Murine Intestinal Tract Research Articles

Identification of a Genetic Determinant in Clinical Enterococcus faecium Strains That Contributes to Intestinal Colonization During Antibiotic Treatment

Intestinal colonization by antibiotic-resistant Enterococcus faecium is the first step in a process that can lead to infections in hospitalized patients. By comparative genome analysis and subsequent polymerase chain reaction screening, we identified a locus that encodes a putative phosphotransferase system (PTS). The PTS locus was widespread in isolates from hospital outbreaks of infection (84.2%) and nonoutbreak clinical infections (66.0%) but absent from human commensal isolates. Deletion of pstD, which is predicted to encode the enzyme IID subunit of this PTS, significantly impaired the ability of E. faecium to colonize the murine intestinal tract during antibiotic treatment. This is the first description of a determinant that contributes to intestinal colonization in clinical E. faecium strains.

Role of Catecholate Siderophores in Gram-Negative Bacterial Colonization of the Mouse Gut

We investigated the importance of the production of catecholate siderophores, and the utilization of their iron (III) complexes, to colonization of the mouse intestinal tract by Escherichia coli. First, a ΔtonB strain was completely unable to colonize mice. Next, we compared wild type E. coli MG1655 to its derivatives carrying site-directed mutations of genes for enterobactin synthesis (ΔentA::Cm; strain CAT0), ferric catecholate transport (Δfiu, ΔfepA, Δcir, ΔfecA::Cm; CAT4), or both (Δfiu, ΔfepA, ΔfecA, Δcir, ΔentA::Cm; CAT40) during colonization of the mouse gut. Competitions between wild type and mutant strains over a 2-week period in vivo showed impairment of all the genetically engineered bacteria relative to MG1655. CAT0, CAT4 and CAT40 colonized mice 101-, 105-, and 102-fold less efficiently, respectively, than MG1655. Unexpectedly, the additional inability of CAT40 to synthesize enterobactin resulted in a 1000-fold better colonization efficiency relative to CAT4. Analyses of gut mucus showed that CAT4 hyperexcreted enterobactin in vivo, effectively rendering the catecholate transport-deficient strain iron-starved. The results demonstrate that, contrary to prior reports, iron acquisition via catecholate siderophores plays a fundamental role in bacterial colonization of the murine intestinal tract.

PWE-089 The roles of CYP2C40 and CYP2C55 in preventing colon cancer

IntroductionCertain Cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolites are thought to induce therapeutic effects in the colon via activation of peroxisome proliferator activated receptors (PPARs), specifically the PPAR α subtype....

Neutrophil Elastase Mediates Pathogenic Effects of Anthrax Lethal Toxin in the Murine Intestinal Tract

Neutrophils isolated from BALB/c or C57BL/6 mice and treated in vitro with anthrax lethal toxin release bioactive neutrophil elastase, a proinflammatory mediator of tissue destruction. Similarly, neutrophils isolated from mice treated with anthrax lethal toxin in vivo and cultured ex vivo release greater amounts of elastase than neutrophils from vehicle-treated controls. Direct measurements from murine intestinal tissue samples demonstrate an anthrax lethal toxin-dependent increase in neutrophil elastase activity in vivo as well. These findings correlate with marked lethal toxin-induced intestinal ulceration and bleeding in neutrophil elastase(+/+) animals, but not in neutrophil elastase(-/-) animals. Moreover, neutrophil elastase(-/-) mice have a significant survival advantage over neutrophil elastase(+/+) animals following exposure to anthrax lethal toxin, thereby establishing a key role for neutrophil elastase in mediating the deleterious effects of anthrax lethal toxin.

Surface area assessment of the murine intestinal tract as a prerequisite for oral dose translation from mouse to man

In many pharmacological and toxicological studies knowledge about the intestinal absorption, which is dependent upon the surface area of absorptive epithelia, is indispensible. Although mice are often used in such preclinical studies, very few quantitative data about their intestinal surface area are available. Especially for locally acting candidate drugs in development, this information is crucial for dose translation towards humans. Therefore, the surface area of the intestinal tract of CD-1 IGS mice was assessed in the present study. The intestinal tracts of 12 mice were collected after euthanasia. From six animals, histological sections from the duodenum, jejunum, ileum, caecum and colon-rectum were made according to common stereological principles. Using these sections, the volumes and surface areas of each intestinal segment were estimated applying stereological counting procedures. In the other six animals, the density and surface area of the microvilli present in each intestinal segment were determined by means of scanning and transmission electron microscopy to assess the increase of the intestinal surface area attributable to the presence of microvilli. The mean total volume and surface area of the intestinal tract were 1.34 cm(3) and 1.41 m(2), respectively. The relative intestinal surface area (intestinal surface area divided by the body surface area) was 119. The relative intestinal surface area of mice is very similar to that of humans. The results of this study are important for the appropriate dose translation of candidate therapeutic compounds in drug development from mouse to humans.

T cell receptor heterodimer (alpha beta/gamma delta) expressions in diffuse panbronchiolitis

Several reports have indicated that T cells bearing gamma delta T cell receptor (TCR) are major effector cells in mucosal immunity of the murine intestinal tract. In humans, abnormal distribution of gamma delta + T cells has also been found in inflammatory bowel diseases. We studied TCR heterodimer expressions in lymphocytes infiltrating the peripheral airways in patients with diffuse panbronchiolitis (DPB), because DPB is histologically characterized by chronic inflammation in bronchioles and could be a model for investigation of the significance of gamma delta + T cells in persistent inflammation of the human respiratory tract. Open lung biopsy specimens were obtained from 7 patients and peripheral blood lymphocytes (PBL) from 9 patients. Immunohistochemically, we detected alpha beta and gamma delta TCR by avidin-biotin complex immunoperoxidase method, utilizing BMA 031 and TCR delta 1 monoclonal antibody, respectively, PBL were analyzed by flow cytometry. Histological analysis revealed that DPB lesions mainly involved the bronchiolar walls with marked infiltration of small round cells. Many T cells were distributed in the submucosal area, although few T cells infiltrated the epithelium. Most of the T cells in the bronchioles expressed alpha beta TCR (98.7%), whereas only 1.3% expressed gamma delta TCR. There was no significant difference in the percentage of gamma delta + T cells in PBL between DPB and age- and sex-matched controls (6.7% vs. 9.1%). These results suggest that the increase of T cells bearing alpha beta TCR played a major role. However, the possibility that the decrease of T cells bearing gamma delta TCR caused reduced mucosal defense against external antigens, leading to persistent inflammation of the lower respiratory tracts in DPB, is not excluded.

Aeromonas caviaestrain induces Th1 cytokine response in mouse intestinal tract

Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus. Microarray profiling of murine small intestinal extracts, 24 h after oral infection with an A. caviae strain, provides evidence of a Th1 type immune response. A large number of gamma-interferon (gamma-IFN) induced genes are up-regulated as well as several tumor necrosis factor-alpha (TNF-alpha) transcripts. Aeromonas caviae has always been considered an opportunistic pathogen because it lacks obvious virulence factors. This current effort suggests that an A. caviae strain can colonize the murine intestinal tract and cause what has been described by others as a dysregulatory cytokine response. This response could explain why a number of diarrheal waterborne disease cases have been attributed to A. caviae even though it lacks obvious enteropathogenic properties.

Role of the Helicobacter hepaticus Flagellar Sigma Factor FliA in Gene Regulation and Murine Colonization

The enterohepatic Helicobacter species Helicobacter hepaticus colonizes the murine intestinal and hepatobiliary tract and is associated with chronic intestinal inflammation, gall stone formation, hepatitis, and hepatocellular carcinoma. Thus far, the role of H. hepaticus motility and flagella in intestinal colonization is unknown. In other, closely related bacteria, late flagellar genes are mainly regulated by the sigma factor FliA (sigma(28)). We investigated the function of the H. hepaticus FliA in gene regulation, flagellar biosynthesis, motility, and murine colonization. Competitive microarray analysis of the wild type versus an isogenic fliA mutant revealed that 11 genes were significantly more highly expressed in wild-type bacteria and 2 genes were significantly more highly expressed in the fliA mutant. Most of these were flagellar genes, but four novel FliA-regulated genes of unknown function were identified. H. hepaticus possesses two identical copies of the gene encoding the FliA-dependent major flagellin subunit FlaA (open reading frames HH1364 and HH1653). We characterized the phenotypes of mutants in which fliA or one or both copies of the flaA gene were knocked out. flaA_1 flaA_2 double mutants and fliA mutants did not synthesize detectable amounts of FlaA and possessed severely truncated flagella. Also, both mutants were nonmotile and unable to colonize mice. Mutants with either flaA gene knocked out produced flagella morphologically similar to those of wild-type bacteria and expressed FlaA and FlaB. flaA_1 mutants which had flagella but displayed reduced motility did not colonize mice, indicating that motility is required for intestinal colonization by H. hepaticus and that the presence of flagella alone is not sufficient.

Self-Regulation of Candida albicans Population Size during GI Colonization

Interactions between colonizing commensal microorganisms and their hosts play important roles in health and disease. The opportunistic fungal pathogen Candida albicans is a common component of human intestinal flora. To gain insight into C. albicans colonization, genes expressed by fungi grown within a host were studied. The EFH1 gene, encoding a putative transcription factor, was highly expressed during growth of C. albicans in the intestinal tract. Counterintuitively, an efh1 null mutant exhibited increased colonization of the murine intestinal tract, a model of commensal colonization, whereas an EFH1 overexpressing strain exhibited reduced colonization of the intestinal tract and of the oral cavity of athymic mice, the latter situation modeling human mucosal candidiasis. When inoculated into the bloodstream of mice, both efh1 null and EFH1 overexpressing strains caused lethal infections. In contrast, other mutants are attenuated in virulence following intravenous inoculation but exhibited normal levels of intestinal colonization. Finally, although expression of several genes is dependent on transcription factor Efg1p during laboratory growth, Efg1p-independent expression of these genes was observed during growth within the murine intestinal tract. These results show that expression of EFH1 regulated the level of colonizing fungi, favoring commensalism as opposed to candidiasis. Also, different genes are required in different host niches and the pathway(s) that regulates gene expression during host colonization can differ from well-characterized pathways used during laboratory growth.

Diversity in secreted PLA2-IIA activity among inbred mouse strains that are resistant or susceptible to Apc Min/+ tumorigenesis.

The secreted phospholipase A2 type IIA (Pla2g2a) gene was previously identified as a modifier of intestinal adenoma multiplicity in Apc Min/+ mice. To determine if intestinal secreted phospholipase A2 (sPLA2) activity was also attenuated in susceptible strains, we developed a sensitive assay to directly quantitate sPLA2 activity in the murine intestinal tract utilizing a fluorescent BODIPY-labeled phospholipid substrate. Here, we report assay conditions that distinguish between secreted and cytosolic PLA2 enzyme activities in extracts of intestinal tissue. The small intestine exhibited higher activity levels than the large intestine. Consistent with predictions from the sPLA2-IIA gene sequence in inbred strains, we detected low levels of enzyme activity in inbred strains containing sPLA2-IIA mutations; these strains were also associated with greater numbers of intestinal polyps. Additionally, the assay was able to distinguish differences in levels of sPLA2 activity between neoplasia-resistant strains, which were then shown by sequencing to carry variant wild-type sPLA2-IIA alleles. Immunohistochemical analyses of intestinal tissues were consistent with sPLA2-IIA activity levels. This approach enables further studies of the mechanisms of sPLA2 action influencing the development and tumorigenesis of the small intestine and colon in both mice and humans.

Differential binding to and biofilm formation on, HEp-2 cells by Salmonella enterica serovar Typhimurium is dependent upon allelic variation in the fimH gene of the fim gene cluster.

Type 1 fimbria-mediated adherence to HEp-2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose-sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp-2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp-2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp-2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp-2 cells using once-flow-through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process.

A Randomised Controlled Trial of a ProbioticLactobacillusStrain in Healthy Adults: Assessment of its Delivery, Transit and Influence on Microbial Flora and Enteric Immunity

In several intestinal disease states, altered microflora, impaired gut barrier and:or intestinal inflammation offer a rationale for the effective therapeut ic use of probiotic microorganisms. However, for most candidate probiotic organisms there is a lack of evidence detailing their characterisation and effects on host flora and immunity. We have previously reported the isolation and characterisation, from surgically resected segments of the human gastrointestinal tract (GIT), of potential probiotic lactic acid bacteria (LAB). We have also described subsequen t animal experiments that evaluated the establishment, persistence and localisation of specific probiotic Lactobacillus strains within the murine intestinal tract, in addition to their ability to in uence the development of murine inflammatory disorders. In these studies, transit and survival of Lactobacillus salivarius UCC118 at the ileum was demonstrated using enteral tube sampling of six healthy volunteer s following consumption of a single dose (150 ml) of fermented milk-borne probiotic (10 8 colony forming units per ml (CFU/ml)). Subsequently, we performed a randomised controlled trial of 80 volunteer s fed strain UCC118 (10 8 CFU/day for 21 d), using two oral delivery vehicles (fresh milk, n= 20 vs. fermented milk, n=2 0; controls, n= 20 for each). Throughout this feeding period, and for up to 100 days following cessation of feeding, the numbers of total culturable lactobacilli and of the administered Lactobacillus UCC118 present in faeces were monitored. Five subjects (5/40; fresh milk, four; fermented milk, one) were still excreting the probiotic lactobacilli 21 days post-cessation of feeding, while one subject (fermented milk) was still colonised up to 100 days after feeding. Consumption of fermented milk-borne UCC118 cells resulted in significantly increased levels of faecal-borne enterococci and lactobacilli. Numbers of bifidobacteria, coliforms and bacteroides were not significantly altered. In addition, changes in salivary IgA levels against UCC118 cells and increased granulocyt e phagocytic activity were observed following consumption of the fermented milk-borne probiotic. In summary, Lactobacillus UCC118 was found to effectively transit (and persist within) the human intestinal tract, to modify the faecal flora and to engage the immune system. Keywords: controlled trial, probiotic, Lactobacillus salivarius , transit, flora, enteric immunity.

CYP2C40, a unique arachidonic acid 16-hydroxylase, is the major CYP2C in murine intestinal tract.

We recently identified five different murine CYP2C cDNAs from a murine cDNA library. When expressed in a bacterial cDNA expression system, all five recombinant proteins metabolized arachidonic acid but produced distinctly different profiles. In addition, some CYP2C mRNAs were found in extrahepatic tissues, as well as in liver. Immunoblots with an antibody raised against recombinant CYP2C38, which recognizes all five murine CYP2Cs, demonstrated that among extrahepatic tissues, colon and cecum contained the highest amount of CYP2Cs. The highest concentration of CYP2Cs occurred in cecum and colon (cecum >/= proximal colon >> distal colon), with lower levels in duodenum, jejunum, and ileum. Immunohistochemical studies revealed that CYP2Cs were localized principally in epithelial cells and autonomic ganglia in gut and colon. Polymerase chain reaction amplification of reverse-transcribed mRNA using murine CYP2C-specific primers followed by cloning and sequencing identified CYP2C40 as the major CYP2C isoform expressed in murine intestinal tract. Recombinant CYP2C40 metabolized arachidonic acid in a regio- and stereospecific manner to 16(R)-HETE (hydroxyeicosatetraenoic acid) as the major product. To our knowledge, CYP2C40 is the first enzyme known to produce primarily 16-HETE. We conclude that CYP2C40 is one of the major cytochrome P450 proteins in the mouse intestinal tract. In the light of vasoactive and anti-neutrophilic effects of 16-HETE, we hypothesize that CYP2C40 may play an important role in endogenous biological functions in intestine.

Colonization Capabilities of Environmental Pseudomonads and anEscherichia coliIntestinal Isolate in Two Different Continuous-Flow Models of the Murine Intestinal Tract

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Colonization Capabilities of Environmental Pseudomonads and anEscherichia coliIntestinal Isolate in Two Different Continuous-Flow Models of the Murine Intestinal Tract

Colonization Capabilities of Environmental Pseudomonads and an<i>Escherichia coli</i>Intestinal Isolate in Two Different Continuous-Flow Models of the Murine Intestinal Tract

Intestinal physiology and pathology in gene-targeted mouse models of cystic fibrosis.

Cystic fibrosis (CF) is a fatal genetic disorder that affects approximately 1 in 2,500 live Caucasian births. The disease can be described as a generalized exocrine disease affecting a variety of epithelial tissues, with early manifestation as meconium ileus in a significant number of neonates. Cloning of the gene causing CF was accomplished in 1989, and the protein product, cystic fibrosis transmembrane conductance regulator (CFTR), has been conclusively shown to be an adenosine 3',5'-cyclic monophosphate (cAMP)-regulated Cl- channel. Subsequently, several mouse models of CF were generated by gene-targeting approaches in an attempt to further understand this disease. The initial excitement generated by the emergence of these mouse models was somewhat tempered by the finding that none of the models developed airway disease, which is currently responsible for most of the morbidity and mortality in the human CF population. However, the various CF mouse models, of which there are now 10, are remarkably similar to their human counterparts with respect to intestinal pathophysiology. Most importantly, the intestinal tract of the CF mouse models demonstrates the absence of cAMP-mediated Cl- transport, which is a hallmark of CF disease. Furthermore, the murine CF intestinal tract also shows an inability to secrete HCO3-, defective cAMP regulation of electroneutral NaCl absorption, and elevated electrogenic Na+ transport in the distal colon, as well as other ion transport perturbations. Besides the fundamental mechanisms of ion transport studied in the murine CF intestinal tract, these models have also been important in understanding other tissues with regard to CF. Mice heterozygous for the CFTR knockout gene have a reduced ability to secret Cl- and fluid and therefore provide further support for the CF "heterozygote advantage" hypothesis. Some CF mouse models maintain a limited ability to secrete Cl-, which may be due to accessory genes that are hypothesized to ameliorate disease severity in the intestines of these mice. This review describes the CF models generated and compares the murine defects in ion transport with observed abnormalities in the human CF intestine.

Impairment of Kit-dependent development of interstitial cells alters contractile responses of murine intestinal tract.

We examined developmental changes in responses of the isolated segment of the ileum of BALB/c mice treated with a monoclonal antibody (ACK2) to the receptor tyrosine kinase (Kit) for 4 days postnatally to pharmacological agents in vitro. Rhythmic contraction and relaxation of the isolated ileum started to appear on day 4 postpartum, and the sensitivity to acetylcholine (ACh) decreased gradually after birth. Treatment with ACK2 induced augmentation of contractile responses and receptor sensitivity of the longitudinal muscle of the ileum to ACh, bradykinin, and prostaglandin F2 alpha. ACh induced larger depolarization in smooth muscle cells of the ileum in the ACK2-treated mice than in the control. Circular muscle responses to these substances, as measured by changes in intraluminal pressure, were not altered by ACK2 treatment. Results suggest that interstitial cells play an important role not only in the development of the pacemaking system of the small intestine but also in the functional development of the contractile properties of the intestinal smooth muscle.

Development of anin vitroContinuous Flow Culture Model of the Murine Intestinal Tract

Enzyme activities and bacterial populations were compared in single stage, two stage (pH=5 and 7), and two stage with cell recycling continuous cultures of mouse faecal microbiota. Results were compared to in vivo findings in the mouse caecum and faeces in order to choose the system which best models the ecology of the murine lower intestinal tract. Fecal homogenates had higher numbers of total aerobes and lactose fermenting enterics than caecal homogenates. The microbial balance of the continuous culture systems more closely resembled the faecal microbiota (the culture inoculum) than the caecal microbiota. Lactobacilli numbers were significantly lower in single stage cultures than in two stage cultures or in vivo samples. Enzyme activities were comparable between culture types, with the exception of β-glucuronidase, which was closest to the in vivo level in single stage cultures. Because no one in vitro system proved superior with regards to all the parameters studied, the choice of which system to use may be based largely on the study purpose. Keywords: continuous culture, intestinal microbiota, enzymes.

Development of an in vitro Continuous Flow Culture Model of the Murine Intestinal Tract

Development of an in vitro Continuous Flow Culture Model of the Murine Intestinal Tract

Development of anin vitroContinuous Flow Culture Model of the Murine Intestinal Tract

Development of an<i>in vitro</i>Continuous Flow Culture Model of the Murine Intestinal Tract