Background We conducted a single-arm interventional study, to explore mucosal changes associated with clinical remission under oral vancomycin (OV) treatment, in primary sclerosing cholangitis-associated inflammatory bowel disease (PSC-IBD); NCT05376228.Methods Fifteen patients with PSC and active colitis (median fecal calprotectin 459 mu g/g; median total Mayo score 5) were treated with OV (125 mg QID) for 4 weeks and followed-up for a further 4 weeks of treatment withdrawal (8 weeks, end-of-study). Colonic biopsies were obtained at baseline and Week 4. Clinical assessments, and serum and stool samples (metagenomics, metatranscriptomics, and metabolomics) were collected at Weeks 0, 2, 4, and 8. The primary efficacy outcome measure was the induction of clinical remission.Results Oral vancomycin resulted in clinical remission in 12/15 patients and significant reductions in fecal calprotectin. Oral vancomycin was associated with reduced abundances of Lachnospiraceae, genera Blautia and Bacteroides; and enrichment of Enterobacteriaceae, and genera Veillonella, Akkermansia, and Escherichia. Oral vancomycin treatment was associated with the downregulation of multiple metatranscriptomic pathways (including short-chain fatty acid [SCFA] metabolism and bile acid [BA] biotransformation), along with host genes and multiple pathways involved in inflammatory responses and antimicrobial defence; and an upregulation of genes associated with extracellular matrix repair. Oral vancomycin use resulted in the loss of specific fecal SCFAs and secondary BAs, including lithocholic acid derivatives. Colitis activity relapsed following OV withdrawal, with host mucosal and microbial changes trending toward baseline.Conclusions Four weeks of OV induces remission in PSC-IBD activity, associated with a reduction in gut bacterial diversity and compositional changes relating to BA and SCFA homeostasis.

The gut microbiota is a reservoir for antimicrobial resistance genes (ARGs). With current sequencing methods, it is difficult to assign ARGs to their microbial hosts, particularly if these ARGs are located on plasmids. Metagenomic chromosome conformation capture approaches (meta3C and Hi-C) have recently been developed to link bacterial genes to phylogenetic markers, thus potentially allowing the assignment of ARGs to their hosts on a microbiome-wide scale. Here, we generated a meta3C dataset of a human stool sample and used previously published meta3C and Hi-C datasets to investigate bacterial hosts of ARGs in the human gut microbiome. Sequence reads mapping to repetitive elements were found to cause problematic noise in, and may importantly skew interpretation of, meta3C and Hi-C data. We provide a strategy to improve the signal-to-noise ratio by discarding reads that map to insertion sequence elements and to the end of contigs. We also show the importance of using spike-in controls to quantify whether the cross-linking step in meta3C and Hi-C protocols has been successful. After filtering to remove artefactual links, 87 ARGs were assigned to their bacterial hosts across all datasets, including 27 ARGs in the meta3C dataset we generated. We show that commensal gut bacteria are an important reservoir for ARGs, with genes coding for aminoglycoside and tetracycline resistance being widespread in anaerobic commensals of the human gut.

Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a “difficult” to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.

*Correspondence - Saaeha Rauz: s.rauz@bham.ac.uk OBJECTIVES: Mucous Membrane Pemphigoid is an orphan multi-system autoimmune scarring disease involving mucosal sites, including the ocular surface (OcMMP) and gut. The gut microbiome plays a critical role in the development of the immune system. This study examines the relationship between gut microbiome diversity and ocular inflammation in patients with OcMMP. METHODS AND ANALYSIS Gut microbiome profiles between OcMMP patients (n=49) and healthy controls (n=40) were compared by extracting DNA from faecal samples and amplified for the V4 region of the 16S rRNA gene followed by Illumina Miseq platform sequencing. Sequencing reads were processed using the bioinformatics pipeline available in the mothur v.1.44.1 software. RESULTS Using multivariable model and adjustment for participant factors, OcMMP cohort was found to be associated with lower number of operational taxonomic units (OTUs) and Shannon Diversity Index when compared to healthy controls. OcMMP OTUs were found to be significantly correlated with both the bulbar conjunctival inflammation score (p=0.03) and the current use of systemic immunotherapy (p=0.02). Linear discriminant analysis effect size scores found Streptococcus and Lachnoclostridium enriched in OcMMP. By contrast, healthy controls were enriched with Oxalobacter, Clostridia uncultured genus-level group (UCG) 014, Christensenellaceae R-7 group and butyrate-producing bacteria such as Ruminococcus, Lachnospiraceae, Coprococcus, Roseburia, Oscillospiraceae UCG 003, 005, NK4A214 group (Log10 LDA score <2, FDR-adjusted p<0.05). CONCLUSION In conclusion, OcMMP patients have gut dysbiosis that correlated with bulbar conjunctival inflammation and the use of systemic immunotherapies. This provides a framework for future longitudinal deep phenotyping studies on the role of the gut microbiome in the pathogenesis of OcMMP. REFERENCE Low L, Suleiman K, Shamdas M, Bassilious K, Poonit N, Rossiter AE, Acharjee A, Loman N, Murray PI, Wallace GR, Rauz S. Gut dysbiosis in ocular mucous membrane pemphigoid. Front. Cell. Infect. Microbiol. 2022;12:780354. https://doi.org/10.3389/fcimb.2022.780354.

Introduction Gut microbiome disruption and 'dysbiosis' is an established characteristic of IBD. The lack of treatment naïve and prospective longitudinal microbiome data makes it challenging to separate the influence of potential confounders. We present our preliminary microbiota analysis from a treatment naïve cohort of IBD patients seen in our rapid-access integrated clinical and research IBD inception clinic. Methods Stool collected in DNA Genotek OM-200 kits is brought to the first face to face pre-diagnosis outpatient review, alongside faecal calprotectin (FCAL). Metadata is collected prospectively with repeat samples collected longitudinally. Healthy controls were recruited separately. Stool microbial DNA was extracted with subsequent 16S rRNA PCR and sequencing. Diversity analysis and taxonomic classifications were performed in line with the QIIME2 workflow. Taxa with linear discriminant analysis (LDA) >2 at a P<0.05 were considered significant on LDA effect size (LEfSe) analysis. Correlation was evaluated with Kendall's Tau coefficient. Results Across sites, our clinics have seen 292 patients, with 153 enrolled in active research. We have banked 107 pre-treatment OM-200 stool samples from this cohort. of the first 49 samples sequenced, 31 were diagnosed with IBD (UC=18; CD=13). An independent cohort of 18 healthy controls were used for comparison. Median FCAL in the IBD cohort was 915ug/g (IQR 1499ug/g). Microbial composition in the IBD patients as assessed by Bray-Curtis distance matrix was significantly different compared to healthy controls (P<0.001). Alpha diversity (Shannon diversity index) was significantly lower in the IBD cohort when compared to healthy controls (P=0.01). These differences in alpha and beta diversity were also observed with IBD subtypes (P<0.01). Patients with IBD had significantly lower relative abundance of multiple taxa belonging to the families Clostridiaceae, Lachnospiraceae, and Coriobacteriaceae and genus Ruminococcus, Butyricicoccus, Bifidobacterium, Blautia and Dorea, as displayed in figure 1 The genuses Fusobacterium, Peptostreptococcus and Subdoligranulum positively correlated with FCAL levels whilst the genus Lachnobacterium negatively correlated (both P<0.05). Conclusions We present our preliminary index analysis of microbiome data in a unique treatment naïve IBD cohort. Our ongoing prospective longitudinal collection of clinical, microbial, immunological and metabolomic datasets will facilitate understanding of potential baseline prognostic indicators, predictive biomarkers for treatment response and relationship with disease activity.

Background Microbial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis. Methods Using full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix™ SK-2S, Sugi® Eyespear, Cotton, Rayon, Dryswab™, Hydraflock®, Albumin-coated, Purflock®, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods. Results We found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (p < 0.001), with the Sugi® Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results. Conclusion We have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing.

The BAM complex in Escherichia coli is composed of five proteins, BamA-E. BamA and BamD are essential for cell viability and are required for the assembly of β-barrel outer membrane proteins. Consequently, BamA and BamD are indispensable for secretion via the classical autotransporter pathway (Type 5a secretion). In contrast, BamB, BamC and BamE are not required for the biogenesis of classical autotransporters. Recently, we demonstrated that TamA, a homologue of BamA, and its partner protein TamB, were required for efficient secretion of proteins via the classical autotransporter pathway. The trimeric autotransporters are a subset of the Type 5-secreted proteins. Unlike the classical autotransporters, they are composed of three identical polypeptide chains which must be assembled together to allow secretion of their cognate passenger domains. In contrast to the classical autotransporters, the role of the Bam and Tam complex components in the biogenesis of the trimeric autotransporters has not been investigated fully. Here, using the Salmonella enterica trimeric autotransporter SadA and the structurally similar YadA protein of Yersinia spp., we identify the importance of BamA and BamD in the biogenesis of the trimeric autotransporters and reveal that BamB, BamC, BamE, TamA and TamB are not required for secretion of functional passenger domain on the cell surface. Importance The secretion of trimeric autotransporters (TAA’s) has yet to be fully understood. Here we show that efficient secretion of TAAs requires the BamA and D proteins, but does not require BamB, C or E. In contrast to classical autotransporter secretion, neither trimeric autotransporter tested required TamA or B proteins to be functionally secreted.

Mucous Membrane Pemphigoid is an orphan multi-system autoimmune scarring disease involving mucosal sites, including the ocular surface (OcMMP) and gut. Loss of tolerance to epithelial basement membrane proteins and generation of autoreactive T cell and/or autoantibodies are central to the disease process. The gut microbiome plays a critical role in the development of the immune system. Alteration in the gut microbiome (gut dysbiosis) affects the generation of autoreactive T cells and B cell autoantibody repertoire in several autoimmune conditions. This study examines the relationship between gut microbiome diversity and ocular inflammation in patients with OcMMP by comparing OcMMP gut microbiome profiles with healthy controls. DNA was extracted from faecal samples (49 OcMMP patients, 40 healthy controls), amplified for the V4 region of the 16S rRNA gene and sequenced using Illumina Miseq platform. Sequencing reads were processed using the bioinformatics pipeline available in the mothur v.1.44.1 software. After adjusting for participant factors in the multivariable model (age, gender, BMI, diet, proton pump inhibitor use), OcMMP cohort was found to be associated with lower number of operational taxonomic units (OTUs) and Shannon Diversity Index when compared to healthy controls. Within the OcMMP cohort, the number of OTUs were found to be significantly correlated with both the bulbar conjunctival inflammation score (p=0.03) and the current use of systemic immunotherapy (p=0.02). The linear discriminant analysis effect size scores indicated that Streptococcus and Lachnoclostridium were enriched in OcMMP patients whilst Oxalobacter, Clostridia uncultured genus-level group (UCG) 014, Christensenellaceae R-7 group and butyrate-producing bacteria such as Ruminococcus, Lachnospiraceae, Coprococcus, Roseburia, Oscillospiraceae UCG 003, 005, NK4A214 group were enriched in healthy controls (Log10 LDA score < 2, FDR-adjusted p <0.05). In conclusion, OcMMP patients have gut dysbiosis correlating with bulbar conjunctival inflammation and the use of systemic immunotherapies. This provides a framework for future longitudinal deep phenotyping studies on the role of the gut microbiome in the pathogenesis of OcMMP.

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S . Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.

The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for Escherichia coli DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

Clearance of intracellular infections caused by Salmonella Typhimurium (STm) requires IFN-γ and the Th1-associated transcription factor T-bet. Nevertheless, whereas IFN-γ-/- mice succumb rapidly to STm infections, T-bet-/- mice do not. In this study, we assess the anatomy of immune responses and the relationship with bacterial localization in the spleens and livers of STm-infected IFN-γ-/- and T-bet-/- mice. In IFN-γ-/- mice, there is deficient granuloma formation and inducible NO synthase (iNOS) induction, increased dissemination of bacteria throughout the organs, and rapid death. The provision of a source of IFN-γ reverses this, coincident with subsequent granuloma formation and substantially extends survival when compared with mice deficient in all sources of IFN-γ. T-bet-/- mice induce significant levels of IFN-γ- after challenge. Moreover, T-bet-/- mice have augmented IL-17 and neutrophil numbers, and neutralizing IL-17 reduces the neutrophilia but does not affect numbers of bacteria detected. Surprisingly, T-bet-/- mice exhibit surprisingly wild-type-like immune cell organization postinfection, including extensive iNOS+ granuloma formation. In wild-type mice, most bacteria are within iNOS+ granulomas, but in T-bet-/- mice, most bacteria are outside these sites. Therefore, Th1 cells act to restrict bacteria within IFN-γ-dependent iNOS+ granulomas and prevent dissemination.

In recent years, multiple studies have examined the bacterial communities present in the gastric microbiota during the progression to gastric cancer (GC). Although Helicobacter pylori is the biggest risk factor for GC, the microbiota of GC comprises a decreased load of H. pylori and an enrichment of bacteria, such as Prevotella spp., Veillonella spp., Actinomyces spp. However, interactions between H. pylori and these cancer-associated bacteria is hugely understudied. Here, we have used hypoxic growth conditions (5 % O2) to investigate polymicrobial interactions between cancer-associated bacteria and H. pylori in vitro. We found that whilst the co-culture of H. pylori with Prevotella spp. and Veillonella spp. had no effect on growth of either bacteria, Actinomyces oris completely inhibited the growth of H. pylori. Moreover, A. oris did not inhibit the growth of other Gram-negative pathogens such as Salmonella Typhimurium and E. coli, whilst there was a slight growth inhibition of Campylobacter jejuni. Furthermore, ultrafiltration of A. oris culture supernatants revealed that inhibition is mediated by a secreted factor larger than 5 kDa, which can be heat inactivated. Interestingly, Actinomyces viscosus can also specifically kill H. pylori suggesting that this inhibition could be conserved across the Actinomyces genus. We are currently identifying the inhibitory factor responsible for inhibiting H. pylori growth. Furthermore, we are investigating whether A. oris can clear gastric H. pylori infection in a mouse model of infection and the implications of this on gastric carcinogenesis. In conclusion, whilst data-rich microbiota studies continue to thrive, it is imperative that we understand the mechanisms underpinning changes to the gastric microbiota and whether these bacteria are drivers or ‘passengers’ of gastric carcinogenesis.

Active efflux due to tripartite RND efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. These pumps are also essential for Gram-negative pathogens to cause infection and form biofilms. They consist of an inner membrane RND transporter; a periplasmic adaptor protein (PAP), and an outer membrane channel. The role of PAPs in assembly, and the identities of specific residues involved in PAP-RND binding, remain poorly understood. Using recent high-resolution structures, four 3D sites involved in PAP-RND binding within each PAP protomer were defined that correspond to nine discrete linear binding sequences or "binding boxes" within the PAP sequence. In the important human pathogen Salmonella enterica, these binding boxes are conserved within phylogenetically-related PAPs, such as AcrA and AcrE, while differing considerably between divergent PAPs such as MdsA and MdtA, despite overall conservation of the PAP structure. By analysing these binding sequences we created a predictive model of PAP-RND interaction, which suggested the determinants that may allow promiscuity between certain PAPs, but discrimination of others. We corroborated these predictions using direct phenotypic data, confirming that only AcrA and AcrE, but not MdtA or MsdA, can function with the major RND pump AcrB. Furthermore, we provide functional validation of the involvement of the binding boxes by disruptive site-directed mutagenesis. These results directly link sequence conservation within identified PAP binding sites with functional data providing mechanistic explanation for assembly of clinically relevant RND-pumps and explain how Salmonella and other pathogens maintain a degree of redundancy in efflux mediated resistance. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a predictive model of pump-contacts that could be exploited in the future for the development of targeted therapeutics and efflux pump inhibitors.

Helicobacter pylori represents an interesting model of bacterial pathogenesis given that most infections are asymptomatic, while a minority of infections cause severe gastric disease. H pylori strain B128 7.13 is used extensively to understand H pylori pathophysiology. Due to extensive restriction‐modification systems, the fact that only some H pylori strains are naturally transformable, the inability of common plasmid and transposon vectors to replicate in this bacterium, as well as the limited number of antibiotic cassettes that are functional in H pylori, there are relatively few genetic tools for the mutagenesis of this bacterium.

Antigen 43 (Ag43) is a cell-surface exposed protein of Escherichia coli secreted by the Type V, subtype a, secretion system (T5aSS) and belonging to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. A phylogenetic network analysis focusing on the passenger revealed for the first time that they actually distribute into four distinct classes, namely C1, C2, C3 and C4. Structural alignment and modelling analyses demonstrated these classes arose from shuffling of two different subdomains within the Ag43 passengers. Functional analyses revealed that homotypic interactions occur for all Ag43 classes but significant differences in the sedimentation kinetics and aggregation state were present when Ag43C3 was expressed. In contrast, heterotypic interaction occurred in a very limited number of cases. Single cell-force spectroscopy demonstrated the importance of specific as well as nonspecific interactions in mediating Ag43-Ag43 recognition. We propose that structural differences in the subdomains of the Ag43 classes account for different autoaggregation dynamics and propensities to co-interact.

Introduction Primary sclerosing cholangitis (PSC) is an idiopathic chronic cholestatic liver disease associated with ulcerative colitis (UC). PSC is thought to be a consequence of a genetically predisposition, dysregulated immune response and unknown factors including the gut microbiome. The colonic mucosal immune response in PSC associated colitis (PSC-UC), however, has been poorly defined. In this study, we analysed the characteristics of colonic mucosal CD4 T cells in patients with PSC-UC. Methods Colon biopsies were collected from patients with PSC-UC (n=13), UC (n=10) and controls (n=20). One patient with PSC-UC and one patient with UC was on biologics. Three patients with PSC and three with UC had colonic inflammation. Lamina propria mononuclear cells were analysed by flow cytometry. Results PSC-UC and UC were characterised by a significantly higher frequency of colonic mucosal CCR6 +CD161+Th17 cells compared to controls (17.5% vs 11.1%; p=0.009% and 21.02% vs 11.1%; p=0.01 respectively). CCR6-CXCR3+CCR5+Th1 cells were significantly lower in PSC-UC compared to controls (15.46% vs 24.50% respectively; p 0.01). CD127-CD25+FoxP3+T regulatory cell frequencies was elevated and CCR6-CCR5-CXCR3- Th2 frequencies were reduced only in UC compared to controls (7.6% vs 4.38%; p=0.007% and 14.84% vs 8.77%; p=0.02 respectively). Significantly increased frequencies of IL17 producing CD4 cells were observed in both PSC-UC and UC compared to controls (7.75% vs 4.7%; p<0.001% and 7.251% vs 4.70%; p=0.006 respectively). Although there were no differences in TNFα and IFNγ producing CD4 cells, patients with PSC-UC had a significantly higher frequency of IL17/IFNγ dual producing CD4 cells compared to controls (2.79% vs 1.76% respectively; p=0.03). Correlation analysis of PSC-UC and controls demonstrated that Th17 frequencies positively correlated with increasing frequencies of IL17 producing cells and negatively with Th1 (p<0.05). Conclusions Our study demonstrates for the first time that the colonic mucosal immune response in PSC-UC is characterised by significantly higher Th17 cells and lower Th1 cells compared to controls. Patients with PSC-UC have higher IL-17 and IL17/IFNγ dual producing CD4 cells. Our findings highlight the need to explore the role of key players such as the gut microbiome in mucosal T cell homeostasis and Th1/Th17 plasticity in PSC.

MCE domains were first reported in Mycobacteria as having a role in Mammalian Cell Entry, with subsequent studies showing their importance during infection. Here, we have examined the function of MCE proteins in Salmonella Typhimurium during mammalian infection. We report that MCE proteins are required for Salmonella virulence, but that this is not related to decreased adherence, entry or survival in mammalian cells. Instead, we reveal that MCE proteins are required for Salmonella bile resistance, in particular to withstand bile salts such as cholate and deoxycholate. Based on our previous work in Escherichia coli, and other studies that have reported roles for MCE proteins in membrane biogenesis, we propose that Salmonella lacking MCE domains have a defective outer membrane that results in bile sensitivity and decreased virulence in vivo. These results suggest that MCE domains mediate fundamental aspects of bacterial membrane physiology as opposed to a proposed direct role in mammalian cell entry, explaining their conservation across both pathogenic and non-pathogenic bacteria.

Introduction Primary sclerosing cholangitis is a hepatobiliary disorder of unknown aetiology that is commonly associated with ulcerative colitis (PSC-UC). We have previously demonstrated that patients with PSC-UC have significant alterations in their gut microbiota compared to patients with UC alone and healthy controls. The mechanisms by which these alterations influence pathogenesis is unclear. Methods Shotgun sequencing was performed on whole transcriptomic libraries of colon biopsies obtained from 10 patients with PSC-UC and 10 healthy controls. Differential gene expression analysis with false discovery rate correction was performed with limma and edgeR. In addition lamina propria mononuclear cells were analysed by multicolour flow cytometry. Results The colonic transcriptome of patients with PSC-UC demonstrated 6774 significantly differentially expressed genes compared to healthy controls. Gene expression in PSC-UC clustered separate to healthy controls on multidimensional scaling (p=0.006). Gene enrichment analysis revealed 280 significantly altered biological processes. PSC-UC was characterised by an overexpression of processes associated with regulation of anti-microbial immune responses compared to healthy controls (p<0.0001). These immunological responses were enriched with pathways associated with innate, cell-mediated and humoral immunity, autophagy, complement activation and chemokine signalling (p<0.0001). Expression of genes associated with microbial handling including defensins, mannose receptors and anti-microbial peptides were significantly upregulated in PSC-UC. Paired CD4 T cell immunophenotyping of colon biopsies revealed a significant increase in the population of Th17 cells and IL-17 producing cells in PSC-UC in comparison to healthy controls (18.6% vs 8.5%; p=0.0007 8.5% vs 5.6%; p=0.005 respectively). Conclusion We have for the first time demonstrated the colonic mucosal transcriptional response in PSC-UC. In comparison to healthy controls, patients with PSC-UC have highly significant expression of genes associated with over-representation of multiple immunological processes, many of which are directed against bacteria. Consistently patients with PSC-UC have a significant increase in colonic mucosal Th17 and IL-17 cell population compared to healthy controls.

Introduction Epidemiological studies have highlighted that the South Asian migrant population in UK have a comparable risk of developing IBD but with a more aggressive phenotype than the white Caucasian population. It remains unclear if this is due to environmental/lifestyle factors or differences in host genetics. The human gut microbiota is impacted by health status and diet and therefore represents a potentially adaptive phenotype that is influenced by the environment. As the gut microbiota has been shown to be different in the native South Asian population compared to those in developed countries, we aimed to investigate if there were ethnic differences in the microbiota in IBD patients. Methods Stool samples were collected from South Asian (n=20) and Caucasian patients (n=46) with IBD attending outpatient clinics at University Hospital Birmingham along with healthy controls (n=17). DNA was extracted and the V4 hyper-variable region of the 16S rRNA gene amplified and sequenced. Analysis was performed on the QIIME pipeline using the GreenGenes database. Diversity analysis was corrected for false discovery rates. Result Patients with IBD had a significantly different gut microbial composition in comparison to healthy controls as expected (p=0.01). Gut microbial diversity was reduced in IBD (p=0.001). A significant decrease in Firmicutes phylum was observed in patients with IBD in comparison to healthy controls which was primarily due a reduced abundance of communities from genus Faecalibacterium praunitizii, Lachnospiracae, Ruminococcus and Blautia (p<0.002). Within the IBD cohort, the alpha or beta diversity of gut microbiota was very similar for South Asians and Caucasian patients. No significant differences were seen at any taxonomic levels. Published literature have previously demonstrated that the gut microbiota in healthy South Asians living in the subcontinent was enriched with populations of Lactobacillus, Ruminococcus, and Bifidobacterium bacteria. In our South Asian IBD cohort these microbial communities were significantly reduced. Conclusion This is the first study to date investigating the gut microbial composition in the migrant population with IBD in UK. The gut microbiota in South Asian IBD patients is similar to Caucasian IBD patients living in UK. Our findings suggest the need to explore further the role environmental factors in the development of IBD associated dysbiosis but also the role of host factors in pathogenesis.