Search

The pre-medicant chosen may influence the ease with which gastroduodenoscopy (GD) is performed. The purpose of this study was to evaluate the relative ease of GD in cats under ketamine and isoflurane anesthesia after IM injection of hydromorphone (H, 0.1 mg kg-1 ), hydromorphone plus glycopyrrolate (HG, 0.1 mg kg-1 (H), 0.01 mg kg-1 (G)), medetomidine (M, 0.03 mg kg-1 ), or butorphanol (B, 0.4 mg kg-1 ). Eight cats were assigned randomly to receive each treatment in a cross-over design with at least 7 days between treatments. Twenty minutes after pre-medication, medetomidine produced greater (p = 0.001) sedation than the other treatments when assessed, using a subjective ordinal scale. The cats were injected with ketamine (10 mg kg-1 IM), orotracheally intubated, connected to a pediatric circle breathing system, and allowed to spontaneously breathe isoflurane in oxygen. Once end-tidal isoflurane concentration was stable at 1.4% for 15 minutes, endoscopy was started. A single endoscopist (REG), who was unaware of the treatment used, performed all endoscopies. The endoscopist scored the difficulty of endoscopy subjectively (0–3). The significance of differences between treatments was evaluated using Friedman's test. Time for entering the stomach was 9.4 (4.7–15.9) (median (minimum–maximum)), 6.6 (5.2–11.7), 8.4 (6.3–16.5), and 7.7 (5.1–14.7) seconds and for entering the duodenum from the stomach was 20.5 (13.8–40.9), 18.2 (10.3–39.8), 20.2 (16.2–119.5), and 22.2 (11.8–83.8) seconds for H, HG, M, and B treatments, respectively. There were no significant differences in the time for, or difficulty of, endoscopy. We conclude that any of these drugs can be used satisfactorily at the doses and combinations tested to pre-medicate cats prior to general anesthesia for GD. [ABSTRACT FROM AUTHOR]

The E. coli strain harboring the polyketide synthase ( Pks) island encodes the genotoxin colibactin, a secondary metabolite reported to have severe implications for human health and for the progression of colorectal cancer. The present study involved whole-genome-wide comparison and phylogenetic analysis of pks harboring E. coli isolates to gain insight into the distribution and evolution of these organism. Fifteen E. coli strains isolated from patients with ulcerative colitis were sequenced, 13 of which harbored pks islands. In addition, 2,654 genomes from the public database were also screened for pks harboring E. coli genomes, 158 of which were pks -positive isolates. Whole-genome-wide comparison and phylogenetic analysis revealed that 171 (158+13) pks -positive isolates belonged to phylogroup B2, and most of the isolates associated to sequence types ST73 and ST95. One isolate from an ulcerative colitis (UC) patient was of the sequence type ST8303. The maximum likelihood tree based on the core genome of pks -positive isolates revealed horizontal gene transfer across sequence types and serotypes. Virulome and resistome analyses revealed the preponderance of virulence genes and a reduced number of antimicrobial genes in Pks -positive isolates. This study strongly contributes to understanding the evolution of pks islands in E. coli .

Hyperthyroidism is the most common feline endocrinopathy. In hyperthyroid humans, untargeted metabolomic analysis identified persistent metabolic derangements despite achieving a euthyroid state. Therefore, we sought to define the metabolome of hyperthyroid cats and identify ongoing metabolic changes after treatment. We prospectively compared privately-owned hyperthyroid cats (n = 7) admitted for radioactive iodine (I-131) treatment and euthyroid privately-owned control (CON) cats (n = 12). Serum samples were collected before (T0), 1-month (T1), and three months after (T3) I-131 therapy for untargeted metabolomic analysis by MS/MS. Hyperthyroid cats (T0) had a distinct metabolic signature with 277 significantly different metabolites than controls (70 increased, 207 decreased). After treatment, 66 (T1 vs. CON) and 64 (T3 vs. CON) metabolite differences persisted. Clustering and data reduction analysis revealed separate clustering of hyperthyroid (T0) and CON cats with intermediate phenotypes after treatment (T1 & T3). Mevalonate/mevalonolactone and creatine phosphate were candidate biomarkers with excellent discrimination between hyperthyroid and healthy cats. We found several metabolic derangements (e.g., decreased carnitine and α-tocopherol) do not entirely resolve after achieving a euthyroid state after treating hyperthyroid cats with I-131. Further investigation is warranted to determine diagnostic and therapeutic implications for candidate biomarkers and persistent metabolic abnormalities., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bechtold et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 H 2 O 2 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 H 2 O 2 concentrations in repeated trials. After two progressive exposures, the ability of L. rhamnosus to grow in the presence of H 2 O 2 increased from 1 mm H 2 O 2 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., (© 2024 Wiley‐VCH GmbH.)

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., (© 2023. The Author(s).)

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., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

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.

Escherichia coli isolates from inflammatory bowel disease (IBD) patients are often multidrug resistant, including to streptomycin. Streptomycin resistance (Str R ) mutations can alter bacterial behavior, which may influence intestinal disease. We generated a spontaneous Str R strain of the intestinal adherent-invasive E. coli (AIEC) strain NC101. Whole-genome sequencing revealed a single missense mutation in rpsL that commonly confers Str R , rpsL -K43N. Str R 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 Str R mutants of 10 clinical E. coli phylotype B2 strains from patients with IBD, colorectal cancer, and urinary tract infection. Out of these 10 Str R 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 Str R , 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 Str R , 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 Str R 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 Str R 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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

Background: An Escherichia coli (E. coli) pathotype with invasive properties, first reported by Darfeuille-Michaud and termed adherent-invasive E. coli (AIEC), was shown to be prevalent in up to half the individuals with Crohn's Disease (CD), suggesting that these bacteria could be involved in the pathophysiology of CD. Among the genes related to AIEC pathogenicity, fim has the potential to generate an inflammatory reaction from the intestinal epithelial cells and macrophages, as it interacts with TLR4, inducing the production of inflammatory cytokines independently of LPS. Therefore, targeting the bacterial adhesion of FimH-expressing bacteria seems a promising therapeutic approach, consisting of disarming bacteria without killing them, representing a selective strategy to suppress a potentially critical trigger of intestinal inflammation, without disturbing the intestinal microbiota., Results: We analyzed the metagenomic composition of the gut microbiome of 358 patients with CD from two different cohorts and characterized the presence of FimH-expressing bacteria. To assess the pathogenic role of FimH, we used human intestinal explants and tested a specific FimH blocker to prevent bacterial adhesion and associated inflammation. We observed a significant and disease activity-dependent enrichment of Enterobacteriaceae in the gut microbiome of patients with CD. Bacterial FimH expression was functionally confirmed in ileal biopsies from 65% of the patients with CD. Using human intestinal explants, we further show that FimH is essential for adhesion and to trigger inflammation. Finally, a specific FimH-blocker, TAK-018, inhibits bacterial adhesion to the intestinal epithelium and prevents inflammation, thus preserving mucosal integrity., Conclusions: We propose that TAK-018, which is safe and well tolerated in humans, is a promising candidate for the treatment of CD and in particular in preventing its recurrence. Video abstract., (© 2021. The Author(s).)

Objectives: To describe the clinical characteristics and long-term outcome of Escherichia coli-associated granulomatous ileocolitis in dogs., Methods: Retrospective review of medical records from dogs with periodic acid-Schiff positive (PAS+) granulomatous ileocolitis and mucosally invasive E. coli in the ileum and colon. Initial bacterial colonisation was evaluated using fluorescence in situ hybridization (FISH) in all dogs and corroborated with colonic and/or ileal culture, when performed., Results: Four boxer dogs and 1 French Bulldog with PAS+ granulomatous ileocolitis (GIC) were evaluated. All dogs had chronic diarrhoea refractory to empirical therapy. Ileocolonoscopy revealed mucosal haemorrhage and ulceration in the ileum (3/4) and colon (5/5). E. coli were visualised as clusters within the ileal and colonic mucosa. Complete (CR, 4/5) or partial (PR, 1/5) clinical response to fluoroquinolones was noted in all dogs within 30 days. CR was sustained in three of four dogs (median disease-free interval 40 months, range 16 to 60). Two dogs relapsed while receiving fluoroquinolones. Repeat biopsy isolated multidrug-resistant, mucosally invasive E. coli in the ileum (1/2) and colon (2/2). Targeted antimicrobial therapy was associated with long-term PR (78 months) in both dogs., Clinical Significance: Concurrent E. coli-associated granulomatous inflammation in the ileum and colon did not impart a poor clinical outcome or lack of response to the conventional standard of care for granulomatous colitis in dogs that were aggressively diagnosed and treated. Clinical outcome was influenced by antimicrobial resistance, with response dependent upon antimicrobial therapy informed by susceptibility testing., (© 2021 British Small Animal Veterinary Association.)

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-MVM 2 ) platform, which is used to produce a wide range of gradient-based antibiotic concentrations quickly (∼10 min). The EL-MVM 2 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-MVM 2 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-MVM 2 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-MVM 2 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.

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 (GM 2 ) 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 ( C max 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 GM 2 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 GM 2 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 GM 2 platform. Lastly, the GM 2 platform tracked the bacterial growth independent of an antibiotic mechanism of action or bacterial species in a robust and easy-to-implement fashion.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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, CX 3 CR1 + mononuclear phagocytes (MNP) are required for PduC-dependent induction of T helper 17 (Th17) cells and interleukin-1β (IL-1β) 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-1β 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC. on behalf of the American College of Veterinary Internal Medicine.)

The building evidence for the contribution of microbiota to human disease has spurred an effort to develop therapies that target the gut microbiota. This is particularly evident in inflammatory bowel diseases (IBDs), where clinical trials of fecal microbiota transplantation have shown some efficacy. To aid the development of novel microbiota-targeted therapies and to better understand the biology underpinning such treatments, we have used gnotobiotic mice to model microbiota manipulations in the context of microbiotas from humans with inflammatory bowel disease. Mice colonized with IBD donor-derived microbiotas exhibit a stereotypical set of phenotypes, characterized by abundant mucosal Th17 cells, a deficit in the tolerogenic RORγt + regulatory T (Treg) cell subset, and susceptibility to disease in colitis models. Transplanting healthy donor-derived microbiotas into mice colonized with human IBD microbiotas led to induction of RORγt + Treg cells, which was associated with an increase in the density of the microbiotas following transplant. Microbiota transplant reduced gut Th17 cells in mice colonized with a microbiota from a donor with Crohn's disease. By culturing strains from this microbiota and screening them in vivo, we identified a specific strain that potently induces Th17 cells. Microbiota transplants reduced the relative abundance of this strain in the gut microbiota, which was correlated with a reduction in Th17 cells and protection from colitis., Competing Interests: Competing interest statement: L.S.M., A.B., A.D. and D.G. are employees of Janssen Research and Development. G.B. is a former employee of Janssen Research and Development. G.J.B. and J.J.F. have filed patents related to the work published in this paper. T.J.B. has an interest in the Centre for Digestive Diseases, where fecal microbiota transplantation is a treatment option for patients, and has filed patents in this field. J.-F.C. has served as consultant, advisory board member, or speaker for AbbVie, Amgen, Boehringer-Ingelheim, Celgene Corporation, Celltrion, Enterome, Ferring, Genentech, Janssen, Lilly, Medimmune, Merck & Co., Pfizer, PPM Services, Protagonist, Second Genome, Seres, Shire, Takeda, Theradiag, and Theravance Biopharma, has stock options in Intestinal Biotech Development and Genfit and research grants from AbbVie, Takeda, and Janssen. M.C.D. has served as a consultant for Janssen. A.G. has received lecture fees from Merck and Takeda. J.J.F. has served as consultant, advisory board member, or speaker for Vedanta Biosciences and Janssen and has research grants from Janssen. The remaining authors declare no competing interests., (Copyright © 2020 the Author(s). Published by PNAS.)

Invasive Escherichia coli is causally associated with granulomatous colitis (GC) of Boxer dogs and French Bulldogs. The virulence determinants of GC E. coli are unclear. E. coli isolated from 16 GC (36 strains) and 17 healthy control (HC: 33 strains) dogs were diverse in phylogeny, genotype, and serotype and lacked diarrheagenic genes. Genes encoding type II ( gsp ), IV ( traC ), and VI ( hcp ) secretion systems, long polar fimbriae ( lpfA 154/141), and iron acquisition ( fyuA , chuA ) were frequent in GC and HC. E. coli from 14/15 GC and 10/11 HC invaded Caco-2 better than non-pathogenic E. coli strain DH5α, with invasion correlated with motility and presence of chuA and colV . E. coli from all GC and 10/11 HC survived better than DH5α in J774 macrophages, with adherent-invasive E. coli (AIEC) in 60% GC and 73% HC. AIEC replicated in monocyte derived macrophages from a GC Boxer with CD48/SLAM risk haplotype but not the HC. Fluroquinolone resistant E. coli were less motile and invasive than fluoroquinolone sensitive ( p < 0.05), and only 1/8 resistant strains met criteria for AIEC. In conclusion GC E. coli are diverse, resemble extraintestinal pathogenic E. coli (ExPEC), including AIEC, and can replicate in GC-susceptible macrophages. They are likely resident pathosymbionts that can opportunistically persist within macrophages of a GC-susceptible dog.

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 < 0.05) inhibit E. coli motility (up to 95%), infectivity (up to 60%), and type 1 fimbria-mediated agglutination (up to 50%). In addition, SCFA significantly ( p < 0.05) inhibit the activation of NF- κ B, and IL-8 production by epithelial cells. Our findings provide novel insights on the role of the regional chemical microenvironment in regulating the growth and virulence of pathosymbiont E. coli and opportunities for therapeutic intervention.

Metabolic reprogramming is associated with the adaptation of host cells to the disease environment, such as inflammation and cancer. However, little is known about microbial metabolic reprogramming or the role it plays in regulating the fitness of commensal and pathogenic bacteria in the gut. Here, we report that intestinal inflammation reprograms the metabolic pathways of Enterobacteriaceae, such as Escherichia coli LF82, in the gut to adapt to the inflammatory environment. We found that E. coli LF82 shifts its metabolism to catabolize L-serine in the inflamed gut in order to maximize its growth potential. However, L-serine catabolism has a minimal effect on its fitness in the healthy gut. In fact, the absence of genes involved in L-serine utilization reduces the competitive fitness of E. coli LF82 and Citrobacter rodentium only during inflammation. The concentration of luminal L-serine is largely dependent on dietary intake. Accordingly, withholding amino acids from the diet markedly reduces their availability in the gut lumen. Hence, inflammation-induced blooms of E. coli LF82 are significantly blunted when amino acids-particularly L-serine-are removed from the diet. Thus, the ability to catabolize L-serine increases bacterial fitness and provides Enterobacteriaceae with a growth advantage against competitors in the inflamed gut.

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 monoassociated with adherent/invasive Escherichia coli (AIEC) that harbors 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 subepithelial tissues and fibrotic lesions that was dependent on Ybt biosynthesis and corresponded with increased fibroblast activation in vitro Together, these findings suggest that Ybt establishes a profibrotic environment in the host in the absence of binding to its cognate receptor and indicate a direct link between intestinal AIEC and the induction of inflammation-associated fibrosis., (Copyright © 2019 Ellermann et al.)

Genomic resources for the domestic dog have improved with the widespread adoption of a 173k SNP array platform and updated reference genome. SNP arrays of this density are sufficient for detecting genetic associations within breeds but are underpowered for finding associations across multiple breeds or in mixed-breed dogs, where linkage disequilibrium rapidly decays between markers, even though such studies would hold particular promise for mapping complex diseases and traits. Here we introduce an imputation reference panel, consisting of 365 diverse, whole-genome sequenced dogs and wolves, which increases the number of markers that can be queried in genome-wide association studies approximately 130-fold. Using previously genotyped dogs, we show the utility of this reference panel in identifying potentially novel associations, including a locus on CFA20 significantly associated with cranial cruciate ligament disease, and fine-mapping for canine body size and blood phenotypes, even when causal loci are not in strong linkage disequilibrium with any single array marker. This reference panel resource will improve future genome-wide association studies for canine complex diseases and other phenotypes., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: ARB is the chief scientific officer of Embark Veterinary Inc.

Objectives: To detect and localise bacteria in gallbladder mucoceles using fluorescence in situ hybridisation (FISH). To report clinical signs, clinicopathologic abnormalities, sonographic findings and histopathological findings in FISH+ and FISH- dogs with gallbladder mucoceles., Materials and Methods: Retrospective review of signalment, clinical signs, clinicopathologic and sonographic findings of 25 cases of histopathologically confirmed gallbladder mucocele. Histopathological sections of gallbladder mucocele were evaluated for cystic mucinous hyperplasia, cystic mucinous hyperplasia with cholecystitis and rupture. The number and spatial distribution of bacteria was determined by eubacterial FISH. Gallbladder contents were cultured in 21 dogs., Results: Bacteria were detected within or adherent to the gallbladder wall in eight of 25 (32%) cases. Bacterial culture was positive in one dog. Cystic mucinous hyperplasia with concurrent cholecystitis was found in 17 of 25 (68%) of dogs with gallbladder mucocele., Clinical Significance: FISH was more sensitive for detection of bacteria in gallbladder mucoceles when compared to bacterial culture of bile. Cholecystitis was common in dogs with gallbladder mucocele. Further study is required to elucidate the relationship of cystic mucinous hyperplasia, bacteria and cholecystitis in the aetiopathogenesis and progression of gallbladder mucocele., (© 2019 British Small Animal Veterinary Association.)

Background: Adherent and invasive Escherichia coli (AIEC) is preferentially associated with ileal Crohn's disease (CD). The role of AIEC in the development of inflammation and its regional tropism is unresolved. The presence of long polar fimbriae (LPF) in 71% of ileal CD AIEC suggests a role for LPF in the tropism and virulence of AIEC. The aim of our study is to determine if AIEC, with or without LpfA, induces intestinal inflammation in monoassociated IL-10-/- mice., Methods: We compared murine AIEC strains NC101 (phylogroup B2, LpfA-) and CUMT8 (phylogroup B1, LpfA+), and isogenic mutant CUMT8 lacking lpfA154, with a non-AIEC (E. coli K12), evaluating histologic inflammation, bacterial colonization, mucosal adherence and invasion, and immune activation., Results: IL-10-/- mice monoassociated with AIEC (either CUMT8, CUMT8:ΔlpfA, or NC101) but not K12 developed diffuse small intestinal and colonic inflammation. There was no difference in the magnitude and distribution of inflammation in mice colonized with CUMT8:ΔlpfA compared with wild-type CUMT8. Bacterial colonization was similar for all E. coli strains. Fluorescence in situ hybridization revealed mucosal adherence and tissue invasion by AIEC but not K12. Production of the cytokines IL-12/23 p40 by the intestinal tissue and IFN-γ and IL-17 by CD4 T cells correlated with inflammation., Conclusions: IL-10-/- mice monoassociated with murine AIEC irrespective of LpfA expression developed chronic inflammation accompanied by IL-12/23 p40 production in the small and large intestines and IFN-γ/IL-17 production by CD4 T cells that model the interplay between enteric pathosymbionts, host susceptibility, and enhanced immune responses in people with IBD., (© 2018 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Microbial metabolites are an emerging class of mediators influencing CD4 + T-cell function. To advance the understanding of direct causal microbial factors contributing to Crohn's disease, we screened 139 predicted Crohn's disease-associated microbial metabolites for their bioactivity on human CD4 + T-cell functions induced by disease-associated T helper 17 (Th17) polarizing conditions. We observed 15 metabolites with CD4 + T-cell bioactivity, 3 previously reported, and 12 unprecedented. A deeper investigation of the microbe-derived metabolite, ascorbate, revealed its selective inhibition on activated human CD4 + effector T cells, including IL-17A-, IL-4-, and IFNγ-producing cells. Mechanistic assessment suggested the apoptosis of activated human CD4 + T cells associated with selective inhibition of energy metabolism. These findings suggest a substantial rate of relevant T-cell bioactivity among Crohn's disease-associated microbial metabolites, and evidence for novel modes of bioactivity, including targeting of T-cell energy metabolism.

Common variable immunodeficiency (CVID) is a rare condition in adult horses characterized by hypogammaglobulinemia and increased susceptibility to parasitic and bacterial infections, including recurrent respiratory diseases, septicemia, and meningitis. Lyme disease is often included as a differential diagnosis in CVID horses with signs of meningitis; however, the Borrelia burgdorferi organism has not been demonstrated previously within central nervous system tissues of CVID horses with neurologic disease, to our knowledge. We report herein a case of neuroborreliosis in a CVID horse, confirmed by combined immunologic testing, histopathology, real-time PCR assay, fluorescent in situ hybridization, and immunohistochemical staining. Implications of these findings include heightened monitoring of CVID horses for Lyme disease in endemic areas and appropriate therapy in the case of neurologic disease.

Background: Proliferative urethritis (PU) is a lower urinary tract disease of dogs characterized by frond-like lesions in the urethra. The etiology of PU is unknown, although an association with bacterial cystitis is reported., Objectives: Deep-seated bacterial cystitis is associated with PU, particularly in dogs with neutrophilic or granulomatous inflammation., Animals: Twenty-two client-owned dogs with PU and 5 control dogs euthanized for non-urinary disease., Methods: In retrospective analysis, medical records of dogs with PU from 1986 to 2016 were reviewed. Signalment, clinical signs, cystoscopic findings, antimicrobial use, and results of urine, bladder, or urethral tissue cultures, if available, were recorded. Histopathology was reviewed and classified as lymphocytic-plasmacytic (LP), neutrophilic, LP-neutrophilic (LPN), granulomatous, or pleocellular. Eubacterial fluorescence in situ hybridization (FISH) was performed on 18 tissue samples (13 cases, 5 controls), with subsequent evaluation of bacterial species., Results: Of the 22 dogs, 9 had LP urethritis, 6 had LPN, 4 had pleocellular, and 3 had neutrophilic urethritis. Of note, 7 of 13 PU samples were FISH+ for adherent or invasive bacteria; 1 of 5 controls were FISH+ for adherent bacteria. Five dogs had negative urine and tissue cultures when FISH was positive. There was no association detected between the type of urethral inflammation and the results of urine and tissue culture or FISH., Conclusions and Clinical Importance: The type of inflammation varied widely in these 22 PU cases. Deep-seated bacterial urethritis could be contributing to the inflammatory process in some dogs, regardless of the inflammation type. Urine and tissue cultures likely underestimate bacterial colonization of the urethra in dogs., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

The rise of antimicrobial resistance is challenging for physicians in clinical practice to prescribe antibiotics that are effective against bacterial infections. Conventional antibiotic susceptibility testing (AST) is labor-intensive and time-consuming (18-24 h). Newly emerging technologies such as microfluidics may enable more rapid AST assay time. In this study, we utilize a nanoliter-sized microchamber/microarray-based microfluidic (N-3M) platform to reduce the AST assay time and rapidly determine the minimum inhibitory concentrations of different antibiotics. Bacterial suspensions, with or without antibiotics, are loaded into small nanoliter-sized chambers, and the change in fluorescent intensity emitted from resazurin reduction, which correlated with bacterial growth, is measured. We demonstrate the reproducibility, functionality, and efficiency of our N-3M platform for numerous wild-type clinical bacterial isolates including Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis. The time-to-result of our N-3M platform varies between ∼1-3 h, depending on growth rates of different bacterial species. We believe that our proposed N-3M platform is robust, is easy-to-implement, has a short time-to-result, and can be applicable for microbial AST in clinical applications.

Colonic inflammation, a hallmark of inflammatory bowel disease, can be influenced by host intrinsic and extrinsic factors. There continues to be a need for models of colonic inflammation that can both provide insights into disease pathogenesis and be used to investigate potential therapies. Herein, we tested the utility of colonoscopic-guided pinch biopsies in mice for studying colonic inflammation and its treatment. Gene expression profiling of colonic wound beds after injury showed marked changes, including increased expression of genes important for the inflammatory response. Interestingly, many of these gene expression changes mimicked those alterations found in inflammatory bowel disease patients. Biopsy-induced inflammation was associated with increases in neutrophils, macrophages, and natural killer cells. Injury also led to elevated levels of sphingosine-1-phosphate (S1P), a bioactive lipid that is an important mediator of inflammation mainly through its receptor, S1P 1 . Genetic deletion of S1P 1 in the endothelium did not alter the inflammatory response but led to increased colonic bleeding. Bacteria invaded into the wound beds, raising the possibility that microbes contributed to the observed changes in mucosal gene expression. In support of this, reducing bacterial abundance markedly attenuated the inflammatory response to wounding. Taken together, this study demonstrates the utility of the pinch biopsy model of colonic injury to elucidate the molecular underpinnings of colonic inflammation and its treatment., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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 were associated with intramacrophage persistence; ibeA and ColV were associated with epithelial invasion and AIEC pathotype (P < 0.05). IBS patients and controls are commonly colonized by E. coli that resemble ExPEC and display pathogen-like behavior in vitro, similar to CD-associated AIEC. The relationship of these resident pathosymbiont E. coli to IBS warrants further investigation.

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.

An 8-year-old male American Staffordshire terrier was admitted for evaluation of chronic episodes of ptyalism and hematemesis after exercise. Abnormalities were not detected on routine clinicopathological tests, thoracic radiography, and abdominal ultrasonography. Endoscopic examination revealed a labyrinthine network of severely distended, hemorrhagic esophageal blood vessels. Computed tomography angiography demonstrated a network of para-esophageal vessels that communicated with the celiac artery caudally and the brachiocephalic trunk cranially, consistent with a diagnosis of non-cirrhotic esophageal varices. This is a report of exercise, ptyalism, and hematemesis secondary to presumptive, non-cirrhotic, bleeding esophageal varices in a dog., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Canine Inflammatory Bowel Disease (IBD) is considered a multifactorial disease caused by complex interactions between the intestinal immune system, intestinal microbiota and environmental factors in genetically susceptible individuals. Although IBD can affect any breed, German shepherd dogs (GSD) in the UK are at increased risk of developing the disease. Based on previous evidence, the aim of the present study was to identify single nucleotide polymorphisms (SNPs), which may confer genetic susceptibility or resistance to IBD using a genome-wide association study (GWAS). Genomic DNA was extracted from EDTA blood or saliva samples of 96 cases and 98 controls. Genotyping of cases and controls was performed on the Canine Illumina HD SNP array and data generated was analyzed using PLINK. Several SNPs and regions on chromosomes 7,9,11 and 13 were detected to be associated with IBD using different SNP-by-SNP association methods and FST windows approach. Searching one Mb up-and down-stream of the most significant SNPs, as identified by single SNP analysis as well as 200Kb before and after the start and the end position of the associated regions identified by FST windows approach, we identified 63 genes. Using a combination of pathways analysis and a list of genes that have been reported to be involved in human IBD, we identified 16 candidate genes potentially associated with IBD in GSD., Competing Interests: We have the following interests: This study was funded in part by Laboklin GmBH. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

The intestinal barrier is vulnerable to damage by microbiota-induced inflammation that is normally restrained through mechanisms promoting homeostasis. Such disruptions contribute to autoimmune and inflammatory diseases including inflammatory bowel disease. We identified a regulatory loop whereby, in the presence of the normal microbiota, intestinal antigen-presenting cells (APCs) expressing the chemokine receptor CX 3 CR1 reduced expansion of intestinal microbe-specific T helper 1 (Th1) cells and promoted generation of regulatory T cells responsive to food antigens and the microbiota itself. We identified that disruption of the microbiota resulted in CX 3 CR1 + APC-dependent inflammatory Th1 cell responses with increased pathology after pathogen infection. Colonization with microbes that can adhere to the epithelium was able to compensate for intestinal microbiota loss, indicating that although microbial interactions with the epithelium can be pathogenic, they can also activate homeostatic regulatory mechanisms. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes tissue homeostasis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Escherichia coli with an adherent and invasive pathotype (AIEC) is implicated in the pathogenesis of Crohn's disease (CD). Rifaximin improves symptoms in mild-to-moderate CD. It is unclear if this outcome is due to its effects on bacteria or intestinal epithelial inflammatory responses. We examined the effects of rifaximin on the growth and virulence of CD-associated E. coli and intestinal epithelial inflammatory responses. Seven well-characterized CD-associated E. coli strains (six AIEC, one non-AIEC; four rifaximin-resistant, three sensitive) were evaluated. We assessed the effects of rifaximin on CD-associated E. coli growth, adhesion to, and invasion of epithelial cells, virulence gene expression, motility, and survival in macrophages. Additionally, we determined the effects of rifaximin on intestinal epithelial inflammatory responses. In vitro rifaximin exerted a dose-dependent effect on the growth of sensitive strains but did not affect the growth of resistant strains. Rifaximin reduced adhesion, invasion, virulence gene expression and motility of CD-associated E. coli in a manner that was independent of its antimicrobial effect. Furthermore, rifaximin reduced IL-8 secretion from pregnane X receptor-expressing T84 colonic epithelial cells. The effect of rifaximin on adhesion was largely attributable to its action on bacteria, whereas decreases in invasion and cytokine secretion were due to its effect on the epithelium. In conclusion, our results show that rifaximin interferes with multiple steps implicated in host-AIEC interactions related to CD, including adhesion to, and invasion of epithelial cells, virulence gene expression, motility, and pro-inflammatory cytokine secretion. Further study is required to determine the relationship of these effects to clinical responses in CD patients.

This case report describes the detection of intrahepatic bacteria in formalin-fixed paraffin-embedded histopathological sections from three dogs with neutrophilic, pyogranulomatous, or lymphoplasmacytic hepatitis and cholangiohepatitis. In each of these cases, eubacterial fluorescence in situ hybridization enabled colocalization of intrahepatic bacteria with neutrophilic and granulomatous inflammation in samples that were negative for bacteria when evaluated by routine hematoxylin and eosin histopathology augmented with histochemical stains. Positive responses to antimicrobial therapy were observed in of 2 out of 2 patients that were treated with antimicrobials. These findings suggest that eubacterial fluorescence in situ hybridization analysis of formalin-fixed paraffin-embedded histopathological sections is more sensitive than conventional histochemical stains for the diagnosis of bacteria-associated canine hepatitis.

Persistent bacterial infections of the gastrointestinal mucosa are causally linked to gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma in people and laboratory animals. We examined the relationship of mucosa-associated bacteria to alimentary lymphoma in cats. Intestinal biopsies from 50 cats with alimentary lymphoma (small cell, n = 33; large cell, n = 17) and 38 controls without lymphoma (normal to minimal change on histopathology, n = 18; lymphocytic-plasmacytic enteritis, n = 20) were evaluated. The number and spatial distribution of bacteria (ie, in luminal cellular debris, villus-associated mucus, adherent to epithelium, mucosal invasion, intravascular, or serosal) were determined by fluorescence in situ hybridization with the eubacterial probe EUB-338. Mucosa-invasive bacteria were more frequently observed in cats with large cell lymphoma (82%, P ≤ .001) than in cats with small cell lymphoma (18%), normal to minimal change on histopathology, and lymphocytic-plasmacytic enteritis (3%). Intravascular bacteria were observed solely in large cell lymphoma (29%), and serosal colonization was more common in cats with large cell lymphoma (57%) than with small cell lymphoma (11%, P ≤ .01), normal to minimal change (8%, P ≤ .01), and lymphocytic-plasmacytic enteritis (6%, P ≤ .001). The high frequency of invasive bacteria within blood vessels and serosa of cats with large cell lymphoma may account for the sepsis-related complications associated with large cell lymphoma and inform clinical management. Further studies are required to determine the role of intramucosal bacteria in the etiopathogenesis of feline alimentary lymphoma.

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 16 S 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 (T H 17) mucosal immunity, which required the virulence-associated metabolic enzyme propanediol dehydratase ( pduC ). Modeling the increase in mucosal and systemic T H 17 immunity we observed in CD-SpA patients, colonization of interleukin-10-deficient or K/BxN mice with CD-SpA-derived E. coli lead to more severe colitis or inflammatory arthritis, respectively. Collectively, these data reveal the power of IgA-seq to identify immunoreactive resident pathosymbionts that link mucosal and systemic T H 17-dependent inflammation and offer microbial and immunophenotype stratification of CD-SpA that may guide medical and biologic therapy., (Copyright © 2017, American Association for the Advancement of Science.)