Richard Hansen

IBD—what role do Proteobacteria play?

The gastrointestinal microbiota has come to the fore in the search for the causes of IBD. This shift has largely been driven by the finding of genetic polymorphisms involved in gastrointestinal innate immunity (particularly polymorphisms in NOD2 and genes involved in autophagy) and alterations in the composition of the microbiota that might result in inflammation (so-called dysbiosis). Microbial diversity studies have continually demonstrated an expansion of the Proteobacteria phylum in patients with IBD. Individual Proteobacteria, in particular (adherent-invasive) Escherichia coli, Campylobacter concisus and enterohepatic Helicobacter, have all been associated with the pathogenesis of IBD. In this Review, we comprehensively describe the various associations of Proteobacteria and IBD. We also examine the importance of pattern recognition in the extracellular innate immune response of the host with particular reference to Proteobacteria, and postulate that Proteobacteria with adherent and invasive properties might exploit host defenses, drive proinflammatory change, alter the intestinal microbiota in favor of dysbiosis and ultimately lead to the development of IBD.

Microbiota of De-Novo Pediatric IBD: Increased Faecalibacterium Prausnitzii and Reduced Bacterial Diversity in Crohn ' s But Not in Ulcerative Colitis

OBJECTIVES:The gastrointestinal microbiota is considered important in inflammatory bowel disease (IBD) pathogenesis. Discoveries from established disease cohorts report reduced bacterial diversity, changes in bacterial composition, and a protective role for Faecalibacterium prausnitzii in Crohns disease (CD). The majority of studies to date are however potentially confounded by the effect of treatment and a reliance on established rather than de-novo disease.METHODS:Microbial changes at diagnosis were examined by biopsying the colonic mucosa of 37 children: 25 with newly presenting, untreated IBD with active colitis (13 CD and 12 ulcerative colitis (UC)), and 12 pediatric controls with a macroscopically and microscopically normal colon. We utilized a dual-methodology approach with pyrosequencing (threshold >10,000 reads) and confirmatory real-time PCR (RT-PCR).RESULTS:Threshold pyrosequencing output was obtained on 34 subjects (11 CD, 11 UC, 12 controls). No significant changes were noted at phylum level among the Bacteroidetes, Firmicutes, or Proteobacteria. A significant reduction in bacterial α-diversity was noted in CD vs. controls by three methods (Shannon, Simpson, and phylogenetic diversity) but not in UC vs. controls. An increase in Faecalibacterium was observed in CD compared with controls by pyrosequencing (mean 16.7% vs. 9.1% of reads, P=0.02) and replicated by specific F. prausnitzii RT-PCR (36.0% vs. 19.0% of total bacteria, P=0.02). No disease-specific clustering was evident on principal components analysis.CONCLUSIONS:Our results offer a comprehensive examination of the IBD mucosal microbiota at diagnosis, unaffected by therapeutic confounders or changes over time. Our results challenge the current model of a protective role for F. prausnitzii in CD, suggesting a more dynamic role for this organism than previously described.

Rising incidence of pediatric inflammatory bowel disease in Scotland

Background: An accurate indication of the changing incidence of pediatric inflammatory bowel disease (PIBD) within a population is useful in understanding concurrent etiological factors. We aimed to compare the current incidence and other demographic attributes of PIBD in the Scottish population to previous data. Methods: A national cohort of prospectively and retrospectively acquired incident cases of PIBD diagnosed less than 16 years old in pediatric services in Scotland was captured for the period 2003–2008; historical Scottish data were used for comparison (1990–1995). Age/sex‐adjusted incidences were calculated and statistical comparisons made using Poisson regression. Results: During the 2003–2008 study period 436 patients were diagnosed with PIBD in Scotland, giving an adjusted incidence of 7.82/100,000/year. The incidence of Crohns disease (CD) was 4.75/100,000/year, ulcerative colitis (UC) 2.06/100,000/year, and inflammatory bowel disease‐unclassified (IBDU) 1.01/100,000/year. Compared with data from 1990–1995 when 260 IBD patients were diagnosed, significant rises in the incidence of IBD (from 4.45/100,000/year, P < 0.0001), CD (from 2.86/100,000/year, P < 0.0001), and UC (from 1.59/100,000/year, P = 0.023) were seen. There was also a significant reduction in the median age at IBD diagnosis from 12.7 years to 11.9 years between the periods (P = 0.003), with a continued male preponderance. Conclusions: The number of Scottish children diagnosed with IBD continues to rise, with a statistically significant 76% increase since the mid‐1990s. Furthermore, PIBD is now being diagnosed at a younger age. The reason for this continued rise is not yet clear; however, new hypotheses regarding disease pathogenesis and other population trends may provide further insights in future years. (Inflamm Bowel Dis 2012;)

Detection of Campylobacter concisus and Other Campylobacter Species in Colonic Biopsies from Adults with Ulcerative Colitis

Introduction The critical role of bacteria in the pathogenesis of ulcerative colitis (UC) is well recognized, but an individual causative microorganism has not been singled out so far. Campylobacter concisus and other non-jejuni species of Campylobacter have been implicated as putative aetiological agents in inflammatory bowel disease in children, but such studies have not been addressed in adults. This study investigated the prevalence of Campylobacter species in colonic biopsy samples from adults with UC and healthy controls. Methods Adult patients who were undergoing diagnostic colonoscopy were recruited for the study, which included 69 patients with histologically proven UC and 65 healthy controls. DNA was extracted from the biopsy samples and subjected to Campylobacter genus specific and Campylobacter concisus specific polymerase chain reaction and sequencing. Results Detection of all Campylobacter DNA utilising genus specific primers was significantly higher in cases of UC, with a prevalence of 73.9% (51/69) compared to 23.1% (15/65) in controls (p = 0.0001). Nested PCR for C. concisus DNA was positive in 33.3% (23/69) of biopsy samples from subjects with UC, which was significantly higher than the prevalence rate of 10.8% (7/65) from controls (p = 0.0019). Sequencing of the remaining Campylobacter positive samples revealed that Campylobacter ureolyticus was positive in 21.7% (15/69) of samples from UC subjects as opposed to 3.1% (2/65) in controls (p = 0.0013). Mixed Campylobacter species were more common in UC patients, 20.3% (14/69) as compared to controls 4.6% (3/65) (p = 0.0084). Conclusion The higher prevalence of Campylobacter genus and more specifically C. concisus and C. ureolyticus in biopsy samples from adults with UC suggests these genera of bacteria may be involved in the chronic inflammation that is characteristically seen in UC. To the best of our knowledge this is the first report of this association of C. concisus and C. ureolyticus with UC in adults.

The role of infection in the aetiology of inflammatory bowel disease

We have greatly increased our understanding of the genetics of inflammatory bowel disease (IBD) in the last decade; however, migrant studies highlight the importance of environment in disease risk. The possibility that IBD is an infection has been debated since the first description of Crohn’s disease. Mycobacterium avium paratuberculosis was the first organism to be suggested as an IBD pathogen, and it has been argued that it fulfils Koch’s postulates and could be designated the cause of Crohn’s disease. Other organisms have been postulated as possible IBD pathogens, including various Helicobacter species, one of which has been identified in primate colitis; others are widely used in animal models of IBD. Adherent-invasive Escherichia coli appear specific to ileal Crohn’s disease and have been shown to induce the release of TNF-α, a key cytokine in IBD inflammation. The aim of this article is to give a concise overview of the infections postulated as being relevant to the onset of IBD. We will also briefly cover the immunology underpinning IBD, in addition to reviewing current knowledge regarding other microorganisms that are associated with modifying the risk of developing IBD. It may be that infectious organisms have an orchestrator role in the development of dysbiosis and subsequently IBD.

Extensive modulation of the fecal metagenome in children With Crohn’s disease during exclusive enteral nutrition

OBJECTIVES:Exploring associations between the gut microbiota and colonic inflammation and assessing sequential changes during exclusive enteral nutrition (EEN) may offer clues into the microbial origins of Crohn’s disease (CD).METHODS:Fecal samples (n=117) were collected from 23 CD and 21 healthy children. From CD children fecal samples were collected before, during EEN, and when patients returned to their habitual diets. Microbiota composition and functional capacity were characterized using sequencing of the 16S rRNA gene and shotgun metagenomics.RESULTS:Microbial diversity was lower in CD than controls before EEN (P=0.006); differences were observed in 36 genera, 141 operational taxonomic units (OTUs), and 44 oligotypes. During EEN, the microbial diversity of CD children further decreased, and the community structure became even more dissimilar than that of controls. Every 10 days on EEN, 0.6 genus diversity equivalents were lost; 34 genera decreased and one increased during EEN. Fecal calprotectin correlated with 35 OTUs, 14 of which accounted for 78% of its variation. OTUs that correlated positively or negatively with calprotectin decreased during EEN. The microbiota of CD patients had a broader functional capacity than healthy controls, but diversity decreased with EEN. Genes involved in membrane transport, sulfur reduction, and nutrient biosynthesis differed between patients and controls. The abundance of genes involved in biotin (P=0.005) and thiamine biosynthesis decreased (P=0.017), whereas those involved in spermidine/putrescine biosynthesis (P=0.031), or the shikimate pathway (P=0.058), increased during EEN.CONCLUSIONS:Disease improvement following treatment with EEN is associated with extensive modulation of the gut microbiome.

Enterohepatic helicobacter in ulcerative colitis: potential pathogenic entities?

Background Changes in bacterial populations termed “dysbiosis” are thought central to ulcerative colitis (UC) pathogenesis. In particular, the possibility that novel Helicobacter organisms play a role in human UC has been debated but not comprehensively investigated. The aim of this study was to develop a molecular approach to investigate the presence of Helicobacter organisms in adults with and without UC. Methodology/Principal Findings A dual molecular approach to detect Helicobacter was developed. Oligonucleotide probes against the genus Helicobacter were designed and optimised alongside a validation of published H. pylori probes. A comprehensive evaluation of Helicobacter genus and H. pylori PCR primers was also undertaken. The combined approach was then assessed in a range of gastrointestinal samples prior to assessment of a UC cohort. Archival colonic samples were available from 106 individuals for FISH analysis (57 with UC and 49 non-IBD controls). A further 118 individuals were collected prospectively for dual FISH and PCR analysis (86 UC and 32 non-IBD controls). An additional 27 non-IBD controls were available for PCR analysis. All Helicobacter PCR-positive samples were sequenced. The association between Helicobacter and each study group was statistically analysed using the Pearson Chi Squared 2 tailed test. Helicobacter genus PCR positivity was significantly higher in UC than controls (32 of 77 versus 11 of 59, p = 0.004). Sequence analysis indicated enterohepatic Helicobacter species prevalence was significantly higher in the UC group compared to the control group (30 of 77 versus 2 of 59, p<0.0001). PCR and FISH results were concordant in 74 (67.9%) of subjects. The majority of discordant results were attributable to a higher positivity rate with FISH than PCR. Conclusions/Significance Helicobacter organisms warrant consideration as potential pathogenic entities in UC. Isolation of these organisms from colonic tissue is needed to enable interrogation of pathogenicity against established criteria.

Could Helicobacter organisms cause inflammatory bowel disease

The discovery of Helicobacter pylori sparked a revolution in the understanding and management of peptic ulcer disease and gastric cancer. Other Helicobacter species are recognized as important pathogenic agents in colitic diseases of rodents and primates, in particular Helicobacter bilis, Helicobacter fennelliae, Helicobacter hepaticus and Helicobacter trogontum. Helicobacter bilis and H. hepaticus are now routinely used to initiate rodent models of inflammatory bowel disease (IBD), particularly in immunocompromised hosts. Molecular evidence exists linking various non-pylori Helicobacter spp. with human IBD; however, attempts to culture organisms in this disease cohort have proved unsuccessful to date. Attributing causation has therefore proved elusive. Seven enterohepatic, non-pylori Helicobacter organisms have been successfully cultured from humans, namely Helicobacter canadensis, Helicobacter canis, Helicobacter cinaedi, H. fennelliae, Helicobacter pullorum, Helicobacter winghamensis and Helicobacter sp. flexispira taxon 8 (now classified as H. bilis). Of these, H. cinaedi and H. fennelliae are the closest to fulfilling Kochs postulates as causative agents in homosexual proctitis. The possibility that novel Helicobacter organisms have a role in the initiation of human IBD warrants further consideration and targeted investigations.

The Microaerophilic Microbiota of De-Novo Paediatric Inflammatory Bowel Disease: The BISCUIT Study

Introduction Children presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study aetiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. We set out to study these and other microaerophilic bacteria in de-novo paediatric IBD. Patients and Methods 100 children undergoing colonoscopy were recruited including 44 treatment naïve de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis. Results 129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312). Conclusions This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon. Trial Registration This study is publically registered on the United Kingdom Clinical Research Network Portfolio (9633).

First-Pass Meconium Samples from Healthy Term Vaginally-Delivered Neonates: An Analysis of the Microbiota

Background Considerable effort has been made to categorise the bacterial composition of the human gut and correlate findings with gastrointestinal disease. The infant gut has long been considered sterile at birth followed by rapid colonisation; however, this view has recently been challenged. We examined first-pass meconium from healthy term infants to confirm or refute sterility. Methods Healthy mothers were approached following vaginal delivery. First-pass meconium stools within 24 hours of delivery were obtained from healthy, breastfed infants with tight inclusion/exclusion criteria including rejecting any known antibiotic exposure - mother within 7 days preceding delivery or infant after birth. Stools were processed in triplicate for fluorescent in-situ hybridisation (FISH) with 16S rRNA-targeted probes including Bifidobacterium; Bacteroides-Prevotella; Lactobacillaceae/Enterococcaceae; Enterobacteriaceae; Streptococcaceae; Staphylococcaceae and Enterococcaceae. Absolute counts of all bacteria and proportional identification of each bacterial group were calculated. Confirmation of bacterial presence by PCR was undertaken on FISH-positive samples. Results The mothers of 31 newborn infants were recruited, 15 met inclusion/exclusion criteria and provided a sample within 24 hours of birth, processed in the lab within 4 hours. All babies were 37–40 weeks gestation. 8/15 were male, mean birth weight was 3.4kg and mean maternal age was 32 years. Meconium samples from 10/15 (66%) infants had evidence of bacteria based on FISH analysis. Of these, PCR was positive in only 1. Positive FISH counts ranged from 2.2 - 41.8 x 104 cells/g with a mean of 15.4 x 104 cells/g. (The limit of detection for automated counting is 106 cells/g). Cell counts were too low to allow formal diversity analysis. Amplification by PCR was not possible despite positive spiked samples demonstrating the feasibility of reaction. One baby was dominated by Enterobacteriaceae. The others contained 2-5 genera, with Bifidobacterium, Enterobacteriaceae, Enterococcaceae and Bacteroides-Prevotella the most prevalent. There was no association between bacterial counts and rupture of membrane duration, time to passage of meconium or time to lab. Conclusion This study provides evidence that low numbers of bacteria are present in first-pass meconium samples from healthy, vaginally-delivered, breastfed term infants. Only two-thirds of meconium samples had detectable bacteria, though at levels too low for automated counting or for reliable confirmation by PCR. This study suggests that gut bacterial colonisation is extremely limited at birth and occurs rapidly thereafter.