Georgina L. Hold

The gut microbiota, bacterial metabolites and colorectal cancer.

Accumulating evidence suggests that the human intestinal microbiota contributes to the aetiology of colorectal cancer (CRC), not only via the pro-carcinogenic activities of specific pathogens but also via the influence of the wider microbial community, particularly its metabolome. Recent data have shown that the short-chain fatty acids acetate, propionate and butyrate function in the suppression of inflammation and cancer, whereas other microbial metabolites, such as secondary bile acids, promote carcinogenesis. In this Review, we discuss the relationship between diet, microbial metabolism and CRC and argue that the cumulative effects of microbial metabolites should be considered in order to better predict and prevent cancer progression.

The gut microbiota and host health: a new clinical frontier

Over the last 10–15 years, our understanding of the composition and functions of the human gut microbiota has increased exponentially. To a large extent, this has been due to new ‘omic’ technologies that have facilitated large-scale analysis of the genetic and metabolic profile of this microbial community, revealing it to be comparable in influence to a new organ in the body and offering the possibility of a new route for therapeutic intervention. Moreover, it might be more accurate to think of it like an immune system: a collection of cells that work in unison with the host and that can promote health but sometimes initiate disease. This review gives an update on the current knowledge in the area of gut disorders, in particular metabolic syndrome and obesity-related disease, liver disease, IBD and colorectal cancer. The potential of manipulating the gut microbiota in these disorders is assessed, with an examination of the latest and most relevant evidence relating to antibiotics, probiotics, prebiotics, polyphenols and faecal microbiota transplantation.

Assessment of microbial diversity in human colonic samples by 16S rDNA sequence analysis

Abstract The bacterial species diversity of three colonic tissue samples from elderly people was investigated by sequence analysis of randomly cloned eubacterial 16S rDNA. The majority of sequences (87%) clustered within three bacterial groups: (1) Bacteroides; (2) low G+C content Gram-positives related to Clostridium coccoides (cluster XIVa); (3) Gram-positives related to Clostridium leptum (cluster IV). These groups have been shown to dominate the human faecal flora. Only 25% of sequences were closely related (>97%) to current species type strains, and 28% were less than 97% related to any database entry. 19% of sequences were most closely related to recently isolated butyrate-producing bacteria belonging to clusters XIVa and IV, with a further 18% of the sequences most closely related to Ruminococcus obeum and Ruminococcus torques (members of cluster XIVa). These results provide the first molecular information on the microbial diversity present in human colonic samples.

Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb. nov.

Two newly isolated strains of obligately anaerobic bacteria from human faeces are shown here to be related to Fusobacterium prausnitzii, which is regarded as one of the most abundant colonizers of the human colon. These strains, along with Fusobacterium prausnitzii ATCC 27768(T) and 27766, are non-motile and produce butyrate, formate and lactate, but not hydrogen as fermentation products. A new finding is that all four strains produce D-lactate, but not L-lactate. The strains have a requirement for acetate in the growth medium and this may account for the previously reported requirement for rumen fluid. The DNA G+C content of the four strains is 47-57 mol%. Together with phylogenetic analysis based on 16S rRNA sequencing, this establishes that Fusobacterium prausnitzii strains are only distantly related to Fusobacterium sensu stricto and are more closely related to members of Clostridium cluster IV (the Clostridium leptum group). It is proposed that a new genus, Faecalibacterium gen. nov. be created; this genus should include Faecalibacterium prausnitzii gen. nov., comb. nov. ATCC 27768(T) (= NCIMB 13872(T)) (formerly Fusobacterium prausnitzii) as the type species together with ATCC 27766 and the newly isolated strains A2-165 and L2-6.

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.

Oligonucleotide Probes That Detect Quantitatively Significant Groups of Butyrate-Producing Bacteria in Human Feces

ABSTRACT 16S rRNA-targeted oligonucleotide probes were designed for butyrate-producing bacteria from human feces. Three new cluster-specific probes detected bacteria related to Roseburia intestinalis, Faecalibacterium prausnitzii, and Eubacterium hallii at mean populations of 2.3, 3.8, and 0.6%, respectively, in samples from 10 individuals. Additional species-level probes accounted for no more than 1%, with a mean of 7.7%, of the total human fecal microbiota identified as butyrate producers in this study. Bacteria related to E. hallii and the genera Roseburia and Faecalibacterium are therefore among the most abundant known butyrate-producing bacteria in human feces.

Polymorphisms in Toll-like receptor genes and risk of cancer

Host genetic factors are emerging as key determinants of disease risk for many cancers. Identifying candidate genes is a major challenge that has to stem from a profound understanding of the pathophysiology of the disease. The Toll-like receptors are important members of the hosts innate immune response and their genes have been found to be polymorphic. This genetic variation allows for a more intricate repertoire that enables the host to withstand microbial challenges. While this may be advantageous on a population level, there may be less favourable outcomes for individuals that harbour certain genotypes associated with excessive immune activation and inflammatory drive. The damage is often collateral and is manifest in organs where this chronic inflammation alters normal physiology. A classic example of this paradigm is the Helicobacter pylori-induced gastric cancer model. Another emerging model is prostate cancer where Toll-like receptor polymorphisms have also been found to play a role. In this review, we discuss polymorphisms in Toll-like receptors and give an insight into how they may influence risk of cancer.

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.

Role of the polymorphic IL‐1B, IL‐1RN and TNF‐A genes in distal gastric cancer in Mexico

Several cytokine gene polymorphisms have been associated with increased risk of distal gastric cancer (GC) and its precursor histological markers in Caucasian, Asian and Portuguese populations although little is known about their role in other ethnic groups. Our study investigates the role of the IL‐1B‐31, IL‐1RN and TNF‐A‐308 gene polymorphisms as risk factors for the development of GC in a Mexican population. We studied 278 patients who were enrolled at the Hospital Universitario Dr. José Eleuterio González, Universidad Autónoma de Nuevo León. The subjects were divided into 2 groups. Sixty‐three patients with histologically confirmed distal GC (mean age = 58.8 years, range = 22–84, F:M = 0.56), and 215 patients with no evidence of distal or proximal GC (mean age = 56.1 years, range = 18–92, F:M = 1.17). The IL‐1B‐31 and the TNF‐A‐308 polymorphisms were determined by PCR‐RFLP and pyrosequencing, respectively, in all cases and controls. The VNTR polymorphism in intron 2 of the 1L‐1RN gene was typed by PCR in 25 cases and 201 controls. The H. pylori status was determined by histology, rapid urease test, culture and serology for non‐cancer controls and by histology for the GC cases. The carriage of the proinflammatory IL‐1B‐31*C allele was associated with increased risk of distal GC (odds ratio [OR] = 7.63, 95% confidence interval [CI] = 1.73‐46.94, p = 0.003). When cases and controls were matched by age and gender, the OR value was higher (OR = 8.05, 95% CI = 1.8–50.22, p = 0.001). When only H. pylori GC cases and controls were compared, the OR value was 7.8 (95% CI = 1.05–161.8, p = 0.04). No association was found between any of the other polymorphisms studied and distal GC. In this Mexican population, the IL‐1B proinflammatory genotype increases the risk of distal GC. These findings are similar to previous reports in Caucasian populations and underscore the importance of cytokine gene polymorphisms in the development of distal GC.

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

Introduction Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Methods Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. Results DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). Conclusion This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples in a study are prepared with the same method, and the need for caution when cross-comparing studies that use different methods.