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 H2O2 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 H2O2 concentrations in repeated trials. After two progressive exposures, the ability of L. rhamnosus to grow in the presence of H2O2 increased from 1 mm H2O2 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.

Defined microbiota transplant restores Th17/RORγt+ regulatory T cell balance in mice colonized with inflammatory bowel disease microbiotas.

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.

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.

Yersiniabactin-Producing Adherent/Invasive Escherichia coli Promotes Inflammation-Associated Fibrosis in Gnotobiotic Il10-/- Mice.

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.

Nanoliter-Sized Microchamber/Microarray Microfluidic Platform for Antibiotic Susceptibility Testing.

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.

The intermediate filament protein, vimentin, is a regulator of NOD2 activity.

OBJECTIVE: Mutations in the nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) gene remain the strongest genetic determinants for Crohn's disease (CD). Having previously identified vimentin as a novel NOD2-interacting protein, the authors aimed to investigate the regulatory effects of vimentin on NOD2 function and the association of variants in Vim with CD susceptibility. DESIGN: Coimmunoprecipitation, fluorescent microscopy and fractionation were used to confirm the interaction between NOD2 and vimentin. HEK293 cells stably expressing wild-type NOD2 or a NOD2 frameshift variant (L1007fs) and SW480 colonic epithelial cells were used alongside the vimentin inhibitor, withaferin A (WFA), to assess effects on NOD2 function using the nuclear factor-kappaB (NF-κB) reporter gene, green fluorescent protein-LC3-based autophagy, and bacterial gentamicin protection assays. International genome-wide association meta-analysis data were used to test for associations of single-nucleotide polymorphisms in Vim with CD susceptibility. RESULTS: The leucine-rich repeat domain of NOD2 contained the elements required for vimentin binding; CD-associated polymorphisms disrupted this interaction. NOD2 and vimentin colocalised at the cell plasma membrane, and cytosolic mislocalisation of the L1007fs and R702W variants correlated with an inability to interact with vimentin. Use of WFA demonstrated that vimentin was required for NOD2-dependent NF-κB activation and muramyl dipeptide-induced autophagy induction, and that NOD2 and vimentin regulated the invasion and survival properties of a CD-associated adherent-invasive Escherichia coli strain. Genetic analysis revealed an association signal across the haplotype block containing Vim. CONCLUSION: Vimentin is an important regulator of NOD2 function and a potential novel therapeutic target in the treatment of CD. In addition, Vim is a candidate susceptibility gene for CD, supporting the functional data.

Rifaximin resistance in Escherichia coli associated with inflammatory bowel disease correlates with prior rifaximin use, mutations in rpoB, and activity of Phe-Arg-ß-naphthylamide-inhibitable efflux pumps.

Escherichia coli is implicated in the pathogenesis of inflammatory bowel disease (IBD). Rifaximin, a nonabsorbable derivative of rifampin effective against E. coli, improves symptoms in mild-to-moderate IBD. However, rifaximin resistance can develop in a single step in vitro. We examined the prevalence and mechanisms of rifaximin resistance in 62 strains of E. coli isolated from the ileal mucosa of 50 patients (19 with ileal Crohn's disease [L1+L3], 6 with colonic Crohn's disease [L2], 13 with ulcerative colitis [UC], 4 with symptomatic non-IBD diagnoses [NI], and 8 healthy [H]). Resistance (MIC > 1,024 mg/liter) was present in 12/48 IBD-associated ileal E. coli strains. Resistance correlated with prior rifaximin treatment (P < 0.00000001) but not with the presence of ileal inflammation (P = 0.73) or E. coli phylogroup. Mutations in a 1,057-bp region of rpoB, which encodes the bacterial target of rifaximin, were identified in 10/12 resistant strains versus 0/50 sensitive strains (P < 0.000000001) and consisted of seven amino acid substitutions. The efflux pump inhibitor Phe-Arg-ß-naphthylamide (PAßN) lowered the MIC of 9/12 resistant strains 8- to 128-fold. Resistance was stable in the absence of rifaximin in 10/12 resistant strains after 30 passages. We conclude that IBD-associated ileal E. coli frequently manifest resistance to rifaximin that correlates with prior rifaximin use, amino acid substitutions in rpoB, and activity of PAßN-inhibitable efflux pumps, but not with the presence of ileal inflammation or E. coli phylogroup. These findings have significant implications for treatment trials targeting IBD-associated E. coli.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Molecular and Phenotypic Characterization of Escherichia coli Associated with Granulomatous Colitis of Boxer Dogs.

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 (lpfA154/141), 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 Modulate the Growth and Virulence of Pathosymbiont Escherichia coli and Host Response.

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-κB, 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.