Diversity of antimicrobial-resistant bacteria isolated from Australian chicken and pork meat.

Antimicrobial-resistant bacteria are frequently isolated from retail meat and may infect humans. To determine the diversity of antimicrobial-resistant bacteria in Australian retail meat, bacteria were cultured on selective media from raw chicken (n = 244) and pork (n = 160) meat samples obtained from all four major supermarket chains in the ACT/NSW, Australia, between March and June 2021. Antimicrobial susceptibility testing (AST) was performed for 13 critically and 4 highly important antibiotics as categorised by the World Health Organization (WHO) for a wide range of species detected in the meat samples. A total of 288 isolates underwent whole-genome sequencing (WGS) to identify the presence of antimicrobial resistance (AMR) genes, virulence genes, and plasmids. AST testing revealed that 35/288 (12%) of the isolates were found to be multidrug-resistant (MDR). Using WGS data, 232/288 (81%) of the isolates were found to harbour resistance genes for critically or highly important antibiotics. This study reveals a greater diversity of AMR genes in bacteria isolated from retail meat in Australia than previous studies have shown, emphasising the importance of monitoring AMR in not only foodborne pathogenic bacteria, but other species that are capable of transferring AMR genes to pathogenic bacteria.

The relationship between extreme inter-individual variation in macrophage gene expression and genetic susceptibility to inflammatory bowel disease.

The differentiation of resident intestinal macrophages from blood monocytes depends upon signals from the macrophage colony-stimulating factor receptor (CSF1R). Analysis of genome-wide association studies (GWAS) indicates that dysregulation of macrophage differentiation and response to microorganisms contributes to susceptibility to chronic inflammatory bowel disease (IBD). Here, we analyzed transcriptomic variation in monocyte-derived macrophages (MDM) from affected and unaffected sib pairs/trios from 22 IBD families and 6 healthy controls. Transcriptional network analysis of the data revealed no overall or inter-sib distinction between affected and unaffected individuals in basal gene expression or the temporal response to lipopolysaccharide (LPS). However, the basal or LPS-inducible expression of individual genes varied independently by as much as 100-fold between subjects. Extreme independent variation in the expression of pairs of HLA-associated transcripts (HLA-B/C, HLA-A/F and HLA-DRB1/DRB5) in macrophages was associated with HLA genotype. Correlation analysis indicated the downstream impacts of variation in the immediate early response to LPS. For example, variation in early expression of IL1B was significantly associated with local SNV genotype and with subsequent peak expression of target genes including IL23A, CXCL1, CXCL3, CXCL8 and NLRP3. Similarly, variation in early IFNB1 expression was correlated with subsequent expression of IFN target genes. Our results support the view that gene-specific dysregulation in macrophage adaptation to the intestinal milieu is associated with genetic susceptibility to IBD.

Comparative genomics and phenotypic studies to determine site-specificity of Escherichia coli in the lower gastrointestinal tract of humans.

Escherichia coli (E. coli) is an important commensal in the human gut; however, it is unknown whether strains show site-specificity in the lower gut. To investigate this, we assessed genotypic and phenotypic differences in 37 clone pairs (two strains with very similar multiple locus variable-number-tandem-repeat analysis [MLVA] profiles) of E. coli isolated from mucosal biopsies of two different gut locations (terminal ileum and rectum). The clone pairs varied at the genomic level; single nucleotide polymorphisms (SNPs) were common, multiple nucleotide polymorphisms (MNPs) were observed but less common, and few indels (insertions and deletions) were detected. The variation was higher in clone pairs that are associated with non-human-associated sequence types (ST) compared to human-associated STs, such as ST95, ST131, and ST73. No gene(s) with non-synonymous mutations were found to be commonly associated with either the terminal ileum or the rectal strains. At the phenotypic level, we identified the metabolic signatures for some STs. Rectum strains of some STs showed consistently higher metabolic activity with particular carbon sources. Clone pairs belonging to specific STs showed distinct growth patterns under different pH conditions. Overall, this study showed that E. coli may exhibit genomic and phenotypic variability at different locations in the gut. Although genomics did not reveal significant information suggesting the site-specificity of strains, some phenotypic studies have suggested that strains may display site-specificity in the lower gut. These results provide insights into the nature and adaptation of E. coli in the lower gut of humans. To the best of our knowledge, no study has investigated or demonstrated the site-specificity of commensal E. coli in the human gut.

Review article: the future of microbiome-based therapeutics.

BACKGROUND: From consumption of fermented foods and probiotics to emerging applications of faecal microbiota transplantation, the health benefit of manipulating the human microbiota has been exploited for millennia. Despite this history, recent technological advances are unlocking the capacity for targeted microbial manipulation as a novel therapeutic. AIM: This review summarises the current developments in microbiome-based medicines and provides insight into the next steps required for therapeutic development. METHODS: Here we review current and emerging approaches and assess the capabilities and weaknesses of these technologies to provide safe and effective clinical interventions. Key literature was identified through Pubmed searches with the following key words, 'microbiome', 'microbiome biomarkers', 'probiotics', 'prebiotics', 'synbiotics', 'faecal microbiota transplant', 'live biotherapeutics', 'microbiome mimetics' and 'postbiotics'. RESULTS: Improved understanding of the human microbiome and recent technological advances provide an opportunity to develop a new generation of therapies. These therapies will range from dietary interventions, prebiotic supplementations, single probiotic bacterial strains, human donor-derived faecal microbiota transplants, rationally selected combinations of bacterial strains as live biotherapeutics, and the beneficial products or effects produced by bacterial strains, termed microbiome mimetics. CONCLUSIONS: Although methods to identify and refine these therapeutics are continually advancing, the rapid emergence of these new approaches necessitates accepted technological and ethical frameworks for measurement, testing, laboratory practices and clinical translation.

Prevalence, diversity and genetic structure of Escherichia coli isolates from septic tanks.

The present study investigated the diversity and genetic structure of Escherichia coli isolates from 100 septic tanks in the Canberra region, Australia. The physicochemical characteristics of the septic tanks were determined to examine the extent to which environmental factors might influence E. coli prevalence, diversity and population structure. The results of this study indicated that the temperature of the septic tank could explain some of the variation observed in the number of E. coli isolates recovered per septic tank, whereas pH was an important driver of E. coli diversity. Conductivity, pH and household size had a significant impact on E. coli population structure, and household size significantly affected the probability of detecting human-associated E. coli lineages [sequence types (STs) 69, 73, 95 and 131] in septic tanks. Phylogroup A and B1 strains were not randomly distributed among septic tanks, and the strong negative association between them may indicate intraspecific competition. The findings of this study suggest that the combination of environmental factors and intraspecific interactions may influence the distribution and genetic structure of E. coli in the environment.

Gut microbiome responses to dietary intake of grain-based fibers with the potential to modulate markers of metabolic disease: a systematic literature review.

CONTEXT: Cereal fiber modulates the gut microbiome and benefits metabolic health. The potential link between these effects is of interest.0. OBJECTIVE: The aim for this systematic review was to assess evidence surrounding the influence of cereal fiber intake on microbiome composition, microbiome diversity, short-chain fatty acid production, and risk factors for metabolic syndrome. DATA SOURCES AND EXTRACTION: The MEDLINE, PubMed, CINAHL, and Cochrane Library databases were searched systematically, and quality of studies was assessed using the Cochrane Risk of Bias 2.0 tool. Evidence relating to study design, dietary data collection, and outcomes was qualitatively synthesized on the basis of fiber type. DATA ANALYSIS: Forty-six primary publications and 2 secondary analyses were included. Cereal fiber modulated the microbiome in most studies; however, taxonomic changes indicated high heterogeneity. Short-chain fatty acid production, microbiome diversity, and metabolic-related outcomes varied and did not always occur in parallel with microbiome changes. Poor dietary data were a further limitation. CONCLUSIONS: Cereal fiber may modulate the gut microbiome; however, evidence of the link between this and metabolic outcomes is limited. Additional research is required with a focus on robust and consistent methodology. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration no. CRD42018107117.

Phylogenetic background and habitat drive the genetic diversification of Escherichia coli.

Escherichia coli is mostly a commensal of birds and mammals, including humans, where it can act as an opportunistic pathogen. It is also found in water and sediments. We investigated the phylogeny, genetic diversification, and habitat-association of 1,294 isolates representative of the phylogenetic diversity of more than 5,000 isolates from the Australian continent. Since many previous studies focused on clinical isolates, we investigated mostly other isolates originating from humans, poultry, wild animals and water. These strains represent the species genetic diversity and reveal widespread associations between phylogroups and isolation sources. The analysis of strains from the same sequence types revealed very rapid change of gene repertoires in the very early stages of divergence, driven by the acquisition of many different types of mobile genetic elements. These elements also lead to rapid variations in genome size, even if few of their genes rise to high frequency in the species. Variations in genome size are associated with phylogroup and isolation sources, but the latter determine the number of MGEs, a marker of recent transfer, suggesting that gene flow reinforces the association of certain genetic backgrounds with specific habitats. After a while, the divergence of gene repertoires becomes linear with phylogenetic distance, presumably reflecting the continuous turnover of mobile element and the occasional acquisition of adaptive genes. Surprisingly, the phylogroups with smallest genomes have the highest rates of gene repertoire diversification and fewer but more diverse mobile genetic elements. This suggests that smaller genomes are associated with higher, not lower, turnover of genetic information. Many of these genomes are from freshwater isolates and have peculiar traits, including a specific capsule, suggesting adaptation to this environment. Altogether, these data contribute to explain why epidemiological clones tend to emerge from specific phylogenetic groups in the presence of pervasive horizontal gene transfer across the species.

Study of a classification algorithm for AIEC identification in geographically distinct E. coli strains.

Adherent-invasive Escherichia coli (AIEC) have been extensively implicated in Crohn's disease pathogenesis. Currently, AIEC is identified phenotypically, since no molecular marker specific for AIEC exists. An algorithm based on single nucleotide polymorphisms was previously presented as a potential molecular tool to classify AIEC/non-AIEC, with 84% accuracy on a collection of 50 strains isolated in Girona (Spain). Herein, our aim was to determine the accuracy of the tool using AIEC/non-AIEC isolates from different geographical origins and extraintestinal pathogenic E. coli (ExPEC) strains. The accuracy of the tool was significantly reduced (61%) when external AIEC/non-AIEC strains from France, Chile, Mallorca (Spain) and Australia (82 AIEC, 57 non-AIEC and 45 ExPEC strains in total) were included. However, the inclusion of only the ExPEC strains showed that the tool was fairly accurate at differentiating these two close pathotypes (84.6% sensitivity; 79% accuracy). Moreover, the accuracy was still high (81%) for those AIEC/non-AIEC strains isolated from Girona and Mallorca (N = 63); two collections obtained from independent studies but geographically close. Our findings indicate that the presented tool is not universal since it would be only applicable for strains from similar geographic origin and demonstrates the need to include strains from different origins to validate such tools.

Phenotypic characteristics contributing to the enhanced growth of Escherichia coli bloom strains.

During bloom events, Escherichia coli cell counts increase to between 10,000 and 100,000 cfu/100 ml of water. The strains responsible for bloom events belong to E. coli phylogenetic groups A and B1, and all have acquired a capsule from Klebsiella. A pan-genome comparison of phylogroup A E. coli revealed that the ferric citrate uptake system (fecIRABCDE) was overrepresented in phylogroup A bloom strains compared with non-bloom E. coli. A series of experiments were carried out to investigate if the capsule together with ferric citrate uptake system could confer a growth rate advantage on E. coli. Capsulated strains had a growth rate advantage regardless of the media composition and the presence/absence of the fec operon, and they had a shorter lag phase compared with capsule-negative strains. The results suggest that the Klebsiella capsule may facilitate nutrient uptake or utilization by a strain. This, together with the protective roles played by the capsule and the shorter lag phase of capsule-positive strains, may explain why it is only capsule-positive strains that produce elevated counts in response to nutrient influx.

Recent advances in gut Microbiota mediated therapeutic targets in inflammatory bowel diseases: Emerging modalities for future pharmacological implications.

The inflammatory bowel diseases (IBDs) are chronic inflammatory conditions, which are increasing in prevalence worldwide. The IBDs are thought to result from an aberrant immune response to gut microbes in genetically susceptible individuals. Dysbiosis of the gut microbiome, both functional and compositional, promotes patient susceptibility to colonization by pathobionts. Manipulating gut microbial communities and gut microbiota-immune system interactions to restore gut homeostasis or reduce inflammation are appealing therapeutic models. We discuss the therapeutic potential of precision microbiota editing, natural and engineered probiotics, the use of gut microbiota-derived metabolites in colitogenic phenotypes, and intestinal stem cells, in maintaining gut microbiota balance, restoring the mucosal barrier, and having positive immunomodulatory effects in experimental IBD. This review highlights that we are only just beginning to understand the complexity of the microbiota and how it can be manipulated for health benefits, including treatment and prevention of the clinical IBDs in future.

Diversity and distribution of Klebsiella capsules in Escherichia coli.

E. coli strains responsible for elevated counts (blooms) in freshwater reservoirs in Australia carry a capsule originating from Klebsiella. The occurrence of Klebsiella capsules in E. coli was about 7% overall and 23 different capsule types were detected. Capsules were observed in strains from phylogroups A, B1 and C, but were absent from phylogroup B2, D, E and F strains. In general, few A, B1 or C lineages were capsule-positive, but when a lineage was encapsulated multiple different capsule types were present. All Klebsiella capsule-positive strains were of serogroups O8, O9 and O89. Regardless of the phylogroup, O9 strains were more likely to be capsule-positive than O8 strains. Given the sequence similarity, it appears that both the capsule region and the O-antigen gene region are transferred to E. coli from Klebsiella as a single block via horizontal gene transfer events. Pan genome analysis indicated that there were only modest differences between encapsulated and non-encapsulated strains belonging to phylogroup A. The possession of a Klebsiella capsule, but not the type of capsule, is likely a key determinant of the bloom status of a strain.

The microbiome of Crohn's disease aphthous ulcers.

BACKGROUND: Reduced intestinal microbial diversity and bacterial imbalance (dysbiosis) are seen in studies of Crohn's disease. As it is difficult to obtain biopsy samples before disease presentation, the earliest mucosal lesions in Crohn's disease, aphthous ulcers, present the best chance at assessing microbial communities at the onset of disease or a new flare. The aim of our study was to compare the microbial communities of aphthous ulcers and adjacent normal mucosa from patients with Crohn's disease with normal mucosa from controls. RESULTS: We did not observe bacterial imbalance or reduced alpha diversity in Crohn's disease aphthous ulcers and adjacent mucosa, relative to control biopsies. Bacteroides were common to all Crohn's disease and control samples, and there were no bacterial taxa unique to aphthous ulcers. The relative abundance of Faecalibacterium was not reduced in aphthous ulcers relative to control mucosa, and was not more likely to be detected in control samples. CONCLUSIONS: In contrast to well-documented changes seen in late-stage Crohn's disease, microbial communities of aphthous ulcers do not display evidence of bacterial imbalance or reduced diversity. Our data suggest that dysbiosis occurs during active disease, and improves when patients are in remission.

The microbiome of translocated bacterial populations in patients with and without inflammatory bowel disease.

BACKGROUND: Using culture-based methods, bacterial translocation from the gut to draining mesenteric lymph nodes is seen in 5% of normal controls and up to 33% of patients with inflammatory bowel diseases (IBD). Many bacteria cannot be cultured, so these methods are unable to capture the full spectrum of bacteria present. AIMS: To detect viable bacteria in lymph nodes of patients with IBD and non-IBD controls using bacterial RNA as a surrogate for viability and to compare them with the same patient's gut microbiome. METHODS: Bacterial RNA was extracted from lymph nodes and mucosa of 20 patients who had undergone intestinal resection (10 IBD, 10 non-IBD). A previous study had detected bacterial DNA in these patients' lymph nodes; 16S rRNA gene high-throughput sequencing was performed. RESULTS: Bacterial RNA was detected in the lymph nodes of five patients with IBD and three controls (volvulus, diverticulitis and bowel obstruction). Lymph nodes had higher alpha (within sample) diversity compared to mucosal samples (Shannon diversity index 2.41 vs 1.81, one-way ANOVA P = 0.035). Beta diversity (inter-sample variation: lymph node vs intestine) was similar within individuals and did not differ between groups. Common gene polymorphisms linked with IBD (NOD2, ATG16L1, IRGM and IL23R) were not associated with bacterial translocation. CONCLUSIONS: Metabolically active bacteria mirroring the individual's gut microbiome were commonly found in the lymph nodes of patients with IBD undergoing resection. An increase in lymph node alpha diversity is likely due to the larger drainage area. The presence of viable bacteria in non-IBD controls is also not unexpected given the underlying pathology in these patients.

Within-host evolution versus immigration as a determinant of Escherichia coli diversity in the human gastrointestinal tract.

When a human host harbors two or more strains of Escherichia coli, the second strain is more likely to be a member of the same phylogroup rather than a different phylogroup. This outcome may be the consequence of a within host evolution event or an independent immigration/establishment event. To determine the relative importance of these two events in determining E. coli diversity in a host, a collection of multiple E. coli isolates recovered from each of 67 patients undergoing colonoscopies was used. Whole genome sequence data were available for one example of every REP-fingerprint type identified in a patient. Sequence type (ST) and single-nucleotide polymorphism (SNP) analyses revealed that 83% of strains observed in the host population were a consequence of immigration/establishment events. Restricting the analysis to hosts harboring two or more strains belonging to the same phylogroup revealed that in about half of these cases, the presence of a second strain belonging to the same phylogroup was the consequence of an independent immigration/establishment event. Thus, the results of this study show that despite hosts being exposed to a diversity of E. coli via their food, factors related to the host also determine what E. coli strains succeed in establishing.

Fine-Scale Structure Analysis Shows Epidemic Patterns of Clonal Complex 95, a Cosmopolitan Escherichia coli Lineage Responsible for Extraintestinal Infection.

The Escherichia coli lineage known as clonal complex 95 (CC95) is a cosmopolitan human-associated lineage responsible for a significant fraction of extraintestinal infections of humans. Whole-genome sequence data of 200 CC95 strains from various origins enabled determination of the CC95 pangenome. The pangenome analysis revealed that strains of the complex could be assigned to one of five subgroups that vary in their serotype, extraintestinal virulence, virulence gene content, and antibiotic resistance gene profile. A total of 511 CC95 strains isolated from humans living in France, Australia, and the United States were screened for their subgroup membership using a PCR-based method. The CC95 subgroups are nonrandomly distributed with respect to their geographic origin. The relative frequency of the subgroups was shown to change through time, although the nature of the changes varies with continent. Strains of the subgroups are also nonrandomly distributed with respect to source of isolation (blood, urine, or feces) and host sex. Collectively, the evidence indicates that although strains belonging to CC95 may be cosmopolitan, human movement patterns have been insufficient to homogenize the distribution of the CC95 subgroups. Rather, the manner in which CC95 strains evolve appears to vary both spatially and temporally. Although CC95 strains appeared globally as pandemic, fine-scale structure analysis shows epidemic patterns of the CC95 subgroups. Furthermore, the observation that the relative frequency of CC95 subgroups at a single locality has changed over time indicates that the relative fitness of the subgroups has changed. IMPORTANCEEscherichia coli clonal complex 95 represents a cosmopolitan, genetically diverse lineage, and the extensive substructure observed in this lineage is epidemiologically and clinically relevant. The frequency with which CC95 strains are responsible for extraintestinal infection appears to have been stable over the past 15 years. However, the different subgroups identified within this lineage have an epidemic structure depending on the host, sample, continent, and time. Thus, the evolution and spread of strains belonging to CC95 are very different from those of another cosmopolitan human-associated clonal complex, CC131, which has increased significantly in frequency as a cause of extraintestinal infection over the past 15 years due to the evolution and spread of two very closely related, nearly monomorphic lineages.

Comparative genomics of Crohn's disease-associated adherent-invasive Escherichia coli.

OBJECTIVE: Adherent-invasive Escherichia coli (AIEC) are a leading candidate bacterial trigger for Crohn's disease (CD). The AIEC pathovar is defined by in vitro cell-line assays examining specific bacteria/cell interactions. No molecular marker exists for their identification. Our aim was to identify a molecular property common to the AIEC phenotype. DESIGN: 41 B2 phylogroup E. coli strains were isolated from 36 Australian subjects: 19 patients with IBD and 17 without. Adherence/invasion assays were conducted using the I-407 epithelial cell line and survival/replication assays using the THP-1 macrophage cell line. Cytokine secretion tumour necrosis factor ((TNF)-α, interleukin (IL) 6, IL-8 and IL-10) was measured using ELISA. The genomes were assembled and annotated, and cluster analysis performed using CD-HIT. The resulting matrices were analysed to identify genes unique/more frequent in AIEC strains compared with non-AIEC strains. Base composition differences and clustered regularly interspaced palindromic repeat (CRISPR) analyses were conducted. RESULTS: Of all B2 phylogroup strains assessed, 79% could survive and replicate in macrophages. Among them, 11/41 strains (5 CD, 2 UCs, 5 non-IBD) also adhere to and invade epithelial cells, a phenotype assigning them to the AIEC pathovar. The AIEC strains were phylogenetically heterogeneous. We did not identify a gene (or nucleic acid base composition differences) common to all, or the majority of, AIEC. Cytokine secretion and CRISPRs were not associated with the AIEC phenotype. CONCLUSIONS: Comparative genomic analysis of AIEC and non-AIEC strains did not identify a molecular property exclusive to the AIEC phenotype. We recommend a broader approach to the identification of the bacteria-host interactions that are important in the pathogenesis of Crohn's disease.

Escherichia coli diversity in the lower intestinal tract of humans.

Previous studies examining the clonal diversity of Escherichia coli populations within humans have been based on faecal isolates. In this study E. coli were isolated from biopsies taken from the terminal ileum, ascending, transverse and descending colon, and rectum of 69 individuals. Multiple isolates from each biopsy were characterized using Rep-PCR. An average of 3.5 genotypes were recovered per host, and in hosts with two or more strains, the phylogroup membership of the second most abundant strain was significantly more likely to be the same as the dominant strain. There was no indication of a non-random distribution of E. coli phylogroups among the regions of the lower intestine. In hosts with multiple genotypes, as defined by Repetitive extragenic palindromic-PCR, genotypes were non-randomly distributed among gut regions in over half the individuals. The phylogroup membership of an individual's numerically dominant strain explained some of the variation in the extent to which strains within an individual were heterogeneously distributed, with most heterogeneity observed when the numerically dominant strain belonged to phylogroups E or F, and the least when the dominant strain belonged to phylogroup B2. The results of this study support previous studies on pigs that demonstrated faecal sampling underestimates the genotype diversity present within a host.

Host litter-associated gut dynamics affect Escherichia coli abundance and adhesion genotype in rats.

The probability of detecting Escherichia coli varies between host species with different diets and body sizes. An experimental study that mimicked the effect of different carnivore body masses found that digesta transit times influence E. coli abundance. In this study, we investigated how the host's gastrointestinal dynamics affected E. coli abundance and genotype in a system that reflected an herbivorous host. Forty rats from nine litters were fed a diet high in fermentable fibre. We found a small effect of fibre concentration on the difference between the liquid and particle digesta retention times. However, the rats' litter membership explained the majority of the retention time differences (79%). In turn, we found that as the difference between liquid and particle retention times increased, E. coli faecal cell densities decreased, while the likelihood that an animal's dominant E. coli strain possessed a gene involved in adhesion (agn43) increased. Thus, this experiment revealed an unanticipated high degree of association between the hosts' litter, their gastrointestinal dynamics and the E. coli genotypes. Furthermore, by comparing our findings to previous work, we show that the presence of fermentable fibre in the diet appears to change the relationship between the host's phenotype and E. coli.

Detection of bacterial DNA in lymph nodes of Crohn's disease patients using high throughput sequencing.

OBJECTIVE: Our aim was to determine whether or not specific microorganisms were transported selectively to lymph nodes in Crohn's disease (CD) by comparing node and mucosal microbial communities in patients and controls. We also sought evidence of dysbiosis and bacterial translocation. DESIGN: Lymph nodes, and involved and uninvolved mucosal samples were obtained from resections of 58 patients (29 CD, eight 'other inflammatory bowel disease' (IBD) and 21 non-IBD). Universal primers targeting V1-V3 regions of bacterial 16S rRNA genes were used to amplify bacterial DNA and amplicons sequenced using high throughput sequencing. 20 patients (eight CD (28%), two other IBD (25%) and 10 non-IBD (48%)) had PCR positive nodes. RESULTS: All samples from an individual were similar: there was no evidence of selective concentration of any microorganism in nodes. No specific microorganism was present in the nodes of all CD samples. Escherichia/Shigella were common in all patient groups but patients with ileal CD had a greater proportion of Escherichia coli reads in their nodes than other CD patients (p=0.0475). Campylobacter, Helicobacter and Yersinia were uncommon; Mycobacterium and Listeria were not detected. Dysbiosis was present in all groups but shifts were specific and no common pattern emerged. CONCLUSIONS: It is unlikely that a single bacterium perpetuates inflammation in late stage CD; dysbiosis was common and we found no evidence of increased bacterial translocation. We believe that future studies should focus on early disease and viable bacteria in nodes, aphthous ulcers and granulomas, as they may be more relevant in the initiation of inflammation in CD.

Impact of colonoscopy bowel preparation on intestinal microbiota.

The gut microbiota is important in maintaining human health, but numerous factors have the potential to alter its composition. Our aim was to examine the impact of a standard bowel preparation on the intestinal microbiota using two different techniques. Fifteen subjects undergoing colonoscopy consumed a bowel preparation comprised of 10 mg bisacodyl and 2 L polyethylene glycol. The microbiota of stool samples, collected one month before, one week before (pre-colonoscopy), and one week, one month, and three to six months after colonoscopy (post-colonoscopy) was evaluated. Two samples were taken three to six months apart from five healthy subjects who did not undergo colonoscopy. Universal primers targeting the V2-V3 region of the 16S rRNA gene were used to PCR amplify all samples for denaturing gradient gel electrophoresis (PCR-DGGE). Pre- and post-colonoscopy samples were compared using Dice's similarity coefficients. Three samples from ten subjects who underwent colonoscopy, and both samples from the five subjects who didn't, were used for high-throughput sequencing of the V1-V3 region of the 16S rRNA gene. Samples were curated and analysed in Mothur. Results of the DGGE analyses show that the fecal microbiota of a small number of subjects had short-term changes. High-throughput sequencing results indicated that the variation between the samples of subjects who underwent colonoscopy was no greater than the variation observed between samples from subjects who did not. We conclude that bowel preparation does not have a lasting effect on the composition of the intestinal microbiota for the majority of subjects.