Extending colonic mucosal microbiome analysis - Assessment of colonic lavage as a proxy for endoscopic colonic biopsies

This study was supported through GI Research funds and MRC Grant Ref: MR/M00533X/1 to GH.Peer reviewedPublisher PD

Cervical intraepithelial neoplasia disease progression is associated with increased vaginal microbiome diversity

Persistent infection with oncogenic Human Papillomavirus (HPV) is necessary for cervical carcinogenesis. Although evidence suggests that the vaginal microbiome plays a functional role in the persistence or regression of HPV infections, this has yet to be described in women with cervical intra-epithelial neoplasia (CIN). We hypothesised that increasing microbiome diversity is associated with increasing CIN severity. llumina MiSeq sequencing of 16S rRNA gene amplicons was used to characterise the vaginal microbiota of women with low-grade squamous intra-epithelial lesions (LSIL; n = 52), high-grade (HSIL; n = 92), invasive cervical cancer (ICC; n = 5) and healthy controls (n = 20). Hierarchical clustering analysis revealed an increased prevalence of microbiomes characterised by high-diversity and low levels of Lactobacillus spp. (community state type-CST IV) with increasing disease severity, irrespective of HPV status (Normal = 2/20,10%; LSIL = 11/52,21%; HSIL = 25/92,27%; ICC = 2/5,40%). Increasing disease severity was associated with decreasing relative abundance of Lactobacillus spp. The vaginal microbiome in HSIL was characterised by higher levels of Sneathia sanguinegens (P < 0.01), Anaerococcus tetradius (P < 0.05) and Peptostreptococcus anaerobius (P < 0.05) and lower levels of Lactobacillus jensenii (P < 0.01) compared to LSIL. Our results suggest advancing CIN disease severity is associated with increasing vaginal microbiota diversity and may be involved in regulating viral persistence and disease progression

Intestinal virome changes precede autoimmunity in type I diabetes-susceptible children

Viruses have long been considered potential triggers of autoimmune diseases. Here we defined the intestinal virome from birth to the development of autoimmunity in children at risk for type 1 diabetes (T1D). A total of 220 virus-enriched preparations from serially collected fecal samples from 11 children (cases) who developed serum autoantibodies associated with T1D (of whom five developed clinical T1D) were compared with samples from controls. Intestinal viromes of case subjects were less diverse than those of controls. Among eukaryotic viruses, we identified significant enrichment of Circoviridae-related sequences in samples from controls in comparison with cases. Enterovirus, kobuvirus, parechovirus, parvovirus, and rotavirus sequences were frequently detected but were not associated with autoimmunity. For bacteriophages, we found higher Shannon diversity and richness in controls compared with cases and observed that changes in the intestinal virome over time differed between cases and controls. Using Random Forests analysis, we identified disease-associated viral bacteriophage contigs after subtraction of age-associated contigs. These disease-associated contigs were statistically linked to specific components of the bacterial microbiome. Thus, changes in the intestinal virome preceded autoimmunity in this cohort. Specific components of the virome were both directly and inversely associated with the development of human autoimmune disease

The gut microbiome in dogs with congestive heart failure: a pilot study

Compromised gut health and dysbiosis in people with heart failure has received a great deal of attention over the last decade. Whether dogs with heart failure have a similar dysbiosis pattern to what is described in people is currently unknown. We hypothesised that dogs with congestive heart failure have quantifiable dysbiosis compared to healthy dogs that are similar in sex and age. A total of 50 dogs (15 healthy dogs and 35 dogs with congestive heart failure) were prospectively recruited, and their faecal gut microbiome was assessed using 16S rRNA sequencing (Illumina MiSeq platform). There was no significant change in the microbial diversity and richness in dogs with congestive heart failure. However, there was an increase in abundance of Proteobacteria in the congestive heart failure group (p = 0.014), particularly due to an increase in the family Enterobacteriaceae (p = 0.002) and Escherichia coli (p = 0.033). We conclude that dogs with congestive heart failure have dysbiosis, and we show additional trends in our data suggesting that dogs may have a similar pattern to that described in people. The results of this study provide useful preliminary information and raise the possibility that dogs represent a clinically relevant animal model of dysbiosis in people with heart failure

The association between environmental exposures during childhood and the subsequent development of Crohn's Disease: A score analysis approach

Background Environmental factors during childhood are thought to play a role in the aetiology of Crohn's Disease (CD). In South Africa, recently published work based on an investigation of 14 childhood environmental exposures during 3 age intervals (0-5, 6-10 and 11-18 years) has provided insight into the role of timing of exposure in the future development of CD. The 'overlapping' contribution of the investigated variables however, remains unclear. The aim of this study was to perform a post hoc analysis using this data and investigate the extent to which each variable contributes to the subsequent development of CD relative to each aforementioned age interval, based on a score analysis approach. Methods Three methods were used for the score analysis. Two methods employed the subgrouping of one or more (similar) variables (methods A and B), with each subgroup assigned a score value weighting equal to one. For comparison, the third approach (method 0) involved no grouping of the 14 variables. Thus, each variable held a score value of one. Results Results of the score analysis (Method 0) for the environmental exposures during 3 age intervals (0-5, 6-10 and 11-18 years) revealed no significant difference between the case and control groups. By contrast, results from Method A and Method B revealed a significant difference during all 3 age intervals between the case and control groups, with cases having significantly lower exposure scores (approximately 30% and 40% lower, respectively). Conclusion Results from the score analysis provide insight into the 'compound' effects from multiple environmental exposures in the aetiology of CD.IS

Regulation of type 1 diabetes development and B-cell activation in nonobese diabetic mice by early life exposure to a diabetogenic environment

Microbes, including viruses, influence type 1 diabetes (T1D) development, but many such influences remain undefined. Previous work on underlying immune mechanisms has focussed on cytokines and T cells. Here, we compared two nonobese diabetic (NOD) mouse colonies, NODlow and NODhigh, differing markedly in their cumulative T1D incidence (22% vs. 90% by 30 weeks in females). NODhigh mice harbored more complex intestinal microbiota, including several pathobionts; both colonies harbored segmented filamentous bacteria (SFB), thought to suppress T1D. Young NODhigh females had increased B-cell activation in their mesenteric lymph nodes. These phenotypes were transmissible. Co-housing of NODlow with NODhigh mice after weaning did not change T1D development, but T1D incidence was increased in female offspring of co-housed NODlow mice, which were exposed to the NODhigh environment both before and after weaning. These offspring also acquired microbiota and B-cell activation approaching those of NODhigh mice. In NODlow females, the low rate of T1D was unaffected by cyclophosphamide but increased by PD-L1 blockade. Thus, environmental exposures that are innocuous later in life may promote T1D progression if acquired early during immune development, possibly by altering B-cell activation and/or PD-L1 function. Moreover, T1D suppression in NOD mice by SFB may depend on the presence of other microbial influences. The complexity of microbial immune regulation revealed in this murine model may also be relevant to the environmental regulation of human T1D

Human Intestinal Lumen and Mucosa-Associated Microbiota in Patients with Colorectal Cancer

Recent reports have suggested the involvement of gut microbiota in the progression of colorectal cancer (CRC). We utilized pyrosequencing based analysis of 16S rRNA genes to determine the overall structure of microbiota in patients with colorectal cancer and healthy controls; we investigated microbiota of the intestinal lumen, the cancerous tissue and matched noncancerous normal tissue. Moreover, we investigated the mucosa-adherent microbial composition using rectal swab samples because the structure of the tissue-adherent bacterial community is potentially altered following bowel cleansing. Our findings indicated that the microbial structure of the intestinal lumen and cancerous tissue differed significantly. Phylotypes that enhance energy harvest from diets or perform metabolic exchange with the host were more abundant in the lumen. There were more abundant Firmicutes and less abundant Bacteroidetes and Proteobacteria in lumen. The overall microbial structures of cancerous tissue and noncancerous tissue were similar; howerer the tumor microbiota exhibited lower diversity. The structures of the intestinal lumen microbiota and mucosa-adherent microbiota were different in CRC patients compared to matched microbiota in healthy individuals. Lactobacillales was enriched in cancerous tissue, whereas Faecalibacterium was reduced. In the mucosa-adherent microbiota, Bifidobacterium, Faecalibacterium, and Blautia were reduced in CRC patients, whereas Fusobacterium, Porphyromonas, Peptostreptococcus, and Mogibacterium were enriched. In the lumen, predominant phylotypes related to metabolic disorders or metabolic exchange with the host, Erysipelotrichaceae, Prevotellaceae, and Coriobacteriaceae were increased in cancer patients. Coupled with previous reports, these results suggest that the intestinal microbiota is associated with CRC risk and that intestinal lumen microflora potentially influence CRC risk via cometabolism or metabolic exchange with the host. However, mucosa-associated microbiota potentially affects CRC risk primarily through direct interaction with the host

IMSA: Integrated metagenomic sequence analysis for identification of exogenous reads in a host genomic background

Metagenomics, the study of microbial genomes within diverse environments, is a rapidly developing field. The identification of microbial sequences within a host organism enables the study of human intestinal, respiratory, and skin microbiota, and has allowed the identification of novel viruses in diseases such as Merkel cell carcinoma. There are few publicly available tools for metagenomic high throughput sequence analysis. We present Integrated Metagenomic Sequence Analysis (IMSA), a flexible, fast, and robust computational analysis pipeline that is available for public use. IMSA takes input sequence from high throughput datasets and uses a user-defined host database to filter out host sequence. IMSA then aligns the filtered reads to a user-defined universal database to characterize exogenous reads within the host background. IMSA assigns a score to each node of the taxonomy based on read frequency, and can output this as a taxonomy report suitable for cluster analysis or as a taxonomy map (TaxMap). IMSA also outputs the specific sequence reads assigned to a taxon of interest for downstream analysis. We demonstrate the use of IMSA to detect pathogens and normal flora within sequence data from a primary human cervical cancer carrying HPV16, a primary human cutaneous squamous cell carcinoma carrying HPV 16, the CaSki cell line carrying HPV16, and the HeLa cell line carrying HPV18