Dietary-Induced Bacterial Metabolites Reduce Inflammation and Inflammation-Associated Cancer via Vitamin D Pathway.

Environmental factors, including westernised diets and alterations to the gut microbiota, are considered risk factors for inflammatory bowel diseases (IBD). The mechanisms underpinning diet-microbiota-host interactions are poorly understood in IBD. We present evidence that feeding a lard-based high-fat (HF) diet can protect mice from developing DSS-induced acute and chronic colitis and colitis-associated cancer (CAC) by significantly reducing tumour burden/incidence, immune cell infiltration, cytokine profile, and cell proliferation. We show that HF protection was associated with increased gut microbial diversity and a significant reduction in Proteobacteria and an increase in Firmicutes and Clostridium cluster XIVa abundance. Microbial functionality was modulated in terms of signalling fatty acids and bile acids (BA). Faecal secondary BAs were significantly induced to include moieties that can activate the vitamin D receptor (VDR), a nuclear receptor richly represented in the intestine and colon. Indeed, colonic VDR downstream target genes were upregulated in HF-fed mice and in combinatorial lipid-BAs-treated intestinal HT29 epithelial cells. Collectively, our data indicate that HF diet protects against colitis and CAC risk through gut microbiota and BA metabolites modulating vitamin D targeting pathways. Our data highlights the complex relationship between dietary fat-induced alterations of microbiota-host interactions in IBD/CAC pathophysiology.

Interpersonal variability of the human gut virome confounds disease signal detection in IBD.

Viruses are increasingly recognised as important components of the human microbiome, fulfilling numerous ecological roles including bacterial predation, immune stimulation, genetic diversification, horizontal gene transfer, microbial interactions, and augmentation of metabolic functions. However, our current view of the human gut virome is tainted by previous sequencing requirements that necessitated the amplification of starting nucleic acids. In this study, we performed an original longitudinal analysis of 40 healthy control, 19 Crohn's disease, and 20 ulcerative colitis viromes over three time points without an amplification bias, which revealed and highlighted the interpersonal individuality of the human gut virome. In contrast to a 16 S rRNA gene analysis of matched samples, we show that α- and ß-diversity metrics of unamplified viromes are not as efficient at discerning controls from patients with inflammatory bowel disease. Additionally, we explored the intrinsic properties of unamplified gut viromes and show there is considerable interpersonal variability in viral taxa, infrequent longitudinal persistence of intrapersonal viruses, and vast fluctuations in the abundance of temporal viruses. Together, these properties of unamplified faecal viromes confound the ability to discern disease associations but significantly advance toward an unbiased and accurate representation of the human gut virome.

An IBD-associated pathobiont synergises with NSAID to promote colitis which is blocked by NLRP3 inflammasome and Caspase-8 inhibitors.

Conflicting evidence exists on the association between consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and symptomatic worsening of inflammatory bowel disease (IBD). We hypothesized that the heterogeneous prevalence of pathobionts [e.g., adherent-invasive Escherichia coli (AIEC)], might explain this inconsistent NSAIDs/IBD correlation. Using IL10-/- mice, we found that NSAID aggravated colitis in AIEC-colonized animals. This was accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis, and pyroptosis, features not seen in mice exposed to AIEC or NSAID alone, revealing an AIEC/NSAID synergistic effect. Inhibition of NLRP3 or Caspase-8 activity ameliorated colitis, with reduction in NLRP3 inflammasome activation, cell death markers, activated T-cells and macrophages, improved histology, and increased abundance of Clostridium cluster XIVa species. Our findings provide new insights into how NSAIDs and an opportunistic gut-pathobiont can synergize to worsen IBD symptoms. Targeting the NLRP3 inflammasome or Caspase-8 could be a potential therapeutic strategy in IBD patients with gut inflammation, which is worsened by NSAIDs.

Comparing In Vitro Faecal Fermentation Methods as Surrogates for Phage Therapy Application.

The human microbiome and its importance in health and disease have been the subject of numerous research articles. Most microbes reside in the digestive tract, with up to 1012 cells per gram of faecal material found in the colon. In terms of gene number, it has been estimated that the gut microbiome harbours >100 times more genes than the human genome. Several human intestinal diseases are strongly associated with disruptions in gut microbiome composition. Less studied components of the gut microbiome are the bacterial viruses called bacteriophages that may be present in numbers equal to or greater than the prokaryotes. Their potential to lyse their bacterial hosts, or to act as agents of horizontal gene transfer makes them important research targets. In this study in vitro faecal fermentation systems were developed and compared for their ability to act as surrogates for the human colon. Changes in bacterial and viral composition occurred after introducing a high-titre single phage preparation both with and without a known bacterial host during the 24 h-long fermentation. We also show that during this timeframe 50 mL plastic tubes can provide data similar to that generated in a sophisticated faecal fermenter system. This knowledge can guide us to a better understanding of the short-term impact of bacteriophage transplants on the bacteriomes and viromes of human recipients.

Stability of the human gut virome and effect of gluten-free diet.

The human gut microbiome consists of bacteria, archaea, eukaryotes, and viruses. The gut viruses are relatively underexplored. Here, we longitudinally analyzed the gut virome composition in 11 healthy adults: its stability, variation, and the effect of a gluten-free diet. Using viral enrichment and a de novo assembly-based approach, we demonstrate the quantitative dynamics of the gut virome, including dsDNA, ssDNA, dsRNA, and ssRNA viruses. We observe highly divergent individual viral communities, carrying on an average 2,143 viral genomes, 13.1% of which were present at all 3 time points. In contrast to previous reports, the Siphoviridae family dominates over Microviridae in studied individual viromes. We also show individual viromes to be stable at the family level but to vary substantially at the genera and species levels. Finally, we demonstrate that lower initial diversity of the human gut virome leads to a more pronounced effect of the dietary intervention on its composition.

Isolation and characterisation of ΦcrAss002, a crAss-like phage from the human gut that infects Bacteroides xylanisolvens.

BACKGROUND: The gut phageome comprises a complex phage community of thousands of individual strains, with a few highly abundant bacteriophages. CrAss-like phages, which infect bacteria of the order Bacteroidales, are the most abundant bacteriophage family in the human gut and make an important contribution to an individual's core virome. Based on metagenomic data, crAss-like phages form a family, with four sub-families and ten candidate genera. To date, only three representatives isolated in pure culture have been reported: ΦcrAss001 and two closely related phages DAC15 and DAC17; all are members of the less abundant candidate genus VI. The persistence at high levels of both crAss-like phage and their Bacteroidales hosts in the human gut has not been explained mechanistically, and this phage-host relationship can only be properly studied with isolated phage-host pairs from as many genera as possible. RESULTS: Faeces from a healthy donor with high levels of crAss-like phage was used to initiate a faecal fermentation in a chemostat, with selected antibiotics chosen to inhibit rapidly growing bacteria and selectively enrich for Gram-negative Bacteroidales. This had the objective of promoting the simultaneous expansion of crAss-like phages on their native hosts. The levels of seven different crAss-like phages expanded during the fermentation, indicating that their hosts were also present in the fermenter. The enriched supernatant was then tested against individual Bacteroidales strains isolated from the same faecal sample. This resulted in the isolation of a previously uncharacterised crAss-like phage of candidate genus IV of the proposed Alphacrassvirinae sub-family, ΦcrAss002, that infects the gut commensal Bacteroides xylanisolvens. ΦcrAss002 does not form plaques or spots on lawns of sensitive cells, nor does it lyse liquid cultures, even at high titres. In keeping with the co-abundance of phage and host in the human gut, ΦcrAss002 and Bacteroides xylanisolvens can also co-exist at high levels when co-cultured in laboratory media. CONCLUSIONS: We report the isolation and characterisation of ΦcrAss002, the first representative of the proposed Alphacrassvirinae sub-family of crAss-like phages. ΦcrAss002 cannot form plaques or spots on bacterial lawns but can co-exist with its host, Bacteroides xylanisolvens, at very high levels in liquid culture without impacting on bacterial numbers. Video abstract.

A postbiotic consisting of heat-treated lactobacilli has a bifidogenic effect in pure culture and in human fermented faecal communities.

The gut microbiota has a significant impact on host health. Dietary interventions using probiotics, prebiotics and postbiotics have the potential to alter microbiota composition and function. Other therapeutic interventions such as antibiotics and faecal microbiota transplantation have also been shown to significantly alter the microbiota and its metabolites. Supplementation of a faecal fermentation model of the human gut with a postbiotic product Lactobacillus LB led to changes in microbiome composition (i.e. increase in beneficial bifidobacteria) and associated metabolic changes (i.e. increased acid production). Lactobacillus LB is a heat-treated preparation of cellular biomass and a fermentate generated by Limosilactobacillus fermentum CNCM MA65/4E-1b (formerly known as Lactobacillus fermentum CNCM MA65/4E-1b) and Lactobacillus delbrueckii ssp. delbrueckii CNCM MA65/4E-2z, medically relevant strains used to produce antidiarrheal preparations. In pure culture, Lactobacillus LB also stimulates the growth of a range of bifidobacterial species and strains. Lactobacillus LB-like preparations generated using other Lactobacillaceae, including commercially available probiotic bacteria, did not have the same impact on a model strain (Bifidobacterium longum subsp. infantis ATCC 15697). This bifidogenic activity is heat- and enzyme-stable and cannot be attributed to lactose, which is a major constituent of Lactobacillus LB. L fermentum CNCM MA65/4E-1b is largely responsible for the observed activity and there is a clear role for compounds smaller than 1 kDa.Importance In general, disruptions to the gut microbiota are associated with multiple disorders in humans. The presence of high levels of Bifidobacterium spp. in the human gut is commonly considered to be beneficial. Bifidobacteria can be supplemented in the diet (as probiotics) or those bifidobacteria already present in the gut can be stimulated by the consumption of prebiotics such as inulin. We demonstrate that Lactobacillus LB (a product consisting of two heat-killed lactic acid bacteria and their metabolites) can stimulate the growth of bifidobacteria in human fermented faecal communities and in pure culture. Given the heat-treatment applied during the production process, there is no risk of the lactic acid bacteria colonising (or causing bacteraemia) in vulnerable consumers (infants, immunocompromised, etc). Lactobacillus LB has the potential to affect human health by selectively promoting the growth of beneficial bacteria.

Ranking microbiome variance in inflammatory bowel disease: a large longitudinal intercontinental study.

OBJECTIVE: The microbiome contributes to the pathogenesis of inflammatory bowel disease (IBD) but the relative contribution of different lifestyle and environmental factors to the compositional variability of the gut microbiota is unclear. DESIGN: Here, we rank the size effect of disease activity, medications, diet and geographic location of the faecal microbiota composition (16S rRNA gene sequencing) in patients with Crohn's disease (CD; n=303), ulcerative colitis (UC; n = 228) and controls (n=161), followed longitudinally (at three time points with 16 weeks intervals). RESULTS: Reduced microbiota diversity but increased variability was confirmed in CD and UC compared with controls. Significant compositional differences between diseases, particularly CD, and controls were evident. Longitudinal analyses revealed reduced temporal microbiota stability in IBD, particularly in patients with changes in disease activity. Machine learning separated disease from controls, and active from inactive disease, when consecutive time points were modelled. Geographic location accounted for most of the microbiota variance, second to the presence or absence of CD, followed by history of surgical resection, alcohol consumption and UC diagnosis, medications and diet with most (90.3%) of the compositional variance stochastic or unexplained. CONCLUSION: The popular concept of precision medicine and rational design of any therapeutic manipulation of the microbiota will have to contend not only with the heterogeneity of the host response, but also with widely differing lifestyles and with much variance still unaccounted for.

Isolation of a Novel Jumbo Bacteriophage Effective Against Klebsiella aerogenes.

Increasing levels of bacterial resistance to many common and last resort antibiotics has increased interest in finding new treatments. The low rate of approval of new antibiotics has led to the search for new and alternative antimicrobial compounds. Bacteriophages (phages) are bacterial viruses found in almost every environment. Phage therapy was historically investigated to control bacterial infections and is still in use in Georgia and as a treatment of last resort. Phage therapy is increasingly recognized as an alternative antimicrobial treatment for antibiotic resistant pathogens. A novel lytic Klebsiella aerogenes phage N1M2 was isolated from maize silage. Klebsiella aerogenes, a member of the ESKAPE bacterial pathogens, is an important target for new antimicrobial therapies. Klebsiella aerogenes can form biofilms on medical devices which aids its environmental persistence and for this reason we tested the effect of phage N1M2 against biofilms. Phage N1M2 successfully removed a pre-formed Klebsiella aerogenes biofilm. Biofilm assays were also carried out with Staphylococcus aureus and Phage K. Phage K successfully removed a preformed Staphylococcus aureus biofilm. Phage N1M2 and Phage K in combination were significantly better at removing a mixed community biofilm of Klebsiella aerogenes and Staphylococcus aureus than either phage alone.

Oral Administration of Heat-Treated Lactobacilli Modifies the Murine Microbiome and Reduces Citrobacter Induced Colitis.

Significant evidence supports a relationship between the gut microbiome, inflammation, host response, and health, including the finding that a number of disorders are associated with disruption of the microbiome. In these disorders, a number of dietary interventions (including prebiotics, live probiotics, or heat-killed microbes) have been proposed to be curative or preventative agents. The use of heat-killed microbes has a number of benefits over living organisms, including reduced infection risk in vulnerable individuals, extended shelf life and the potential for use in combination with antimicrobial agents. We previously reported that murine chow supplemented with 5% ADR-159, a heat-treated fermentate generated by two Lactobacillus strains, altered both behavior and the microbiome of male mice. Now we show that ADR-159 fed female mice also display a similar microbiome shift as determined by 16S rDNA analysis. In particular, we observed a reduction of levels of Turicibacter and Clostridium sensu stricto. These subtle changes in the bacterial component of the microbiome were mirrored by changes in the virome. Extended consumption of the ADR-159 diet had no negative effect on general health and lipocalin 2 levels (LCN2; a proxy for inflammation), but we observed increased IL-17f and decreased IL-12α expression in the colon and decreased short chain fatty acid levels in the ADR-159 fed animals. Four weeks into the diet, half of the animals were dosed with Citrobacter to determine the effect of ADR-159 on infection and on pathogen induced colitis. Overall, our results suggest that while the ADR-159 diet does not prevent Citrobacter infection, it had an effect on Citrobacter-induced inflammation. In contrast to animals fed standard chow, ADR-159 fed animals did not show a reduction of small intestine length and increase of colon crypt depth, which occurred in control mice. These microbiological, histological, and immunological results provide evidence to support the impact of heat-treated microorganisms and their metabolites on the murine microbiome and health.

Whole-Virome Analysis Sheds Light on Viral Dark Matter in Inflammatory Bowel Disease.

The human gut virome is thought to significantly impact the microbiome and human health. However, most virome analyses have been performed on a limited fraction of known viruses. Using whole-virome analysis on a published keystone inflammatory bowel disease (IBD) cohort and an in-house ulcerative colitis dataset, we shed light on the composition of the human gut virome in IBD beyond this identifiable minority. We observe IBD-specific changes to the virome and increased numbers of temperate phage sequences in individuals with Crohn's disease. Unlike prior database-dependent methods, no changes in viral richness were observed. Among IBD subjects, the changes in virome composition reflected alterations in bacterial composition. Furthermore, incorporating both bacteriome and virome composition offered greater classification power between health and disease. This approach to analyzing whole virome across cohorts highlights significant IBD signals, which may be crucial for developing future biomarkers and therapeutics.

The Human Gut Virome Is Highly Diverse, Stable, and Individual Specific.

The human gut contains a vast array of viruses, mostly bacteriophages. The majority remain uncharacterized, and their roles in shaping the gut microbiome and in impacting on human health remain poorly understood. We performed longitudinal metagenomic analysis of fecal viruses in healthy adults that reveal high temporal stability, individual specificity, and correlation with the bacterial microbiome. Using a database-independent approach that uses most of the sequencing data, we uncovered the existence of a stable, numerically predominant individual-specific persistent personal virome. Clustering of viral genomes and de novo taxonomic annotation identified several groups of crAss-like and Microviridae bacteriophages as the most stable colonizers of the human gut. CRISPR-based host prediction highlighted connections between these stable viral communities and highly predominant gut bacterial taxa such as Bacteroides, Prevotella, and Faecalibacterium. This study provides insights into the structure of the human gut virome and serves as an important baseline for hypothesis-driven research.

Evaluation of Phage Therapy in the Context of Enterococcus faecalis and Its Associated Diseases.

Bacteriophages (phages) or bacterial viruses have been proposed as natural antimicrobial agents to fight against antibiotic-resistant bacteria associated with human infections. Enterococcus faecalis is a gut commensal, which is occasionally found in the mouth and vaginal tract, and does not usually cause clinical problems. However, it can spread to other areas of the body and cause life-threatening infections, such as septicemia, endocarditis, or meningitis, in immunocompromised hosts. Although E. faecalis phage cocktails are not commercially available within the EU or USA, there is an accumulated evidence from in vitro and in vivo studies that have shown phage efficacy, which supports the idea of applying phage therapy to overcome infections associated with E. faecalis. In this review, we discuss the potency of bacteriophages in controlling E. faecalis, in both in vitro and in vivo scenarios. E. faecalis associated bacteriophages were compared at the genome level and an attempt was made to categorize phages with respect to their suitability for therapeutic application, using orthocluster analysis. In addition, E. faecalis phages have been examined for the presence of antibiotic-resistant genes, to ensure their safe use in clinical conditions. Finally, the domain architecture of E. faecalis phage-encoded endolysins are discussed.

Choice of assembly software has a critical impact on virome characterisation.

BACKGROUND: The viral component of microbial communities plays a vital role in driving bacterial diversity, facilitating nutrient turnover and shaping community composition. Despite their importance, the vast majority of viral sequences are poorly annotated and share little or no homology to reference databases. As a result, investigation of the viral metagenome (virome) relies heavily on de novo assembly of short sequencing reads to recover compositional and functional information. Metagenomic assembly is particularly challenging for virome data, often resulting in fragmented assemblies and poor recovery of viral community members. Despite the essential role of assembly in virome analysis and difficulties posed by these data, current assembly comparisons have been limited to subsections of virome studies or bacterial datasets. DESIGN: This study presents the most comprehensive virome assembly comparison to date, featuring 16 metagenomic assembly approaches which have featured in human virome studies. Assemblers were assessed using four independent virome datasets, namely, simulated reads, two mock communities, viromes spiked with a known phage and human gut viromes. RESULTS: Assembly performance varied significantly across all test datasets, with SPAdes (meta) performing consistently well. Performance of MIRA and VICUNA varied, highlighting the importance of using a range of datasets when comparing assembly programs. It was also found that while some assemblers addressed the challenges of virome data better than others, all assemblers had limitations. Low read coverage and genomic repeats resulted in assemblies with poor genome recovery, high degrees of fragmentation and low-accuracy contigs across all assemblers. These limitations must be considered when setting thresholds for downstream analysis and when drawing conclusions from virome data.

Short-term consumption of a high-fat diet increases host susceptibility to Listeria monocytogenes infection.

BACKGROUND: A westernized diet comprising a high caloric intake from animal fats is known to influence the development of pathological inflammatory conditions. However, there has been relatively little focus upon the implications of such diets for the progression of infectious disease. Here, we investigated the influence of a high-fat (HF) diet upon parameters that influence Listeria monocytogenes infection in mice. RESULTS: We determined that short-term administration of a HF diet increases the number of goblet cells, a known binding site for the pathogen, in the gut and also induces profound changes to the microbiota and promotes a pro-inflammatory gene expression profile in the host. Host physiological changes were concordant with significantly increased susceptibility to oral L. monocytogenes infection in mice fed a HF diet relative to low fat (LF)- or chow-fed animals. Prior to Listeria infection, short-term consumption of HF diet elevated levels of Firmicutes including Coprococcus, Butyricicoccus, Turicibacter and Clostridium XIVa species. During active infection with L. monocytogenes, microbiota changes were further exaggerated but host inflammatory responses were significantly downregulated relative to Listeria-infected LF- or chow-fed groups, suggestive of a profound tempering of the host response influenced by infection in the context of a HF diet. The effects of diet were seen beyond the gut, as a HF diet also increased the sensitivity of mice to systemic infection and altered gene expression profiles in the liver. CONCLUSIONS: We adopted a systems approach to identify the effects of HF diet upon L. monocytogenes infection through analysis of host responses and microbiota changes (both pre- and post-infection). Overall, the results indicate that short-term consumption of a westernized diet has the capacity to significantly alter host susceptibility to L. monocytogenes infection concomitant with changes to the host physiological landscape. The findings suggest that diet should be a consideration when developing models that reflect human infectious disease.

Biology and Taxonomy of crAss-like Bacteriophages, the Most Abundant Virus in the Human Gut.

CrAssphages represent the most abundant virus in the human gut microbiota, but the lack of available genome sequences for comparison has kept them enigmatic. Recently, sequence-based classification of distantly related crAss-like phages from multiple environments was reported, leading to a proposed familial-level taxonomic group. Here, we assembled the metagenomic sequencing reads from 702 human fecal virome/phageome samples and analyzed 99 complete circular crAss-like phage genomes and 150 contigs ≥70 kb. In silico comparative genomics and taxonomic analysis enabled a classification scheme of crAss-like phages from human fecal microbiomes into four candidate subfamilies composed of ten candidate genera. Laboratory analysis was performed on fecal samples from an individual harboring seven distinct crAss-like phages. We achieved crAss-like phage propagation in ex vivo human fecal fermentations and visualized short-tailed podoviruses by electron microscopy. Mass spectrometry of a crAss-like phage capsid protein could be linked to metagenomic sequencing data, confirming crAss-like phage structural annotations.

Determinants of Reduced Genetic Capacity for Butyrate Synthesis by the Gut Microbiome in Crohn's Disease and Ulcerative Colitis.

BACKGROUND AND AIMS: Alterations in short chain fatty acid metabolism, particularly butyrate, have been reported in inflammatory bowel disease, but results have been conflicting because of small study numbers and failure to distinguish disease type, activity or other variables such as diet. We performed a comparative assessment of the capacity of the microbiota for butyrate synthesis, by quantifying butyryl-CoA:acetate CoA-transferase [BCoAT] gene content in stool from patients with Crohn's disease [CD; n = 71], ulcerative colitis [UC; n = 58] and controls [n = 75], and determined whether it was related to active vs inactive inflammation, microbial diversity, and composition and/or dietary habits. METHODS: BCoAT gene content was quantified by quantitative polymerase chain reaction [qPCR]. Disease activity was assessed clinically and faecal calprotectin concentration measured. Microbial composition was determined by sequencing 16S rRNA gene. Dietary data were collected using an established food frequency questionnaire. RESULTS: Reduced butyrate-synthetic capacity was found in patients with active and inactive CD [p < 0.001 and p < 0.01, respectively], but only in active UC [p < 0.05]. In CD, low BCoAT gene content was associated with ileal location, stenotic behaviour, increased inflammation, lower microbial diversity, greater microbiota compositional change, and decreased butyrogenic taxa. Reduced BCoAT gene content in patients with CD was linked with a different regimen characterised by lower dietary fibre. CONCLUSIONS: Reduced butyrate-synthetic capacity of the microbiota is more evident in CD than UC and may relate to reduced fibre intake. The results suggest that simple replacement of butyrate per se may be therapeutically inadequate, whereas manipulation of microbial synthesis, perhaps by dietary means, may be more appropriate.

A clinician's guide to microbiome analysis.

Microbiome analysis involves determining the composition and function of a community of microorganisms in a particular location. For the gastroenterologist, this technology opens up a rapidly evolving set of challenges and opportunities for generating novel insights into the health of patients on the basis of microbiota characterizations from intestinal, hepatic or extraintestinal samples. Alterations in gut microbiota composition correlate with intestinal and extraintestinal disease and, although only a few mechanisms are known, the microbiota are still an attractive target for developing biomarkers for disease detection and management as well as potential therapeutic applications. In this Review, we summarize the major decision points confronting new entrants to the field or for those designing new projects in microbiome research. We provide recommendations based on current technology options and our experience of sequencing platform choices. We also offer perspectives on future applications of microbiome research, which we hope convey the promise of this technology for clinical applications.

16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform.

BACKGROUND: Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA. RESULTS: The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar. CONCLUSIONS: Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.