Cellular and Molecular Gastroenterology and Hepatology, 2021
The incidence of inflammatory bowel diseases is increasing worldwide, suggesting a potential role... more The incidence of inflammatory bowel diseases is increasing worldwide, suggesting a potential role for dietary factors. Here, we demonstrate that a high fructose diet worsens colitis in a microbiota-dependent manner. Overall, this study highlights the importance of diet-microbe interactions in intestinal inflammation. BACKGROUND & AIMS: The incidence of inflammatory bowel diseases has increased over the last half century, suggesting a role for dietary factors. Fructose consumption has increased in recent years. Recently, a high fructose diet (HFrD) was shown to enhance dextran sodium sulfate (DSS)-induced colitis in mice. The primary objectives of the current study were to elucidate the mechanism(s) underlying the pro-colitic effects of dietary fructose and to determine whether this effect occurs in both microbially driven and genetic models of colitis. METHODS: Antibiotics and germ-free mice were used to determine the relevance of microbes for HFrD-induced worsening of colitis. Mucus thickness and quality were determined by histologic analyses. 16S rRNA profiling, in situ hybridization, metatranscriptomic analyses, and fecal metabolomics were used to determine microbial composition, spatial distribution, and metabolism. The significance of HFrD on pathogen and genetic-driven models of colitis was determined by using Citrobacter rodentium infection and Il10-/mice, respectively. RESULTS: Reducing or eliminating bacteria attenuated HFrDmediated worsening of DSS-induced colitis. HFrD feeding enhanced access of gut luminal microbes to the colonic mucosa by reducing thickness and altering the quality of colonic mucus. Feeding an HFrD also altered gut microbial populations and metabolism including reduced protective commensal and bile salt hydrolase-expressing microbes and increased luminal conjugated bile acids. Administration of conjugated bile acids to mice worsened DSS-induced colitis. The HFrD also worsened colitis in Il10-/mice and mice infected with C rodentium. CONCLUSIONS: Excess dietary fructose consumption has a procolitic effect that can be explained by changes in the composition, distribution, and metabolic function of resident enteric microbiota.
Short chain fatty acids (SCFA), principally acetate, propionate, and butyrate, are produced by fe... more Short chain fatty acids (SCFA), principally acetate, propionate, and butyrate, are produced by fermentation of dietary fibers by the gut microbiota. SCFA regulate the growth and virulence of enteric pathogens, such as enterohemorrhagic E. coli (EHEC), Klebsiella and Salmonella. We sought to investigate the impact of SCFA on growth and virulence of pathosymbiont E. coli associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC), and their role in regulating host responses to bacterial infection in vitro. We found that under ileal conditions (pH = 7.4; 12 mM total SCFA), SCFA significantly (p < 0.05) potentiate the growth and motility of pathosymbiont E. coli. However, under colonic conditions (pH = 6.5; 65 to 123 mM total SCFA), SCFA significantly (p < 0.05) inhibit growth in a pH dependent fashion (up to 60%), and down-regulate virulence gene expression (e.g., fliC, fimH, htrA, chuA, pks). Functional analysis reveals that colonic SCFA significantly (p < 0....
Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IB... more Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IBD), especially Crohn’s disease, and colorectal cancer. AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by in vitro functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated E. coli in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohn’s disease and non-Crohn’s tissues. We show these strains can colonize the intestine of IBD susceptible mice ...
independent techniques to evaluate the microbial composition of pouch and afferent limb tissue bi... more independent techniques to evaluate the microbial composition of pouch and afferent limb tissue biopsies from patients with variable pouch outcomes. Methods: Patients with confirmed UC or familial adenomatous polyposis(FAP) who had a colectomy and IPAA, with ileostomy closure ≥1 year prior to study enrollment were recruited from Mount Sinai Hospital at the time of follow-up endoscopy. Retrospective and cross-sectional clinical/endoscopic information was collected at the time of enrollment and patients were classified into 4 groups: FAP/ no pouchitis(FAP), UC/no pouchitis(NP), UC/pouchitis(P) andUC/CD-like phenotype(CDL). P was defined based on the mPDAI. CDL diagnosis was based on presence of afferent limb inflammation or fistula. 2 biopsies were collected and snap-frozen in liquid nitrogen (1 afferent limb and 1 pouch). Microbial DNA was extracted and sequenced using the 454 GS FLX Titanium Sequencing system using primers specific for the V1-V3 and V2-V3 hypervariable regions of 16S rRNA genes in 2 batches. Sequences were quality trimmed and aligned to a SILVA reference database using mothur, with a minimum bootstrap cutoff of 80%. A threshold level of 0.05 was used to assign sequence clusters to a specific genus and patients were considered positive for genera with counts >0. Dichotomized results were analyzed using Fisher's exact test and Bonferroni correction for multiple testing. Results: 245 patients were recruited to the study of whom 79 were selected for analysis with complete data available (18 FAP, 21 NP, 20 P, 20 CDL). Analysis at the genus level showed that Blautia was detected significantly less often in the P group in both afferent limb (p=0.002) and pouch samples (p=0.008) compared to the other groups. Bacteroides and Sutterella were significantly less frequent among P and CDL groups in the pouch samples only (p=0.001 and 0.01 respectively). There was no significant difference in the proportions of these genera between outcome groups in the different batches. Conclusion: Differences in microbial composition are associated with outcomes following IPAA. Specifically, the genera Blautia, Bacteroides and Sutterella were found less frequently in patients with P and CDL compared to those without pouchitis. Further study may allow us to identify the role these organisms play in pouch pathogenesis, and to identify interactions occurring between microorganisms and host endogenous elements such as genotype and development of anti-microbial antibodies.
Fibrosis is a significant complication of intestinal disorders associated with microbial dysbiosi... more Fibrosis is a significant complication of intestinal disorders associated with microbial dysbiosis and pathobiont expansion, notably Crohn’s disease (CD). Mechanisms that favor fibrosis are not well understood and therapeutic strategies are limited. Here we demonstrate that colitis susceptible Il10-deficient mice develop inflammation-associated fibrosis when mono-associated with adherent/invasive Escherichia coli (AIEC) that harbor the yersiniabactin (Ybt) pathogenicity island. Inactivation of Ybt siderophore production in AIEC nearly abrogated fibrosis development in inflamed mice. In contrast, inactivation of Ybt import through its cognate receptor FyuA enhanced fibrosis severity. This corresponded with increased colonic expression of profibrogenic genes prior to the development of histological disease, therefore suggesting causality. FyuA-deficient AIEC also exhibited greater localization within sub-epithelial tissues and fibrotic lesions that was dependent on Ybt biosynthesis an...
5-aminosalicylate (5-ASA) is widely prescribed for the treatment of inflammatory bowel disease (I... more 5-aminosalicylate (5-ASA) is widely prescribed for the treatment of inflammatory bowel disease (IBD) and prevention of inflammation-associated colorectal cancer (CRC). Its clinical effect is widely attributed to modulation of host inflammatory responses. However, the recent association of intestinal dysbiosis and selective enrichment in Escherichia coli in patients with IBD and CRC raises the possibility that 5-ASA might also affect the enteric microflora. The aim of this study was to investigate the effect of 5-ASA on the growth and virulence of E. coli associated with IBD and CRC, and its impact on host cell inflammatory responses. Our results show that 5-ASA inhibited E. coli growth in a dose-dependent manner and downregulated the expression of bacterial virulence genes associated with IBD (fliC, fimH, ompC, yfgL, nlpL, lpfA, htrA, dsbA, fyuA, and chuA) and CRC (pks). 5-ASA inhibited E. coli motility (30-70%), epithelial adherence and invasion, and IL-8 secretion (p < 0.05). 5-ASA reduced E. coli survival in J774A.1 macrophages by 20 to 50% (p < 0.01) and TNF-α secretion by infected macrophages up to 30% (p < 0.05). In addition, 5-ASA reduced DNA damage in epithelial cells (Caco-2) induced by pks-positive E. coli. Our results reveal a multifaceted and previously unrecognized effect of 5-ASA on the growth and virulence of IBD-and CRC-associated E. coli, in addition to its inhibitory effect on host cell inflammatory responses. These results suggest that 5-ASA may abrogate the proinflammatory and oncogenic effects of E. coli in patients with IBD and CRC.
Streptococcus uberis is an important environmental pathogen associated with bovine mastitis as we... more Streptococcus uberis is an important environmental pathogen associated with bovine mastitis as well as with high total bacterial numbers in bulk tank milk. This study was conducted to determine whether S. uberis reproduction is likely to contribute to high bacterial numbers in bulk tank milk. Four S. uberis raw milk isolates were individually inoculated into UHT-treated milk and incubated at 4.4 or 7°C for up to 5 d to simulate appropriate cooling; at 10°C for 5 d to simulate marginally inadequate cooling; at 21 or 25°C for 7 h to simulate ambient temperatures; or at 32°C for 7 h to simulate elevated temperature conditions. None of the S. uberis isolates grew at either 4.4 or 7°C. Streptococcus uberis growth at 10°C appeared to be ribotype-specific. Although ribotype 116-520-S-1 isolates did not grow at 10°C, ribotype 116-520-S-2 isolate numbers increased up to 3.5 log 10 cfu/mL within 5 d. Generation times were calculated as 2.7 ± 0.1 h, 2.1 ± 0.1 h, and 1.0 ± 0.1 h for 116-520-S-1 isolates and 1.8 ± 0.4 h, 1.3 ± 0.3 h, and 0.8 ± 0.1 h for 116-520-S-2 isolates at 21, 25, and 32°C, respectively. Our results suggest that high numbers of S. uberis in bulk tank milk are more likely to reflect high numbers of S. uberis shed by mastitic cows, rather than multiplication of these organisms under cooling conditions required for production of Grade A milk.
Irritable bowel syndrome (IBS) affects 10%-20% of people. Increased numbers of Escherichia coli (... more Irritable bowel syndrome (IBS) affects 10%-20% of people. Increased numbers of Escherichia coli (E. coli) correlate with symptoms, and patients respond to antimicrobials targeting E. coli. We examined whether specific E. coli strains, phylogroups and pathotypes are associated with IBS. We evaluated 218 E. coli isolates from 33 IBS patients and 23 healthy controls. RAPD analysis revealed 89 E. coli strains (29 controls, 60 IBS), spanning the A, B1, B2 and D phylogroups. Strains were similarly enriched in virulence genes associated with extraintestinal pathogenic E. coli (ExPEC) and/or adherent-invasive E. coli (AIEC). Three strains harbored a diarrheagenic virulence gene (2 IBS, 1 control). Escherichia coli capable of invading epithelial cells or replicating in macrophages were detected in 53% of IBS and 50% controls, and 67% IBS and 45% controls respectively (P > 0.05). AIEC were identified in 33% of IBS patients vs 20% of controls (P = 0.35). Virulence genes ibeA, ColV and pduC ...
Perturbation of the microbiome is implicated in the pathogenesis of many human ailments, includin... more Perturbation of the microbiome is implicated in the pathogenesis of many human ailments, including inflammatory bowel diseases such as Crohn's disease (CD). Recapitulating the microbiome associated with health and disease necessitates controlling the composition of multiple bacterial species. This is difficult to achieve in vitro due to the overgrowth of bacterial species over time. Here, a microfluidic‐based model incorporating bacteria‐embedded hydrogel microfibers for the coculture of human enteric bacteria is introduced. Employing bacterial species and strains associated with CD, it is shown that the hydrogel‐based bacteria‐embedded microfiber model is physically and mechanically robust, and tunable. Metabolite analysis of the medium in both mono‐ and coculture revealed the interfiber exchange of soluble mediators and their impact on the growth of different bacterial species. This novel approach should enhance the ability to decipher contact‐independent cross‐talk within the polymicrobial intestinal luminal environment, and its impact on the intestinal epithelium.
mucosal surface of the Sigirr-/mouse. While p19A WT infection caused only minimal pathology on it... more mucosal surface of the Sigirr-/mouse. While p19A WT infection caused only minimal pathology on its own, it dramatically worsened the course of DSS colitis, in concert with deep penetration of the damaged colonic mucosa. Notably, intestinal tissue histology shows that the p19A hemolysin deletion mutant was severely attenuated in its ability to promote DSS colitis in Sigirr-/mice. Conclusion Our findings provide evidence that UC associated phylogroup B2 E. coli strains p19A WT can readily and persistently colonize the intestines of susceptible hosts, and significantly worsen the course of colitis. The intestinal tissue damages in DSS colitis Sigirr-/mice colonized with p19A hemolysin deletion mutant were severely attenuated, which indicates that p19A E. coli harbouring two alpha-hemolysin gene, might be responsible for barrier dysfunction and intestinal tissue damages in susceptible hosts. This model thus facilitates research into the role played by UC associated E. coli pathobionts in colitis pathogenesis.
Peripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with act... more Peripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with active inflammatory bowel disease (IBD) characterized by inflammatory enthesitis, dactylitis, or synovitis of nonaxial joints. However, a mechanistic understanding of the link between intestinal inflammation and SpA has yet to emerge. We evaluated and functionally characterized the fecal microbiome of IBD patients with or without peripheral SpA. Coupling the sorting of immunoglobulin A (IgA)-coated microbiota with 16S ribosomal RNA-based analysis (IgA-seq) revealed a selective enrichment in IgA-coated Escherichia coli in patients with Crohn's disease-associated SpA (CD-SpA) compared to CD alone. E. coli isolates from CD-SpA-derived IgA-coated bacteria were similar in genotype and phenotype to an adherent-invasive E. coli (AIEC) pathotype. In comparison to non-AIEC E. coli, colonization of germ-free mice with CD-SpA E. coli isolates induced T helper 17 cell (TH17) mucosal immunity, which...
The use of point-of-care (POC) devices in limited resource settings where access to commonly used... more The use of point-of-care (POC) devices in limited resource settings where access to commonly used infrastructure, such as water and electricity, can be restricted represents simultaneously one of the best application fits for POC systems as well as one of the most challenging places to deploy them. Of the many challenges involved in these systems, the preparation and processing of complex samples like stool, vomit, and biopsies are particularly difficult due to the high number and varied nature of mechanical and chemical interferents present in the sample. Previously we have demonstrated the ability to use solar-thermal energy to perform PCR based nucleic acid amplifications. In this work demonstrate how the technique, using similar infrastructure, can also be used to perform solar-thermal based sample processing system for extracting and isolating Vibrio Cholerae nucleic acids from fecal samples. The use of opto-thermal energy enables the use of sunlight to drive thermal lysing rea...
Escherichia coli is an important cause of bovine mastitis and can cause both severe inflammation ... more Escherichia coli is an important cause of bovine mastitis and can cause both severe inflammation with a short-term transient infection, as well as less severe, but more chronic inflammation and infection persistence. E. coli is a highly diverse organism that has been classified into a number of different pathotypes or pathovars, and mammary pathogenic E. coli (MPEC) has been proposed as a new such pathotype. The purpose of this study was PLOS ONE |
Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection... more Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection. However, E. coli can also cause clonal persistent intramammary infection (IMI) in dairy cows. In this study, we explored the possibility that E. coli strains associated with persistent IMI are better able to adhere to, invade, survive and replicate in cultured mammary epithelial cells (MAC-T) than transient strains, and examined their serotype, overall genotype, phylogenetic group, and the presence of known virulence genes. Both transient and persistent E. coli strains adhered to MAC-T cells, but persistent strains invaded MAC-T cells 2.6-63.5 times more than transient strains. Blocking the adhesin/invasin FimH with mannose diminished but did not eliminate adhesion and invasion of any strain. Cytoskeletal and protein kinase inhibitors cytochalasin D, colchicine, genistein and wortmannin dramatically reduced invasion of MAC-T cells by both strains. All of the persistent strains, but only one transient strain, were able to survive and replicate intracellularly in MAC-T cells over 48 h. Transient and persistent strains displayed heterogeneous serotypes and overall genotypes, but similar phylogeny (group A), and lacked virulence genes of invasive E. coli. We have found that E. coli strains associated with persistent IMI are better able to invade and replicate within cultured mammary epithelial cells than transient strains. The invasion process involves the host cytoskeleton and signaling cascades and is not FimH dependent. Our findings suggest that the invasion of mammary epithelial cells and intracellular survival play an important role in the pathogenesis of persistent E. coli mastitis.
Bovine mastitis is the most economically important disease of the dairy industry and Escherichia ... more Bovine mastitis is the most economically important disease of the dairy industry and Escherichia coli is one of the most common causes of bovine mastitis in well managed dairies with low milk somatic cell counts (SCC) (Bradley, 2002). E. coli isolates associated with bovine mastitis are traditionally considered random environmental opportunistic pathogens. The observation that lipopolysaccharide (LPS) can induce a mammary immune response in the absence of live bacteria (Bannerman et al., 2003) supports the idea of strain variation having little effect on the severity of bovine clinical mastitis (Burvenich et al., 2003). However, live bacteria but not LPS, can induce mastitis in animals lacking the TLR4 ligand, that Veterinary Microbiology 159 (2012) 163-170
The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathoge... more The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathogenesis of inflammatory bowel disease. This study explored the possibility that an abnormal mucosal flora is involved in the etiopathogenesis of granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected dogs ( n = 13) and controls ( n = 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. GCB-associated Escherichia coli isolates were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages as well as for serotype, phylogenetic group, genome size, overall genotype, and presence of virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E. coli . Three of four GCB-associated E. coli isolates adh...
Objective-Mutations in nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) rema... more Objective-Mutations in nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) remain the strongest genetic determinants for Crohn's disease (CD). Having previously identified vimentin as a novel NOD2-interacting protein, we aimed to investigate the regulatory effects of vimentin on NOD2 function and the association of variants in Vim to CD susceptibility. Design-Co-immunoprecipitation, fluorescent microscopy and fractionation were used to confirm the interaction between NOD2 and vimentin. HEK293 cells stably expressing wild-type NOD2 or NOD2-frameshift variant (L1007fs) and SW480 colonic epithelial cells were used alongside the vimentin inhibitor Withaferin-A (WFA) to assess effects on NOD2 function using nuclear factor-kappaB (NF-κB) reporter gene, GFP-LC3-based autophagy, and bacterial
Cellular and Molecular Gastroenterology and Hepatology, 2021
The incidence of inflammatory bowel diseases is increasing worldwide, suggesting a potential role... more The incidence of inflammatory bowel diseases is increasing worldwide, suggesting a potential role for dietary factors. Here, we demonstrate that a high fructose diet worsens colitis in a microbiota-dependent manner. Overall, this study highlights the importance of diet-microbe interactions in intestinal inflammation. BACKGROUND & AIMS: The incidence of inflammatory bowel diseases has increased over the last half century, suggesting a role for dietary factors. Fructose consumption has increased in recent years. Recently, a high fructose diet (HFrD) was shown to enhance dextran sodium sulfate (DSS)-induced colitis in mice. The primary objectives of the current study were to elucidate the mechanism(s) underlying the pro-colitic effects of dietary fructose and to determine whether this effect occurs in both microbially driven and genetic models of colitis. METHODS: Antibiotics and germ-free mice were used to determine the relevance of microbes for HFrD-induced worsening of colitis. Mucus thickness and quality were determined by histologic analyses. 16S rRNA profiling, in situ hybridization, metatranscriptomic analyses, and fecal metabolomics were used to determine microbial composition, spatial distribution, and metabolism. The significance of HFrD on pathogen and genetic-driven models of colitis was determined by using Citrobacter rodentium infection and Il10-/mice, respectively. RESULTS: Reducing or eliminating bacteria attenuated HFrDmediated worsening of DSS-induced colitis. HFrD feeding enhanced access of gut luminal microbes to the colonic mucosa by reducing thickness and altering the quality of colonic mucus. Feeding an HFrD also altered gut microbial populations and metabolism including reduced protective commensal and bile salt hydrolase-expressing microbes and increased luminal conjugated bile acids. Administration of conjugated bile acids to mice worsened DSS-induced colitis. The HFrD also worsened colitis in Il10-/mice and mice infected with C rodentium. CONCLUSIONS: Excess dietary fructose consumption has a procolitic effect that can be explained by changes in the composition, distribution, and metabolic function of resident enteric microbiota.
Short chain fatty acids (SCFA), principally acetate, propionate, and butyrate, are produced by fe... more Short chain fatty acids (SCFA), principally acetate, propionate, and butyrate, are produced by fermentation of dietary fibers by the gut microbiota. SCFA regulate the growth and virulence of enteric pathogens, such as enterohemorrhagic E. coli (EHEC), Klebsiella and Salmonella. We sought to investigate the impact of SCFA on growth and virulence of pathosymbiont E. coli associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC), and their role in regulating host responses to bacterial infection in vitro. We found that under ileal conditions (pH = 7.4; 12 mM total SCFA), SCFA significantly (p < 0.05) potentiate the growth and motility of pathosymbiont E. coli. However, under colonic conditions (pH = 6.5; 65 to 123 mM total SCFA), SCFA significantly (p < 0.05) inhibit growth in a pH dependent fashion (up to 60%), and down-regulate virulence gene expression (e.g., fliC, fimH, htrA, chuA, pks). Functional analysis reveals that colonic SCFA significantly (p < 0....
Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IB... more Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IBD), especially Crohn’s disease, and colorectal cancer. AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by in vitro functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated E. coli in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohn’s disease and non-Crohn’s tissues. We show these strains can colonize the intestine of IBD susceptible mice ...
independent techniques to evaluate the microbial composition of pouch and afferent limb tissue bi... more independent techniques to evaluate the microbial composition of pouch and afferent limb tissue biopsies from patients with variable pouch outcomes. Methods: Patients with confirmed UC or familial adenomatous polyposis(FAP) who had a colectomy and IPAA, with ileostomy closure ≥1 year prior to study enrollment were recruited from Mount Sinai Hospital at the time of follow-up endoscopy. Retrospective and cross-sectional clinical/endoscopic information was collected at the time of enrollment and patients were classified into 4 groups: FAP/ no pouchitis(FAP), UC/no pouchitis(NP), UC/pouchitis(P) andUC/CD-like phenotype(CDL). P was defined based on the mPDAI. CDL diagnosis was based on presence of afferent limb inflammation or fistula. 2 biopsies were collected and snap-frozen in liquid nitrogen (1 afferent limb and 1 pouch). Microbial DNA was extracted and sequenced using the 454 GS FLX Titanium Sequencing system using primers specific for the V1-V3 and V2-V3 hypervariable regions of 16S rRNA genes in 2 batches. Sequences were quality trimmed and aligned to a SILVA reference database using mothur, with a minimum bootstrap cutoff of 80%. A threshold level of 0.05 was used to assign sequence clusters to a specific genus and patients were considered positive for genera with counts >0. Dichotomized results were analyzed using Fisher's exact test and Bonferroni correction for multiple testing. Results: 245 patients were recruited to the study of whom 79 were selected for analysis with complete data available (18 FAP, 21 NP, 20 P, 20 CDL). Analysis at the genus level showed that Blautia was detected significantly less often in the P group in both afferent limb (p=0.002) and pouch samples (p=0.008) compared to the other groups. Bacteroides and Sutterella were significantly less frequent among P and CDL groups in the pouch samples only (p=0.001 and 0.01 respectively). There was no significant difference in the proportions of these genera between outcome groups in the different batches. Conclusion: Differences in microbial composition are associated with outcomes following IPAA. Specifically, the genera Blautia, Bacteroides and Sutterella were found less frequently in patients with P and CDL compared to those without pouchitis. Further study may allow us to identify the role these organisms play in pouch pathogenesis, and to identify interactions occurring between microorganisms and host endogenous elements such as genotype and development of anti-microbial antibodies.
Fibrosis is a significant complication of intestinal disorders associated with microbial dysbiosi... more Fibrosis is a significant complication of intestinal disorders associated with microbial dysbiosis and pathobiont expansion, notably Crohn’s disease (CD). Mechanisms that favor fibrosis are not well understood and therapeutic strategies are limited. Here we demonstrate that colitis susceptible Il10-deficient mice develop inflammation-associated fibrosis when mono-associated with adherent/invasive Escherichia coli (AIEC) that harbor the yersiniabactin (Ybt) pathogenicity island. Inactivation of Ybt siderophore production in AIEC nearly abrogated fibrosis development in inflamed mice. In contrast, inactivation of Ybt import through its cognate receptor FyuA enhanced fibrosis severity. This corresponded with increased colonic expression of profibrogenic genes prior to the development of histological disease, therefore suggesting causality. FyuA-deficient AIEC also exhibited greater localization within sub-epithelial tissues and fibrotic lesions that was dependent on Ybt biosynthesis an...
5-aminosalicylate (5-ASA) is widely prescribed for the treatment of inflammatory bowel disease (I... more 5-aminosalicylate (5-ASA) is widely prescribed for the treatment of inflammatory bowel disease (IBD) and prevention of inflammation-associated colorectal cancer (CRC). Its clinical effect is widely attributed to modulation of host inflammatory responses. However, the recent association of intestinal dysbiosis and selective enrichment in Escherichia coli in patients with IBD and CRC raises the possibility that 5-ASA might also affect the enteric microflora. The aim of this study was to investigate the effect of 5-ASA on the growth and virulence of E. coli associated with IBD and CRC, and its impact on host cell inflammatory responses. Our results show that 5-ASA inhibited E. coli growth in a dose-dependent manner and downregulated the expression of bacterial virulence genes associated with IBD (fliC, fimH, ompC, yfgL, nlpL, lpfA, htrA, dsbA, fyuA, and chuA) and CRC (pks). 5-ASA inhibited E. coli motility (30-70%), epithelial adherence and invasion, and IL-8 secretion (p < 0.05). 5-ASA reduced E. coli survival in J774A.1 macrophages by 20 to 50% (p < 0.01) and TNF-α secretion by infected macrophages up to 30% (p < 0.05). In addition, 5-ASA reduced DNA damage in epithelial cells (Caco-2) induced by pks-positive E. coli. Our results reveal a multifaceted and previously unrecognized effect of 5-ASA on the growth and virulence of IBD-and CRC-associated E. coli, in addition to its inhibitory effect on host cell inflammatory responses. These results suggest that 5-ASA may abrogate the proinflammatory and oncogenic effects of E. coli in patients with IBD and CRC.
Streptococcus uberis is an important environmental pathogen associated with bovine mastitis as we... more Streptococcus uberis is an important environmental pathogen associated with bovine mastitis as well as with high total bacterial numbers in bulk tank milk. This study was conducted to determine whether S. uberis reproduction is likely to contribute to high bacterial numbers in bulk tank milk. Four S. uberis raw milk isolates were individually inoculated into UHT-treated milk and incubated at 4.4 or 7°C for up to 5 d to simulate appropriate cooling; at 10°C for 5 d to simulate marginally inadequate cooling; at 21 or 25°C for 7 h to simulate ambient temperatures; or at 32°C for 7 h to simulate elevated temperature conditions. None of the S. uberis isolates grew at either 4.4 or 7°C. Streptococcus uberis growth at 10°C appeared to be ribotype-specific. Although ribotype 116-520-S-1 isolates did not grow at 10°C, ribotype 116-520-S-2 isolate numbers increased up to 3.5 log 10 cfu/mL within 5 d. Generation times were calculated as 2.7 ± 0.1 h, 2.1 ± 0.1 h, and 1.0 ± 0.1 h for 116-520-S-1 isolates and 1.8 ± 0.4 h, 1.3 ± 0.3 h, and 0.8 ± 0.1 h for 116-520-S-2 isolates at 21, 25, and 32°C, respectively. Our results suggest that high numbers of S. uberis in bulk tank milk are more likely to reflect high numbers of S. uberis shed by mastitic cows, rather than multiplication of these organisms under cooling conditions required for production of Grade A milk.
Irritable bowel syndrome (IBS) affects 10%-20% of people. Increased numbers of Escherichia coli (... more Irritable bowel syndrome (IBS) affects 10%-20% of people. Increased numbers of Escherichia coli (E. coli) correlate with symptoms, and patients respond to antimicrobials targeting E. coli. We examined whether specific E. coli strains, phylogroups and pathotypes are associated with IBS. We evaluated 218 E. coli isolates from 33 IBS patients and 23 healthy controls. RAPD analysis revealed 89 E. coli strains (29 controls, 60 IBS), spanning the A, B1, B2 and D phylogroups. Strains were similarly enriched in virulence genes associated with extraintestinal pathogenic E. coli (ExPEC) and/or adherent-invasive E. coli (AIEC). Three strains harbored a diarrheagenic virulence gene (2 IBS, 1 control). Escherichia coli capable of invading epithelial cells or replicating in macrophages were detected in 53% of IBS and 50% controls, and 67% IBS and 45% controls respectively (P > 0.05). AIEC were identified in 33% of IBS patients vs 20% of controls (P = 0.35). Virulence genes ibeA, ColV and pduC ...
Perturbation of the microbiome is implicated in the pathogenesis of many human ailments, includin... more Perturbation of the microbiome is implicated in the pathogenesis of many human ailments, including inflammatory bowel diseases such as Crohn's disease (CD). Recapitulating the microbiome associated with health and disease necessitates controlling the composition of multiple bacterial species. This is difficult to achieve in vitro due to the overgrowth of bacterial species over time. Here, a microfluidic‐based model incorporating bacteria‐embedded hydrogel microfibers for the coculture of human enteric bacteria is introduced. Employing bacterial species and strains associated with CD, it is shown that the hydrogel‐based bacteria‐embedded microfiber model is physically and mechanically robust, and tunable. Metabolite analysis of the medium in both mono‐ and coculture revealed the interfiber exchange of soluble mediators and their impact on the growth of different bacterial species. This novel approach should enhance the ability to decipher contact‐independent cross‐talk within the polymicrobial intestinal luminal environment, and its impact on the intestinal epithelium.
mucosal surface of the Sigirr-/mouse. While p19A WT infection caused only minimal pathology on it... more mucosal surface of the Sigirr-/mouse. While p19A WT infection caused only minimal pathology on its own, it dramatically worsened the course of DSS colitis, in concert with deep penetration of the damaged colonic mucosa. Notably, intestinal tissue histology shows that the p19A hemolysin deletion mutant was severely attenuated in its ability to promote DSS colitis in Sigirr-/mice. Conclusion Our findings provide evidence that UC associated phylogroup B2 E. coli strains p19A WT can readily and persistently colonize the intestines of susceptible hosts, and significantly worsen the course of colitis. The intestinal tissue damages in DSS colitis Sigirr-/mice colonized with p19A hemolysin deletion mutant were severely attenuated, which indicates that p19A E. coli harbouring two alpha-hemolysin gene, might be responsible for barrier dysfunction and intestinal tissue damages in susceptible hosts. This model thus facilitates research into the role played by UC associated E. coli pathobionts in colitis pathogenesis.
Peripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with act... more Peripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with active inflammatory bowel disease (IBD) characterized by inflammatory enthesitis, dactylitis, or synovitis of nonaxial joints. However, a mechanistic understanding of the link between intestinal inflammation and SpA has yet to emerge. We evaluated and functionally characterized the fecal microbiome of IBD patients with or without peripheral SpA. Coupling the sorting of immunoglobulin A (IgA)-coated microbiota with 16S ribosomal RNA-based analysis (IgA-seq) revealed a selective enrichment in IgA-coated Escherichia coli in patients with Crohn's disease-associated SpA (CD-SpA) compared to CD alone. E. coli isolates from CD-SpA-derived IgA-coated bacteria were similar in genotype and phenotype to an adherent-invasive E. coli (AIEC) pathotype. In comparison to non-AIEC E. coli, colonization of germ-free mice with CD-SpA E. coli isolates induced T helper 17 cell (TH17) mucosal immunity, which...
The use of point-of-care (POC) devices in limited resource settings where access to commonly used... more The use of point-of-care (POC) devices in limited resource settings where access to commonly used infrastructure, such as water and electricity, can be restricted represents simultaneously one of the best application fits for POC systems as well as one of the most challenging places to deploy them. Of the many challenges involved in these systems, the preparation and processing of complex samples like stool, vomit, and biopsies are particularly difficult due to the high number and varied nature of mechanical and chemical interferents present in the sample. Previously we have demonstrated the ability to use solar-thermal energy to perform PCR based nucleic acid amplifications. In this work demonstrate how the technique, using similar infrastructure, can also be used to perform solar-thermal based sample processing system for extracting and isolating Vibrio Cholerae nucleic acids from fecal samples. The use of opto-thermal energy enables the use of sunlight to drive thermal lysing rea...
Escherichia coli is an important cause of bovine mastitis and can cause both severe inflammation ... more Escherichia coli is an important cause of bovine mastitis and can cause both severe inflammation with a short-term transient infection, as well as less severe, but more chronic inflammation and infection persistence. E. coli is a highly diverse organism that has been classified into a number of different pathotypes or pathovars, and mammary pathogenic E. coli (MPEC) has been proposed as a new such pathotype. The purpose of this study was PLOS ONE |
Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection... more Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection. However, E. coli can also cause clonal persistent intramammary infection (IMI) in dairy cows. In this study, we explored the possibility that E. coli strains associated with persistent IMI are better able to adhere to, invade, survive and replicate in cultured mammary epithelial cells (MAC-T) than transient strains, and examined their serotype, overall genotype, phylogenetic group, and the presence of known virulence genes. Both transient and persistent E. coli strains adhered to MAC-T cells, but persistent strains invaded MAC-T cells 2.6-63.5 times more than transient strains. Blocking the adhesin/invasin FimH with mannose diminished but did not eliminate adhesion and invasion of any strain. Cytoskeletal and protein kinase inhibitors cytochalasin D, colchicine, genistein and wortmannin dramatically reduced invasion of MAC-T cells by both strains. All of the persistent strains, but only one transient strain, were able to survive and replicate intracellularly in MAC-T cells over 48 h. Transient and persistent strains displayed heterogeneous serotypes and overall genotypes, but similar phylogeny (group A), and lacked virulence genes of invasive E. coli. We have found that E. coli strains associated with persistent IMI are better able to invade and replicate within cultured mammary epithelial cells than transient strains. The invasion process involves the host cytoskeleton and signaling cascades and is not FimH dependent. Our findings suggest that the invasion of mammary epithelial cells and intracellular survival play an important role in the pathogenesis of persistent E. coli mastitis.
Bovine mastitis is the most economically important disease of the dairy industry and Escherichia ... more Bovine mastitis is the most economically important disease of the dairy industry and Escherichia coli is one of the most common causes of bovine mastitis in well managed dairies with low milk somatic cell counts (SCC) (Bradley, 2002). E. coli isolates associated with bovine mastitis are traditionally considered random environmental opportunistic pathogens. The observation that lipopolysaccharide (LPS) can induce a mammary immune response in the absence of live bacteria (Bannerman et al., 2003) supports the idea of strain variation having little effect on the severity of bovine clinical mastitis (Burvenich et al., 2003). However, live bacteria but not LPS, can induce mastitis in animals lacking the TLR4 ligand, that Veterinary Microbiology 159 (2012) 163-170
The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathoge... more The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathogenesis of inflammatory bowel disease. This study explored the possibility that an abnormal mucosal flora is involved in the etiopathogenesis of granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected dogs ( n = 13) and controls ( n = 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. GCB-associated Escherichia coli isolates were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages as well as for serotype, phylogenetic group, genome size, overall genotype, and presence of virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E. coli . Three of four GCB-associated E. coli isolates adh...
Objective-Mutations in nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) rema... more Objective-Mutations in nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) remain the strongest genetic determinants for Crohn's disease (CD). Having previously identified vimentin as a novel NOD2-interacting protein, we aimed to investigate the regulatory effects of vimentin on NOD2 function and the association of variants in Vim to CD susceptibility. Design-Co-immunoprecipitation, fluorescent microscopy and fractionation were used to confirm the interaction between NOD2 and vimentin. HEK293 cells stably expressing wild-type NOD2 or NOD2-frameshift variant (L1007fs) and SW480 colonic epithelial cells were used alongside the vimentin inhibitor Withaferin-A (WFA) to assess effects on NOD2 function using nuclear factor-kappaB (NF-κB) reporter gene, GFP-LC3-based autophagy, and bacterial
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Papers by Belgin Dogan