atrophy, intestinal metaplasia, dysplasia, and finally intestinal-type GC. Germ-line encoded receptors known as pattern-recognition receptors (PRRs) are critical for generating mature pro-inflammatory cytokines that are crucial for both Th1 and Th2 responses. Given that H. pylori is initially targeted by PRRs, it is conceivable that dysfunction within genes of this arm of the immune system could modulate the host response against H. pylori infection, and subsequently influence the emergence of GC. Current evidence suggests that Toll-like receptors (TLRs) (TLR2, TLR3, TLR4, TLR5, and TLR9), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (NOD1, NOD2, and NLRP3), a C-type lectin receptor (DC-SIGN), and retinoic acid-inducible gene (RIG)-I-like receptors (RIG-I and MDA-5), are involved in both the recognition of H. pylori and gastric carcinogenesis. In addition, polymorphisms in genes involved in the TLR (TLR1, TLR2, TLR4, TLR5, TLR9, and CD14) and NLR (NOD1, NOD2, NLRP3, NLRP12, NLRX1, CASP1, ASC, and CARD8) signaling pathways have been shown to modulate the risk of H. pylori infection, gastric precancerous lesions, and/or GC. Further, the modulation of PRRs has been suggested to suppress H. pylori-induced inflammation and enhance GC cell apoptosis, highlighting their potential relevance in GC therapeutics. In this review, we present current advances in our understanding of the role of the TLR and NLR signaling pathways in the pathogenesis of GC, address the involvement of other recently identified PRRs in GC, and discuss the potential implications of PRRs in GC immunotherapy.

BackgroundCurrently, it is well established that cancer arises in chronically inflamed tissue. A number of NOD-like receptors (NLRs) form inflammasomes, intracellular multiprotein complexes critical for generating mature pro-inflammatory cytokines (IL-1β and IL-18). As chronic inflammation of the gastric mucosa is a consequence of Helicobacter pylori infection, we investigated the role of genetic polymorphisms and expression of genes involved in the NLR signalling pathway in H. pylori infection and related gastric cancer (GC).Materials and methodsFifty-one genetic polymorphisms were genotyped in 310 ethnic Chinese (87 non-cardia GC cases and 223 controls with functional dyspepsia). In addition, gene expression of 84 molecules involved in the NLR signalling pathway was assessed in THP-1 cells challenged with two H. pylori strains, GC026 (GC) and 26695 (gastritis).ResultsCARD8-rs11672725, NLRP3-rs10754558, NLRP3-rs4612666, NLRP12-rs199475867 and NLRX1-rs10790286 showed significant associations with GC. On multivariate analysis, CARD8-rs11672725 remained a risk factor (OR: 4.80, 95% CI: 1.39-16.58). Further, NLRP12-rs2866112 increased the risk of H. pylori infection (OR: 2.13, 95% CI: 1.22-3.71). Statistical analyses assessing the joint effect of H. pylori infection and the selected polymorphisms revealed strong associations with GC (CARD8, NLRP3, CASP1 and NLRP12 polymorphisms). In gene expression analyses, five genes encoding NLRs were significantly regulated in H. pylori-challenged cells (NLRC4, NLRC5, NLRP9, NLRP12 and NLRX1). Interestingly, persistent up-regulation of NFKB1 with simultaneous down-regulation of NLRP12 and NLRX1 was observed in H. pylori GC026-challenged cells. Further, NF-κB target genes encoding pro-inflammatory cytokines, chemokines and molecules involved in carcinogenesis were markedly up-regulated in H. pylori GC026-challenged cells.ConclusionsNovel associations between polymorphisms in the NLR signalling pathway (CARD8, NLRP3, NLRP12, NLRX1, and CASP1) and GC were identified in Chinese individuals. Our genetic polymorphisms and gene expression results highlight the relevance of the NLR signalling pathway in gastric carcinogenesis and its close interaction with NF-κB.

Background Gastric cancer (GC) is a progressive process initiated by Helicobacter pylori -induced inflammation. Initial recognition of H. pylori involves Toll-like receptors (TLRs), central molecules in the host inflammatory response. Here, we investigated the association between novel polymorphisms in genes involved in the TLR signalling pathway, including TLR2 , TLR4 , LBP , MD-2, CD14 and TIRAP , and risk of H. pylori infection and related GC. Methods A case-control study comprising 310 ethnic Chinese individuals (87 non-cardia GC cases and 223 controls with functional dyspepsia) was conducted. Twenty-five polymorphisms were detected by MALDI-TOF mass spectrometry, PCR, PCR–RFLP and real-time PCR. Results Seven polymorphisms showed significant associations with GC ( TLR4 rs11536889, TLR4 rs10759931, TLR4 rs1927911, TLR4 rs10116253, TLR4 rs10759932, TLR4 rs2149356 and CD14 −260 C/T). In multivariate analyses, TLR4 rs11536889 remained a risk factor for GC (OR: 3.58, 95% CI: 1.20–10.65). TLR4 rs10759932 decreased the risk of H. pylori infection (OR: 0.59, 95% CI: 0.41–0.86). Statistical analyses assessing the joint effect of H. pylori infection and the selected polymorphisms revealed strong associations with GC ( TLR2 , TLR4 , MD-2 , LBP and TIRAP polymorphisms). Conclusions Novel polymorphisms in TLR2 , TLR4 , MD-2, LBP , CD14 and TIRAP , genes encoding important molecules of the TLR signalling pathway, showed clear associations with H. pylori -related GC in Chinese.

Helicobacter pylori causes human gastroduodenal diseases, including chronic gastritis and peptic ulcer disease. It is also a major microbial risk factor for the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Twenty-one strains with different ethnicity, disease, and antimicrobial susceptibility backgrounds were sequenced by use of Illumina HiSeq and PacBio RS platforms.

Direct links between proteomic and genomic/transcriptomic data are not frequently made, partly because of lack of appropriate bioinformatics tools. To help address this, we have developed the PG Nexus pipeline. The PG Nexus allows users to covisualize peptides in the context of genomes or genomic contigs, along with RNA-seq reads. This is done in the Integrated Genome Viewer (IGV). A Results Analyzer reports the precise base position where LC-MS/MS-derived peptides cover genes or gene isoforms, on the chromosomes or contigs where this occurs. In prokaryotes, the PG Nexus pipeline facilitates the validation of genes, where annotation or gene prediction is available, or the discovery of genes using a "virtual protein"-based unbiased approach. We illustrate this with a comprehensive proteogenomics analysis of two strains of Campylobacter concisus . For higher eukaryotes, the PG Nexus facilitates gene validation and supports the identification of mRNA splice junction boundaries and splice variants that are protein-coding. This is illustrated with an analysis of splice junctions covered by human phosphopeptides, and other examples of relevance to the Chromosome-Centric Human Proteome Project. The PG Nexus is open-source and available from https://github.com/IntersectAustralia/ap11_Samifier . It has been integrated into Galaxy and made available in the Galaxy tool shed.

Infection-related preterm birth is a leading cause of infant mortality and morbidity; knowledge of bacterial populations invading the amniotic cavity and the routes of invasion is required to make progress in the prevention of preterm birth. Significant advances have been made in understanding bacterial communities in the vagina, but much less studied are intra-uterine bacterial populations during pregnancy. A systematic review of data published on the intra-uterine microbiome was performed; molecular information and summaries of species found in healthy individuals and in women with diagnosed infections served to construct a database and to analyse results to date. Thirteen studies fulfilled the review's inclusion criteria. The data of various investigations were collated, organized, and re-analyzed to achieve a more comprehensive understanding of microbial populations in the intra-amniotic space. The most common intra-amniotic bacterial taxa were species that can colonies the vagina in health and disease; there were others associated with the habitats of the mouth, gastrointestinal tract, and respiratory tract. The results suggest a central role for the ascending route of infections during pregnancy, and point to a possible secondary contribution via haematogenous invasion of the intra-amniotic space. The complete census of the intra-uterine microbiome awaits completion.

There is mounting evidence for a possible role for Campylobacter concisus in Crohn’s disease (CD). However, the pathogenic potential of C. concisus remains disputed due to its presence in healthy subjects. It is documented that genetic diversity exists within this species, with some strains possessing putative virulence determinants such as exotoxin 9/DnaI that may enable them to persist intracellularly in host cells. In order to clarify this, we employed real-time PCR to determine C. concisus and exotoxin 9 levels within faecal samples of CD patients and healthy controls, and correlated these levels with abundances of microbial taxa identified in a subset of subjects. Both C. concisus and exotoxin 9 levels were found to be higher in CD patients than healthy controls, suggesting not only that CD patients had a greater abundance of C. concisus but also that their strains were likely to be more virulent. Moreover, C. concisus levels correlated with the exotoxin 9 levels in CD patients but not in healthy controls, indicating that healthy controls were colonized by non-virulent C. concisus strains. Correlations with the intestinal microbiota found C. concisus levels to correlate with Eubacterium, Subdoligranulum and Blautia, while exotoxin 9 levels correlated with Dialister, Oscillospira, Lachnospira and Prevotella. This suggests that either the composition of the intestinal microbial flora has the ability to influence levels of both virulent and non-virulent C. concisus strains, or infection with C. concisus may modulate the levels of specific bacterial taxa within the gastrointestinal tract.

Background In spite of its association with gastroenteritis and inflammatory bowel diseases, the isolation of Campylobacter concisus from both diseased and healthy individuals has led to controversy regarding its role as an intestinal pathogen. One proposed reason for this is the presence of high genetic diversity among the genomes of C. concisus strains. Results In this study the genomes of six C. concisus strains were sequenced, assembled and annotated including two strains isolated from Crohn’s disease patients (UNSW2 and UNSW3), three from gastroenteritis patients (UNSW1, UNSWCS and ATCC 51562) and one from a healthy individual (ATCC 51561). The genomes of C. concisus BAA-1457 and UNSWCD, available from NCBI, were included in subsequent comparative genomic analyses. The Pan and Core genomes for the sequenced C. concisus strains consisted of 3254 and 1556 protein coding genes, respectively. Conclusion Genes were identified with specific conservation in C. concisus strains grouped by phenotypes such as invasiveness, adherence, motility and diseased states. Phylogenetic trees based on ribosomal RNA sequences and concatenated host-related pathways for the eight C. concisus strains were generated using the neighbor-joining method, of which the 16S rRNA gene and peptidoglycan biosynthesis grouped the C. concisus strains according to their pathogenic phenotypes. Furthermore, 25 non-synonymous amino acid changes with 14 affecting functional domains, were identified within proteins of conserved host-related pathways, which had possible associations with the pathogenic potential of C. concisus strains. Finally, the genomes of the eight C. concisus strains were compared to the nine available genomes of the well-established pathogen Campylobacter jejuni, which identified several important differences in the respiration pathways of these two species. Our findings indicate that C. concisus strains are genetically diverse, and suggest the genomes of this bacterium contain respiration pathways and modifications in the peptidoglycan layer that may play an important role in its virulence.

The stomach acts as a barrier to ingested microbes, thereby influencing the microbial ecology of the entire gastrointestinal (GI) tract. The stomach microbiota and the role of human host and environmental factors, such as health status or medications, in shaping its composition remain largely unknown. We sought to characterize the bacterial and fungal microbiota in the stomach fluid in order to gain insights into the role of the stomach in GI homeostasis. Gastric fluid was collected from 25 patients undergoing clinically indicated upper endoscopy. DNA isolates were used for PCR amplification of bacterial 16S ribosomal RNA (rRNA) genes and fungal internal transcribed spacers (ITS). RNA isolates were used for 16S rRNA cDNA generation and subsequent PCR amplification. While all stomach fluid samples are dominated by the phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Fusobacteria (>99% of sequence reads), the transcriptionally active microbiota shows significant reduction in Actinobacteria (34%) and increase in Campylobacter (444%) (P4 (70% P

Background Helicobacter trogontum is a putative enterohepatic pathogen, which following infection of IL-10 knock-out mice, results in severe clinical signs and typhlocolitis. Materials and Methods The pathogenic potential of H. trogontum Type strain LRB 8581 was investigated using proteomics coupled with mass spectrometry to characterize the secretome of H. trogontum and scanning electron microscopy to visualize H. trogontum adherence and invasion. Results One hundred and four proteins were identified and bioinformatically predicted to be secreted. Further functional classifications revealed proteins involved in motility, virulence, and colonization factors and the type VI secretion system. Microscopy showed that H. trogontum can adhere to host cells through flagella–microvillus interactions and invade causing a membrane ruffling-like effect and severe cell damage. Conclusions This indicated H. trogontum has the ability to adhere to and invade human cells and secrete factors that may contribute to disease development.

Background. Exclusive enteral nutrition (EEN) is a well-established approach to the management of Crohn’s disease.Aim. To determine effects of EEN upon inflammation and gut barrier function in a colitis mouse model.Methods. Interleukin-10-deficient mice (IL-10−/−) were inoculated withHelicobacter trogontumand then treated with EEN, metronidazole, hydrocortisone, or EEN and metronidazole combination. Blood and tissue were collected at 2 and 4 weeks with histology, mucosal integrity, tight junction integrity, inflammation, andH. trogontumload evaluated.Results.H. trogontuminduced colitis in IL-10−/−mice with histological changes in the cecum and colon. Elevated mucosal IL-8 mRNA in infected mice was associated with intestinal barrier dysfunction indicated by decreased transepithelial electrical resistance and mRNA of tight junction proteins and increased short-circuit current, myosin light chain kinase mRNA, paracellular permeability, and tumor necrosis factor-αand myeloperoxidase plasma levels (P<0.01for all comparisons). EEN and metronidazole, but not hydrocortisone, treatments restored barrier function, maintained gut barrier integrity, and reversed inflammatory changes along with reduction ofH. trogontumload (versus infected controlsP<0.05).Conclusion.H. trogontuminfection in IL-10−/−mice induced typhlocolitis with intestinal barrier dysfunction. EEN and metronidazole, but not hydrocortisone, modulate barrier dysfunction and reversal of inflammatory changes.

Campylobacter showae UNSWCD was isolated from a patient with Crohn's disease. Here we present a 2.1 Mb draft assembly of its genome.

The influence of diets rich in saturated fats and simple sugars on the intestinal microbiota plays a central role in obesity. Being overweight or obese predisposes individuals to several diseases including oesophageal adenocarcinoma (OAC), which develops through a cascade of events starting with gastro-oesophageal reflux disease, progressing to Barrett's oesophagus (BO), and then OAC. A range of mechanisms for the increased risk of OAC in obese individuals have been proposed; however, a role for the oesophageal microbiota has been largely ignored. This is despite the fact that it is clear that the composition of the oesophageal microbiota shifts with the development of OAC. Given the well-established impact that unhealthy diets have on the intestinal microbiota, it is plausible that exposure to unhealthy foods, and the ensuing obesity, would result in an imbalance in the oesophageal microbiota. It is also likely that these changes may mimic the changes observed in the intestinal microbiota (i.e. increase in short-chain fatty acid (SCFA) producers and bile acid biosynthesis). The modulation of SCFAs and bile acids in the oesophagus by diet could promote the transdifferentiation from squamous to intestinal-like columnar cells observed in BO, given that intestinal cells proliferate in the presence of SCFAs.

Helicobacter pylori is the main bacterial causative agent of gastroduodenal disorders and a risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. The draft genomes of 10 closely related H. pylori isolates from the multiracial Malaysian population will provide an insight into the genetic diversity of isolates in Southeast Asia. These isolates were cultured from gastric biopsy samples from patients with functional dyspepsia and gastric cancer. The availability of this genomic information will provide an opportunity for examining the evolution and population structure of H. pylori isolates from Southeast Asia, where the East meets the West.

PAR-1 has been involved in inflammation of the gastrointestinal tract, tumour cell growth and invasion of gastric carcinoma cells. Thus, we aimed at determining, for the first time, the association between PAR-1 - 506 ins/del and -IVSn-14 A/T and risk of Helicobacter pylori-related gastric cancer (GC) in an ethnic Chinese population.A case-control study comprising of 225 ethnic Chinese individuals (77 non-cardia GC cases and 148 controls with functional dyspepsia) was conducted. PAR-1 IVSn-14 A/T and 506 ins/del were genotyped by means of real-time PCR and MALDI-TOF mass spectrometry, respectively.H. pylori infection, male gender and the PAR-1 IVSn-14 TT genotype increased GC risk (OR:3.15, 95% CI:1.54-6.45, OR:2.44, 95% CI:1.35-4.42 and OR:2.58, 95% CI:1.09-6.13, respectively). PAR-1 -506 ins/del did not provide significant results.PAR-1 IVSn-14 T allele is a risk factor for H. pylori-related GC in ethnic Chinese subjects. PAR-1 -506 ins/del polymorphism is not involved in gastric carcinogenesis.

Over the last decade Campylobacter concisus, a highly fastidious member of the Campylobacter genus has been described as an emergent pathogen of the human intestinal tract. Historically, C. concisus was associated with the human oral cavity and has been linked with periodontal lesions, including gingivitis and periodontitis, although currently its role as an oral pathogen remains contentious. Evidence to support the role of C. concisus in acute intestinal disease has come from studies that have detected or isolated C. concisus as sole pathogen in fecal samples from diarrheic patients. C. concisus has also been associated with chronic intestinal disease, its prevalence being significantly higher in children with newly diagnosed Crohn’s disease and adults with ulcerative colitis than in controls. Further C. concisus has been isolated from biopsy specimens of patients with Crohn’s disease. While such studies support the role of C. concisus as an intestinal pathogen, its isolation from healthy individuals, and failure of some studies to show a significant difference in C. concisus prevalence in subjects with diarrhea and healthy controls has raised contention as to its role in intestinal disease. Such findings could argue against the role of C. concisus in intestinal disease, however, the fact that C. concisus strains are genetically diverse raises the possibility that differences exist in their pathogenic potential. Evidence to support this view comes from studies showing strain specific differences in the ability of C. concisus to attach to and invade cells and produce virulence factors, including toxins and hemolytic phospholipase A. Further, sequencing of the genome of a C. concisus strain isolated from a child with Crohn’s disease (UNSWCD) and comparison of this with the only other fully sequenced strain (BAA-1457) would suggest that major differences exist in the genetic make-up of this species which could explain different outcomes of C. concisus infection.

IntroductionCrohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD). A high prevalence of Campylobacter concisus was previously detected in paediatric CD and adult UC. Currently, the prevalence of C. concisus in adult CD and the preferential colonization sites of Campylobacter species in the human intestine are unknown. In this study, we examined the prevalence of Campylobacter species in biopsies collected from multiple anatomic sites of adult patients with IBD and controls.MethodsThree hundred and one biopsies collected from ileum, caecum, descending colon and rectum of 28 patients IBD (15 CD and 13 UC) and 33 controls were studied. Biopsies were used for DNA extraction and detection of Campylobacter species by PCR-sequencing and Campylobacter cultivation.ResultsA significantly higher prevalence of C. concisus in colonic biopsies of patients with CD (53%) was detected as compared with the controls (18%). Campylobacter genus-PCR positivity and C. concisus positivity in patients with UC were 85% and 77% respectively, being significantly higher than that in the controls (48% and 36%). C. concisus was more often detected in descending colonic and rectal biopsies from patients with IBD in comparison to the controls. C. concisus was isolated from patients with IBD.ConclusionThe high intestinal prevalence of C. concisus in patients with IBD, particularly in the proximal large intestine, suggests that future studies are needed to investigate the possible involvement of C. concisus in a subgroup of human IBD. To our knowledge, this is the first report of the association between adult CD and C. concisus as well as the first study of the preferential colonization sites of C. concisus in the human intestine.

Campylobacter concisus is an emerging pathogen of the human gastrointestinal tract. Recently, a significantly higher prevalence of C. concisus DNA and higher levels of antibodies specific to C. concisus was detected in children with Crohn's disease when compared with controls. The aim of this study was to identify C. concisus immunoreactive antigens. Proteins from C. concisus were separated using two-dimensional gel electrophoresis, and sera from 10 C. concisus-positive children with Crohn's disease were employed for immunoprobing. The patients' sera reacted with 69 spots, which corresponded to 31 proteins identified by mass spectrometry. The proteins were functionally classified as involved in chemotaxis, signal transduction, flagellar motility, surface binding and membrane protein assembly. Although the individual patients' sera reacted to different sets of proteins, common antigens that were recognized by all patients were flagellin B, ATP synthase F1 alpha subunit, and outer membrane protein 18. Cross-reactivity between proteins of the Campylobacter genus was tested using patients' sera absorbed with Campylobacter showae, Campylobacter jejuni and Campylobacter ureolyticus. Most of the C. concisus immunoreactive proteins identified in this study showed cross-reactivity with other species except for three antigens. In conclusion, this study has identified C. concisus proteins that are immunoreactive within patients with Crohn's disease.

Crohn's disease is widely regarded as a multifactorial disease, and evidence from human and animal studies suggests that bacteria have an instrumental role in its pathogenesis. Comparison of the intestinal microbiota of patients with Crohn's disease to that of healthy controls has revealed compositional changes. In most studies these changes are characterized by an increase in the abundance of Bacteroidetes and Proteobacteria and a decrease in that of Firmicutes. In addition, a number of specific mucosa-associated bacteria have been postulated to have a role in Crohn's disease, including Mycobacterium avium subspecies paratuberculosis, adherent and invasive Escherichia coli, Campylobacter and Helicobacter species. The association between mutations in pattern-recognition receptors (Toll-like receptors and Nod-like receptors) and autophagy proteins and Crohn's disease provides further evidence to suggest that defective sensing and killing of bacteria may drive the onset of disease. In this Review, we present recent advances in understanding the role of bacteria and the contribution of pattern-recognition receptors and autophagy in the pathogenesis of Crohn's disease.

Since the discovery that Helicobacter pylori causes a range of pathologies in the stomachs of infected humans, it has become apparent that Helicobacters are found in a diverse range of animal species where they are frequently associated with disease. In 2003 and 2004, there were two outbreaks of increased mortality associated with gastric bleeding and weight-loss in a captive colony of the Australian marsupial, the Stripe-faced Dunnart (Sminthopsis macroura). The presence of gastric pathology led to an investigation of potential Helicobacter pathogenesis in these animals. Histological examination revealed the presence of gastritis, and PCR analysis confirmed the presence of Helicobacter infection in the stomachs of these marsupials. Surprisingly, sequencing of 16S rRNA from these bacteria identified the species as H. pylori and PCR confirmed the strain to be positive for the important pathogenesis factor, cagA. We therefore describe, for the first time, an apparent reverse zoonotic infection of Stripe-faced Dunnarts with H. pylori. Already prone to pathological effects of stress (as experienced during breeding season), concomitant H. pylori infection appears to be a possible essential but not sufficient co-factor in prototypic gastric bleeding and weight loss in these marsupials. The Stripe-faced Dunnart could represent a new model for investigating Helicobacter-driven gastric pathology. Infections from their human handlers, specifically of H. pylori, may be a potential risk to captive colonies of marsupials.

Campylobacter concisus has garnered increasing attention due to its association with intestinal disease, thus, the pathogenic potential of strains isolated from different intestinal diseases was investigated. A method to isolate C. concisus was developed and the ability of eight strains from chronic and acute intestinal diseases to adhere to and invade intestinal epithelial cells was determined. Features associated with bacterial invasion were investigated using comparative genomic analyses and the effect of C. concisus on host protein expression was examined using proteomics. Our isolation method from intestinal biopsies resulted in the isolation of three C. concisus strains from children with Crohn's disease or chronic gastroenteritis. Four C. concisus strains from patients with chronic intestinal diseases can attach to and invade host cells using mechanisms such as chemoattraction to mucin, aggregation, flagellum-mediated attachment, "membrane ruffling", cell penetration and damage. C. concisus strains isolated from patients with chronic intestinal diseases have significantly higher invasive potential than those from acute intestinal diseases. Investigation of the cause of this increased pathogenic potential revealed a plasmid to be responsible. 78 and 47 proteins were upregulated and downregulated in cells infected with C. concisus, respectively. Functional analysis of these proteins showed that C. concisus infection regulated processes related to interleukin-12 production, proteasome activation and NF-κB activation. Infection with all eight C. concisus strains resulted in host cells producing high levels of interleukin-12, however, only strains capable of invading host cells resulted in interferon-γ production as confirmed by ELISA. These findings considerably support the emergence of C. concisus as an intestinal pathogen, but more significantly, provide novel insights into the host immune response and an explanation for the heterogeneity observed in the outcome of C. concisus infection. Moreover, response to infection with invasive strains has substantial similarities to that observed in the inflamed mucosa of Crohn's disease patients.

Given that members of Helicobacteraceae family colonize the intestinal mucus layer, it has been hypothesized that they may play a role in Crohn's disease. This study investigated the presence of Helicobacteraceae DNA in biopsies collected from children with Crohn's disease and controls.The presence of Helicobacteraceae DNA was investigated in intestinal biopsies collected from 179 children undergoing colonoscopy (Crohn's disease n = 77, controls n = 102) using a Helicobacteraceae-specific PCR.Members of the Helicobacteraceae were detected in 32/77 children with Crohn's disease (41.5%) and 23/102 controls (22.5%). Statistical analysis showed the prevalence of Helicobacteraceae detected in patients to be significantly higher than that in controls (p = .0062). Analysis of non-pylori Helicobacteraceae showed that their prevalence was also significantly higher in patients than in controls (p = .04). Helicobacter pylori was detected in 14.0% of the biopsies across all groups. Given that all children tested were negative for gastric H. pylori, this was a surprising finding. Phylogenetic analysis of H. pylori sequences detected in the biopsies showed that the H. pylori strains identified in the patients did not group with gastric H. pylori included in the analysis, but rather with other H. pylori strains detected in the intestine, gall bladder, and liver.The higher prevalence of Helicobacteraceae DNA in Crohn's disease patients would suggest that members of this family may be involved in this disease. In addition, phylogenetic analysis of H. pylori strains showed that extragastric sequences clustered together, indicating that different H. pylori strains may adapt to colonize extragastric niches.

The Campylobacter genus consists of a number of important human and animal pathogens. Although the 16S rRNA gene has been used extensively for detection and identification of Campylobacter species, there is currently limited information on the 23S rRNA gene and the internal transcribed spacer (ITS) region that lies between the 16S and 23S rRNA genes. We examined the potential of the 23S rRNA gene and the ITS region to be used in species differentiation and delineation of systematic relationships for 30 taxa within the Campylobacter genus. The ITS region produced the highest mean pairwise percentage difference (35.94%) compared to the 16S (5.34%) and 23S (7.29%) rRNA genes. The discriminatory power for each region was further validated using Simpson's index of diversity ( D value). The D values were 0.968, 0.995, and 0.766 for the ITS region and the 23S and 16S rRNA genes, respectively. A closer examination of the ITS region revealed that Campylobacter concisus , Campylobacter showae , and Campylobacter fetus subsp. fetus harbored tRNA configurations not previously reported for other members of the Campylobacter genus. We also observed the presence of strain-dependent intervening sequences in the 23S rRNA genes. Neighbor-joining trees using the ITS region revealed that Campylobacter jejuni and Campylobacter coli strains clustered in subgroups, which was not observed in trees derived from the 16S or 23S rRNA gene. Of the three regions examined, the ITS region is by far the most cost-effective region for the differentiation and delineation of systematic relationships within the Campylobacter genus.

A higher prevalence of Campylobacter concisus and higher levels of IgG antibodies specific to C. concisus in Crohn's disease patients than in controls were recently detected. In this study, 1D and 2D gel electrophoresis coupled with LTQ FT-MS and QStar tandem MS, respectively, were performed to characterize the secretome of a C. concisus strain isolated from a Crohn's disease patient. Two hundred and one secreted proteins were identified, of which 86 were bioinformatically predicted to be secreted. Searches were performed on the genome of C. concisus strain 13826, and 25 genes that have been associated with virulence or colonization in other organisms were identified. The zonula occludens toxin was found only in C. concisus among the Campylobacterales, although expanded searches revealed that this protein was present in two epsilon-proteobacterial species from extreme marine environments. Alignments and structural threading indicated that this toxin shared features with that of other virulent pathogens, including Neisseria meningitidis and Vibrio cholerae. Further comparative analyses identified several associations between the secretome of C. consisus and putative virulence factors of this bacterium. This study has identified several factors putatively associated with disease outcome, suggesting that C. concisus is a pathogen of the gastrointestinal tract.

This chapter discusses the association between Helicobacter pylori infection and several extragastric diseases, including idiopathic thrombocytopenic purpura, iron deficiency anaemia, cardiovascular disease, asthma, inflammatory bowel disease, and hepatobiliary and pancreatic diseases.

Helicobacter pylori-related disease is at least partially attributable to the genotype of the infecting strain, particularly the presence of specific virulence factors. We investigated the prevalence of a novel combination of H. pylori virulence factors, including the cag pathogenicity island (PAI), and their association with severe disease in isolates from the three major ethnicities in Malaysia and Singapore, and evaluated whether the cag PAI was intact and functional in vitro. Polymerase chain reaction (PCR) was used to detect dupA, cagA, cagE, cagT, cagL and babA, and to type vacA, the EPIYA motifs, HP0521 alleles and oipA ON status in 159 H. pylori clinical isolates. Twenty-two strains were investigated for IL-8 induction and CagA translocation in vitro. The prevalence of cagA, cagE, cagL, cagT, babA, oipA ON and vacA s1 and i1 was >85%, irrespective of the disease state or ethnicity. The prevalence of dupA and the predominant HP0521 allele and EPIYA motif varied significantly with ethnicity (p < 0.05). A high prevalence of an intact cag PAI was found in all ethnic groups; however, no association was observed between any virulence factor and disease state. The novel association between the HP0521 alleles, EPIYA motifs and host ethnicity indicates that further studies to determine the function of this gene are important.

Helicobacter pylori has been shown to degrade two phosphonates, N-phosphonoacetyl-L: -aspartate and phosphonoacetate; however, the bacterium does not have any genes homologous to those of the known phosphonate metabolism pathways suggesting that H. pylori may have a novel phosphonate metabolism pathway. Growth of H. pylori on phosphonates was studied and the catabolism of these compounds was measured employing (1)H-nuclear magnetic resonance spectroscopy. The specificity of the catabolic enzymes was elucidated by assaying the degradation of several phosphonates and through substrate competition studies. H. pylori was able to utilise phenylphosphonate as a sole source of phosphate for growth. Three strains of H. pylori showed sigmoidal enzyme kinetics of phenylphosphonate catabolism. Allosteric kinetics were removed when lysates were fractionated into cytosolic and membrane fractions. Catabolic rates increased with the addition of DTT, Mg(2+) and phosphate and decreased with the addition of EDTA. The physiological properties of H. pylori phosphonate metabolism were characterised and the presence of at least two novel phosphonate catabolism pathways that do not require phosphate starvation growth conditions for activity has been established.

Background The putative H. pylori pathogenicity-associated factor dupA has been associated with IL-8 induction in vitro, and duodenal ulcer (DU) and gastric cancer (GC) development in certain populations, but this association is inconsistent between studies. We aimed to investigate dupA prevalence in clinical isolates from Sweden, Australia and from ethnic Chinese, Indians and Malays resident in Malaysia and Singapore and to examine the association with DU and GC. In addition we investigated the sequence diversity between isolates from these diverse groups and compared the level of IL-8 secretion in isolates possessing and lacking dupA. Methods PCR primers were designed to amplify over the C/T insertion denoting a continuous dupA. PCR products from 29 clinical isolates were sequenced and compared with sequences from three additional strains obtained from GenBank. Clinical isolates from 21 Malaysian patients (8 dupA-positive, 14 dupA-negative) were assessed for their ability to induce IL-8 in AGS cells in vitro. Statistical analysis was performed using Fisher's exact test. Results The prevalence of dupA in isolates from Swedish functional dyspepsia (FD) control patients (65%, 13/20) was higher and in isolates from Indian FD patients (7.1%, 3/42) was lower as compared with isolates from Chinese (28.9%, 13/49, P = 0.005, P = 0.025), Malay (35.7%, 5/14, P = 0.16, P = 0.018) and Australian (37.8%, 17/45, P = 0.060, P < 0.001) FD patients. dupA was associated with DU and GC development in Chinese with 62.5% (10/16) and 54.6% (12/22) of isolates possessing dupA respectively as compared with FD controls (28.9%) (P = 0.015, P = 0.032). No significant difference in prevalence of dupA between FD controls, DU (63.6%, 7/11) and GC (61.9%, 13/21) cases (P = 1.000) was observed in the Swedish population. Sequence analysis revealed a pairwise variation of 1.9% and all isolates possessed the C/T insertion. The average IL-8 induction was 1330 pg/mL for dupA-positive isolates and 1378 pg/mL for dupA-negative isolates. Conclusion Although dupA is highly conserved when present, we identified no consistent association between dupA and DU or GC development across the ethnic groups investigated, with the dupA prevalence in control groups varying significantly. Our results would suggest that in the clinical isolates investigated dupA is not associated with IL-8 induction in vitro.

Metronidazole resistance in Helicobacter pylori has been attributed to mutations in rdxA or frxA . Insufficient data correlating RdxA and/or FrxA with the resistant phenotype, and the emergence of resistant strains with no mutations in either rdxA or frxA , indicated that the molecular basis of H. pylori resistance to metronidazole required further characterization. The rdxA and frxA genes of four matched pairs of metronidazole-susceptible and -resistant strains were sequenced. The resistant strains had mutations in either rdxA , frxA , neither gene, or both genes. The reduction rates of five substrates suggested that metabolic differences between susceptible and resistant strains cannot be explained only by mutations in rdxA and/or frxA . A more global approach to understanding the resistance phenotype was taken by employing two-dimensional gel electrophoresis combined with tandem mass spectrometry analyses to identify proteins differentially expressed by the matched pair of strains with no mutations in rdxA or frxA . Proteins involved in the oxireduction of ferredoxin were downregulated in the resistant strain. Other redox enzymes, such as thioredoxin reductase, alkyl hydroperoxide reductase, and superoxide dismutase, showed a pI change in the resistant strain. The data suggested that metronidazole resistance involved more complex metabolic changes than specific gene mutations, and they provided evidence of a role for the intracellular redox potential in the development of resistance.