Adaptive Laboratory Evolution of Probiotics toward Oxidative Stress Using a Microfluidic-Based Platform.

Adaptive laboratory evolution (ALE) can be used to make bacteria less susceptible to oxidative stress. An alternative to large batch scale ALE cultures is to use microfluidic platforms, which are often more economical and more efficient. Microfluidic ALE platforms have shown promise, but many have suffered from subpar cell passaging mechanisms and poor spatial definition. A new approach is presented using a microfluidic Evolution on a Chip (EVoc) design which progressively drives microbial cells from areas of lower H2 O2 concentration to areas of higher concentration. Prolonged exposure, up to 72 h, revealed the survival of adaptive strains of Lacticaseibacillus rhamnosus GG, a beneficial probiotic often included in food products. After performing ALE on this microfluidic platform, the bacteria persisted under high H2 O2 concentrations in repeated trials. After two progressive exposures, the ability of L. rhamnosus to grow in the presence of H2 O2 increased from 1 mm H2 O2 after a lag time of 31 h to 1 mm after 21 h, 2 mm after 28 h, and 3 mm after 42 h. The adaptive strains have different morphology, and gene expression compared to wild type, and genome sequencing revealed a potentially meaningful single nucleotide mutation in the protein omega-amidase.

Measurement of feline-specific pancreatic lipase aids in the diagnosis of pancreatitis in cats.

OBJECTIVE: To establish a reference interval for a feline-specific pancreatic lipase assay (Spec fPL test; Idexx Laboratories Inc) in healthy cats and determine the sensitivity and specificity of the Spec fPL test in a large group of ill cats with and without pancreatitis. ANIMALS: 41 healthy cats, 141 cats with clinical signs consistent with pancreatitis, and 786 stored sera with known feline pancreatic lipase immunoreactivity (fPLI) concentrations. METHODS: This was a prospective, cross-sectional, nonrandomized study. Based on a detailed review of the medical history and results of physical examination, CBC, serum biochemical profile, urinalysis, abdominal ultrasonography, and clinical outcome, each cat was categorized by 2 board-certified internists masked to the fPLI test results into 1 of 6 categories from definitely pancreatitis to definitely not pancreatitis. RESULTS: The reference interval for the Spec fPL test, determined from the central 95th percentile of results from healthy cats, was fPLI of 0.7 to 3.5 µg/L. An fPLI concentration of ≥ 5.4 µg/L was determined to be consistent with pancreatitis. With an fPLI of 5.4 µg/L as the diagnostic cutoff, the sensitivity of the Spec fPL test for feline pancreatitis (definitely pancreatitis and probably pancreatitis) was 79.4%, the specificity for cats characterized as probably not pancreatitis and definitely not pancreatitis was 79.7%, and positive and negative predictive values were 69% and 87%, respectively. CLINICAL RELEVANCE: These findings support the use of the Spec fPL test as a valuable diagnostic test for feline pancreatitis.

A consortia of clinical E. coli strains with distinct in vitro adherent/invasive properties establish their own co-colonization niche and shape the intestinal microbiota in inflammation-susceptible mice.

BACKGROUND: Inflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in vitro definition fully predicts mucosal colonization in vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis. RESULTS: Germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortium of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. CONCLUSIONS: Our findings establish the in vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in vivo colonization dynamics of patient-derived bacteria in murine models. Video Abstract.

Randomized controlled trial of hydrolyzed fish diets in dogs with chronic enteropathy.

BACKGROUND: The role of diet in the pathogenesis and treatment of chronic enteropathies (CE) in dogs is unresolved. OBJECTIVES: To compare the ability of diets composed of hydrolyzed fish, rice starch, and fish oil without (HF) or with prebiotics, turmeric, and high cobalamin (HF+) against a limited ingredient diet containing mixed nonhydrolyzed antigens and oils (control) to resolve clinical signs and maintain serum cobalamin and folate concentrations in dogs with nonprotein losing CE (non-PLE). To determine the ability of hydrolyzed fish diets to support recovery and remission in dogs with PLE. ANIMALS: Thirty-one client-owned dogs with CE: 23 non-PLE, 8 PLE. METHODS: Randomized, blinded, controlled trial. Diets were fed for 2 weeks; responders continued for 12 weeks. Nonresponders were crossed over to another diet for 12 weeks. Response was determined by standardized clinical evaluation with long-term follow-up at 26 weeks. Concurrent medications were allowed in PLE. RESULTS: Nineteen of 23 (83%; 95% confidence interval [CI], 60%-94%) non-PLE CE responded clinically to their initial diet, with no difference between diets (P > .05). Four nonresponders responded to another diet, with sustained remission of 18/18 (100%; 95%CI, 78%-100%) at 26 weeks. Serum cobalamin concentration was increased (P < .05) and maintained by diet. Serum folate concentration decreased posttreatment (P < .05) but was restored by dietary supplementation. Hydrolyzed fish diets supported weight gain, serum albumin concentration, and recovery (P < .05) in dogs with PLE. CONCLUSIONS AND CLINICAL IMPORTANCE: Changing diet, independent of antigen restriction or supplemental ingredients, induced long-term remission in dogs with non-PLE CE. Serum cobalamin and folate concentrations were maintained by diet. Hydrolyzed fish diets supported clinical recovery and remission in PLE.

Heterogeneity among Clinical Intestinal Escherichia coli Isolates upon Acquired Streptomycin Resistance.

Escherichia coli isolates from inflammatory bowel disease (IBD) patients are often multidrug resistant, including to streptomycin. Streptomycin resistance (StrR) mutations can alter bacterial behavior, which may influence intestinal disease. We generated a spontaneous StrR strain of the intestinal adherent-invasive E. coli (AIEC) strain NC101. Whole-genome sequencing revealed a single missense mutation in rpsL that commonly confers StrR, rpsL-K43N. StrR NC101 exhibited a striking loss of aggregation and significantly increased motility, behaviors that can impact host-microbe interactions. Behavioral changes were associated with reduced transcription of csgA, encoding the biofilm component curli, and increased transcription of fliC, encoding flagellin. Scanning electron microscopy (SEM) detailed morphologic changes consistent with the observed alterations in multicellular behavior. Because intestinal E. coli isolates exhibit remarkable strain-specific differences, we generated spontaneous StrR mutants of 10 clinical E. coli phylotype B2 strains from patients with IBD, colorectal cancer, and urinary tract infection. Out of these 10 StrR clinical strains, two had altered colony morphology on Congo red agar (suggesting changes in extracellular products), and three had significant changes in motility. These changes were not associated with a particular rpsL mutation nor with the presence of virulence genes encoding the inflammation-associated E. coli metabolites yersiniabactin or colibactin. We conclude that common mutations in rpsL, which confer StrR, can differentially alter disease-associated phenotypes across intestinal E. coli strains. These findings highlight the heterogeneity among seemingly similar intestinal E. coli strains and reveal the need to carefully study the strain-specific effects of antibiotic resistance mutations, particularly when using these mutations during strain selection studies. IMPORTANCE We demonstrate that StrR, commonly acquired through a single point mutation in rpsL (a gene encoding part of the 30S bacterial ribosome), strikingly alters the morphology and behavior of a key intestinal AIEC strain, NC101. These changes include remarkably diminished aggregation and significantly increased motility, traits that are linked to AIEC-defining features and disease development. Phenotypic changes were heterogeneous among other StrR clinical E. coli strains, underscoring the need to evaluate the strain-specific effects of commonly acquired antibiotic resistance mutations. This is important, as the results of studies using mutant StrR Enterobacteriaceae strains (e.g., for cloning or in vivo selection) may be confounded beyond our demonstrated effects. Long term, these findings can help researchers better distinguish the contribution of specific E. coli traits to functional changes in the microbiota. Evaluating these strain-level differences could provide insight into the diversity of IBD symptoms and lead to improved therapies for microbiota-driven intestinal disorders.

A consortia of clinical E. coli strains with distinct in-vitro adherent/invasive properties establish their own co-colonization niche and shape the intestinal microbiota in inflammation-susceptible mice.

Background: Inflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in-vitro definition fully predicts mucosal colonization in-vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis. Results: Germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortia of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. Conclusions: Our findings establish the in-vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in-vivo colonization dynamics of patient-derived bacteria in murine models.

Agr2-associated ER stress promotes adherent-invasive E. coli dysbiosis and triggers CD103+ dendritic cell IL-23-dependent ileocolitis.

Endoplasmic reticulum (ER) stress is associated with Crohn's disease (CD), but its impact on host-microbe interaction in disease pathogenesis is not well defined. Functional deficiency in the protein disulfide isomerase anterior gradient 2 (AGR2) has been linked with CD and leads to epithelial cell ER stress and ileocolitis in mice and humans. Here, we show that ileal expression of AGR2 correlates with mucosal Enterobactericeae abundance in human inflammatory bowel disease (IBD) and that Agr2 deletion leads to ER-stress-dependent expansion of mucosal-associated adherent-invasive Escherichia coli (AIEC), which drives Th17 cell ileocolitis in mice. Mechanistically, our data reveal that AIEC-induced epithelial cell ER stress triggers CD103+ dendritic cell production of interleukin-23 (IL-23) and that IL-23R is required for ileocolitis in Agr2-/- mice. Overall, these data reveal a specific and reciprocal interaction of the expansion of the CD pathobiont AIEC with ER-stress-associated ileocolitis and highlight a distinct cellular mechanism for IL-23-dependent ileocolitis.

A high-throughput integrated biofilm-on-a-chip platform for the investigation of combinatory physicochemical responses to chemical and fluid shear stress.

Physicochemical conditions play a key role in the development of biofilm removal strategies. This study presents an integrated, double-layer, high-throughput microfluidic chip for real-time screening of the combined effect of antibiotic concentration and fluid shear stress (FSS) on biofilms. Biofilms of Escherichia coli LF82 and Pseudomonas aeruginosa were tested against gentamicin and streptomycin to examine the time dependent effects of concentration and FSS on the integrity of the biofilm. A MatLab image analysis method was developed to measure the bacterial surface coverage and total fluorescent intensity of the biofilms before and after each treatment. The chip consists of two layers. The top layer contains the concentration gradient generator (CGG) capable of diluting the input drug linearly into four concentrations. The bottom layer contains four expanding FSS chambers imposing three different FSSs on cultured biofilms. As a result, 12 combinatorial states of concentration and FSS can be investigated on the biofilm simultaneously. Our proof-of-concept study revealed that the reduction of E. coli biofilms was directly dependent upon both antibacterial dose and shear intensity, whereas the P. aeruginosa biofilms were not impacted as significantly. This confirmed that the effectiveness of biofilm removal is dependent on bacterial species and the environment. Our experimental system could be used to investigate the physicochemical responses of other biofilms or to assess the effectiveness of biofilm removal methods.

Long-Term Lacticaseibacillus rhamnosus GG Storage at Ambient Temperature in Vegetable Oil: Viability and Functional Assessments.

Vegetable oils with varying saturated fat levels were inoculated with Lacticaseibacillus rhamnosus GG (LGG), subjected to different heat treatments in the absence and presence of inulin and stored for 12 months at room temperature. After storage, the heat-treated probiotics actively grew to high concentrations after removal of the oils and reculturing. The bacterial samples, regardless of aerobic or anaerobic conditions and treatment methods, showed no changes in their growth behavior. The random amplified polymorphic DNA-polymerase chain reaction, antimicrobial, morphology, and motility tests also showed no major differences. Samples of LGG treated with a higher antioxidant content (Gal400) showed reduced inflammatory and anti-inflammatory properties. These findings have been confirmed by metabolite and genome sequencing studies, indicating that Gal400 showed lower concentrations and secretion percentages and the highest number of single nucleotide polymorphisms. We have shown proof of concept that LGG can be stored in oil with minimum impact on probiotic in vitro viability.

Mucosal metabolites fuel the growth and virulence of E. coli linked to Crohn's disease.

Elucidating how resident enteric bacteria interact with their hosts to promote health or inflammation is of central importance to diarrheal and inflammatory bowel diseases across species. Here, we integrated the microbial and chemical microenvironment of a patient's ileal mucosa with their clinical phenotype and genotype to identify factors favoring the growth and virulence of adherent and invasive E. coli (AIEC) linked to Crohn's disease. We determined that the ileal niche of AIEC was characterized by inflammation, dysbiosis, coculture of Enterococcus, and oxidative stress. We discovered that mucosal metabolites supported general growth of ileal E. coli, with a selective effect of ethanolamine on AIEC that was augmented by cometabolism of ileitis-associated amino acids and glutathione and by symbiosis-associated fucose. This metabolic plasticity was facilitated by the eut and pdu microcompartments, amino acid metabolism, γ-glutamyl-cycle, and pleiotropic stress responses. We linked metabolism to virulence and found that ethanolamine and glutamine enhanced AIEC motility, infectivity, and proinflammatory responses in vitro. We connected use of ethanolamine to intestinal inflammation and L-fuculose phosphate aldolase (fucA) to symbiosis in AIEC monoassociated IL10-/- mice. Collectively, we established that AIEC were pathoadapted to utilize mucosal metabolites associated with health and inflammation for growth and virulence, enabling the transition from symbiont to pathogen in a susceptible host.

Cytologic, histopathologic, and clinical features of granulomatous colitis in a French Bulldog.

A young French Bulldog was presented with clinical signs of chronic gastrointestinal disease, unresponsive to medical therapies. Parasite screenings and abdominal ultrasound failed to identify the etiology of the clinical signs. Cytologic evaluation of a rectal scraping sample diagnosed presumptive granulomatous colitis (GC) based on the presence of numerous macrophages with characteristic abundant, pink, granular cytoplasm, which showed an intense pink color when stained with periodic acid-Schiff. Tissue biopsy samples and Escherichia coli fluorescence in situ hybridization analysis confirmed the cytologic diagnosis. The cytologic, histopathologic, and clinical features and staining properties of GC in a French Bulldog are reported. Rectal scraping should be considered a part of the diagnostic evaluation in patients with suspected GC.

Long-read sequencing to interrogate strain-level variation among adherent-invasive Escherichia coli isolated from human intestinal tissue.

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 and induce inflammatory cytokines from cultured macrophages. We demonstrate that these strains can be quantified and distinguished in the presence of 99.5% mammalian DNA and from within a fecal population. Analysis of global genomic structure and specific sequence variation within the ribosomal RNA operon provides a framework for efficiently tracking strain-level variation of closely-related E. coli and likely other commensal/pathogenic bacteria impacting intestinal inflammation in experimental settings and IBD patients.

Antimicrobial Susceptibility Testing in a Rapid Single Test via an Egg-like Multivolume Microchamber-Based Microfluidic Platform.

Fast determination of antimicrobial agents' effectiveness (susceptibility/resistance pattern) is an essential diagnostic step for treating bacterial infections and stopping world-wide outbreaks. Here, we report an egg-like multivolume microchamber-based microfluidic (EL-MVM2) platform, which is used to produce a wide range of gradient-based antibiotic concentrations quickly (∼10 min). The EL-MVM2 platform works based upon testing a bacterial suspension in multivolume microchambers (microchamber sizes that range from a volume of 12.56 to 153.86 nL). Antibiotic molecules from a stock solution diffuse into the microchambers of various volumes at the same loading rate, leading to different concentrations among the microchambers. Therefore, we can quickly and easily produce a robust antibiotic gradient-based concentration profile. The EL-MVM2 platform's diffusion (loading) pattern was investigated for different antibiotic drugs using both computational fluid dynamics simulations and experimental approaches. With an easy-to-follow protocol for sample loading and operation, the EL-MVM2 platform was also found to be of high precision with respect to predicting the susceptibility/resistance outcome (>97%; surpassing the FDA-approval criterion for technology-based antimicrobial susceptibility testing instruments). These features indicate that the EL-MVM2 is an effective, time-saving, and precise alternative to conventional antibiotic susceptibility testing platforms currently being used in clinical diagnostics and point-of-care settings.

Gradient-Based Microfluidic Platform for One Single Rapid Antimicrobial Susceptibility Testing.

Antimicrobial resistance is a growing problem, necessitating rapid antimicrobial susceptibility testing (AST) to enable effective in-clinic diagnostic testing and treatment. Conventional AST using broth microdilution or the Kirby-Bauer disk diffusion are time-consuming (e.g., 24-72 h), labor-intensive, and costly and consume reagents. Here, we propose a novel gradient-based microchamber microfluidic (GM2) platform to perform AST assay for a wide range of antibiotic concentrations plus zero (positive control) and maximum (negative control) concentrations all in a single test. Antibiotic lateral diffusion within enriched to depleted (Cmax and zero, respectively) cocurrent flowing fluids, moving alongside a micron-sized main channel, is led to form an antibiotic concentration profile in microchambers, connected to the depleted side of the main channel. We examined the tunability of the GM2 platform, in terms of producing a wide range of antibiotic concentrations in a gradient mode between two consecutive microchambers with changing either the loading fluids' flow rates or their initial concentrations. We also tested the GM2 platform for profiling bacteria associated with human Crohn's disease and bovine mastitis. Time to result for performing a complete AST assay was ∼ 3-4 h in the GM2 platform. Lastly, the GM2 platform tracked the bacterial growth independent of an antibiotic mechanism of action or bacterial species in a robust and easy-to-implement fashion.

Diffusion-Convection Hybrid Microfluidic Platform for Rapid Antibiotic Susceptibility Testing.

Conventional antibiotic susceptibility testing (AST) assays such as broth microdilution and Kirby-Bauer disk diffusion are time-consuming (e.g., 24-72 h) and labor-intensive. Here, we present a microfluidic platform to perform AST assays with a broad range of antibiotic concentrations and controls. A culture medium stream was serially enriched with antibiotics along the length of the platform via diffusion and flow-directing mass convection mechanisms, generating a concentration gradient captured in a series of microchamber duplicates. We observed an agreement between the simulated and experimental concentration gradients and applicability to a variety of different molecules by changing the loading time according to a simple linear equation. The AST assay in our platform is based on bacterial metabolism, indicated by resazurin fluorescence. The small reaction volume enabled a minimum inhibitory concentration (MIC) to be determined in 4-5 h. Proof-of-concept functionality testing, using human isolates and clinically important antibiotics from different classes, indicated a high rate of agreement (94%: MIC within ±1 two-fold dilution of the reference method) of on-chip MICs and conventional broth microdilution. Overall, our results showed that this microfluidic platform is capable of determining antibiotic susceptibility in a rapid and reliable manner.

Biological small-molecule assays using gradient-based microfluidics.

Studying the potency of small-molecules on eukaryotic and prokaryotic cells using conventional biological settings requires time-consuming procedures and large volumes of expensive small-molecules. Microfluidics could significantly expedite these assays by enabling operation in high-throughput and (semi)automated modes. Here, we introduce a microfluidics platform based on multi-volume microchamber arrays that can produce a wide range of small-molecule concentrations with a desired gradient-based profile for rapid and precise biological testing within a single device with minimal hands-on time. The concept behind this device is based on introducing the same amount of a small-molecule into microchambers of different volumes to spontaneously generate a gradient concentration profile via diffusion. This design enables to obtain an unprecedented concentration range (e.g., three orders of magnitude) that can be easily adjusted, allowing us to pinpoint the precise effect of small-molecules on pre-loaded prokaryotic and eukaryotic cells. We also propose a comprehensive relationship for determining the loading time (the only required parameter for implementing this platform) in order to study the effects of any small-molecule on a biological species in a desired test. We demonstrate the versatility of this microfluidics platform by conducting two small-molecule assays-antimicrobial resistance and sugar-phosphate toxicity for both eukaryotic and prokaryotic biological systems.

Adherent-invasive E. coli metabolism of propanediol in Crohn's disease regulates phagocytes to drive intestinal inflammation.

Adherent-invasive E. coli (AIEC) are enriched in the intestinal microbiota of patients with Crohn's disease (CD) and promote intestinal inflammation. Yet, how AIEC metabolism of nutrients impacts intestinal homeostasis is poorly defined. Here, we show that AIEC encoding the large subunit of propanediol dehydratase (PduC), which facilitates the utilization of fucose fermentation product 1,2-propanediol, are increased in the microbiome of CD patients and drive AIEC-induced intestinal T cell inflammation. In murine models, CX3CR1+ mononuclear phagocytes (MNP) are required for PduC-dependent induction of T helper 17 (Th17) cells and interleukin-1ß (IL-1ß) production that leads to AIEC-induced inflammatory colitis. Activation of this inflammatory cascade requires the catalytic activity of PduC to generate propionate, which synergizes with lipopolysaccharide (LPS) to induce IL-1ß by MNPs. Disrupting fucose availability limits AIEC-induced propionate production and intestinal inflammation. These findings identify MNPs as metabolic sensors linking AIEC metabolism with intestinal inflammation and identify microbial metabolism as a potential therapeutic target in Crohn's disease treatment.

Dietary Fructose Alters the Composition, Localization, and Metabolism of Gut Microbiota in Association With Worsening Colitis.

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 HFrD-mediated 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 a 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 pro-colitic effect that can be explained by changes in the composition, distribution, and metabolic function of resident enteric microbiota.

Escherichia coli-associated granulomatous colitis in dogs treated according to antimicrobial susceptibility profiling.

BACKGROUND: Eradication of intramucosal Escherichia coli correlates with remission of periodic acid-Schiff-positive E coli-associated granulomatous colitis (GC). Treatment failures attributed to multidrug resistant (MDR) bacteria necessitate alternative approaches. HYPOTHESIS/OBJECTIVES: Determine clinical outcome of E coli-associated GC in dogs treated based on antimicrobial susceptibility profiling and characterize E coli phylogeny and resistance mechanisms. ANIMALS: Twenty Boxers and 4 French Bulldogs with E coli-associated GC. METHODS: Culture, antimicrobial susceptibility profiling, and molecular characterization of E coli were performed and response to treatment was evaluated. RESULTS: Initial biopsy sample culture yielded fluoroquinolone-sensitive (FQ-S) E coli from 9/24 dogs and fluoroquinolone-resistant (FQ-R) E coli from 15/24. All but 1 FQ-R E coli were MDR with susceptibility to macrophage-penetrating antimicrobials restricted to carbapenems in 13/15 dogs. Of 22/24 treated based on susceptibility profiling, 8/9 FQ-S dogs had complete initial clinical response (CR) during fluoroquinolone (FQ) treatment, whereas 9/13 FQ-R dogs had complete or partial response (PR) during meropenem or doxycycline treatment. In 5/9 FQ-S and 12/13 FQ-R dogs with follow-up ≥3 months, CR was sustained in 5/5 FQ-S (median, 25 months; range, 4-46) whereas 6/12 FQ-R had long-term CR (median, 59 months; range 15-102), 4/12 PR (median, 19 months; range, 5-65), and 2/12 had no response (NR). Four dogs with long-term follow-up died within 4 years of diagnosis, including 2 euthanized for refractory colitis. Escherichia coli were genetically diverse. Fluoroquinolone resistance was associated with mutations in gyrA and parC, with plasmid-mediated resistance less common. CONCLUSIONS AND CLINICAL IMPORTANCE: Antimicrobial treatment guided by susceptibility profiling was associated with positive long-term outcomes in >80% of cases. Fluoroquinolone-resistance was widespread and not clonal. Further study is required to optimize treatment for dogs with MDR E coli-associated GC.

Acute Ulcerative Enterocolitis With Severe Protein Loss Due to Mucosal Invasion With Enterococcus spp. in a Dog With Exocrine Pancreatic Insufficiency: A Case Report.

We describe an unusual case of severe acute protein-losing enteropathy in a dog, which presented with a systemic inflammatory response syndrome. This dog's condition could not be categorized as any well-known canine intestinal condition. Instead, components of several enteropathies like acute hemorrhagic diarrhea syndrome (AHDS), chronic inflammatory enteropathy (CIE), and ulcerative and granulomatous colitis were present. Thorough investigations identified concurrent exocrine pancreatic insufficiency (EPI) and hypocobalaminemia. On histopathology, marked diffuse chronic-active ileitis and ulcerative colitis with fibroplasia and neovascularization were present. Intestinal biopsy cultures identified E.coli and multiresistant Enterococcus spp. The latter was identified as mucosally invasive using fluorescent in situ hybridization (FISH). Protracted clinical signs following the acute presentation required intensive care including enteral and parenteral feeding for a successful outcome, but eventually stabilized with antibiotics and immunosuppressive doses of glucocorticoids. This case highlights a potentially previously unrecognized condition, suspected to be a form of CIE manifesting acutely after bacterial mucosal invasion. In this case, this might have been facilitated by EPI-induced dysbiosis. The use of FISH and mucosal culture in this context provided important clinical information and should be considered more frequently in CIE and non-responsive AHDS.