Fecal Colonization Research Articles

Efficacy of topical versus oral 5-aminosalicylate for treatment of 2,4,6-trinitrobenzene sulfonic acid-induced ulcerative colitis in rats

5-aminosalicylic acid (5-ASA) is drug of choice for the treatment of ulcerative colitis (UC). In this study, the efficacy of topical versus oral 5-ASA for the treatment of UC was examined as well as the action mechanism of this medication. A flexible tube was inserted into the rat cecum to establish a topical administration model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced UC. A total of 60 rats were divided into sham operation group (receiving an enema of 0.9% saline solution instead of the TNBS solution via the tube), model group, topical 5-ASA group, oral Etiasa group (a release agent of mesalazine used as positive control) and oral 5-ASA group (n=12 each). Different treatments were administered 1 day after UC induction. The normal saline (2 mL) was instilled twice a day through the tube in the sham operation group and model group. 5-ASA was given via the tube in the topical 5-ASA group (7.5 g/L, twice per day, 100 mg/kg), and rats in the oral Etiasa group and oral 5-ASA group intragastrically received Etiasa (7.5 g/L, twice per day, 100 mg/kg) and 5-ASA (7.5 g/L, twice per day, 100 mg/kg), respectively. The body weight was recorded every day. After 7 days of treatment, blood samples were drawn from the heart to harvest the sera. Colonic tissues were separated and prepared for pathological and related molecular biological examinations. The concentrations of 5-ASA were detected at different time points in the colonic tissues, feces and sera in different groups by using the high pressure liquid chromatography (HPLC). The results showed that the symptoms of acute UC, including bloody diarrhea and weight loss, were significantly improved in topical 5-ASA-treated rats. The colonic mucosal damage, both macroscopical and histological, was significantly relieved and the myeloperoxidase activity was markedly decreased in rats topically treated with 5-ASA compared with those treated with oral 5-ASA or Etiasa. The mRNA and protein expression of IL-1β, IL-6, and TNF-α was down-regulated in the colonic tissue of rats topically treated with 5-ASA, significantly lower than those from rats treated with oral 5-ASA or Etiasa. The concentrations of 5-ASA in the colonic tissue were significantly higher in the topical 5-ASA group than in the oral 5-ASA and oral Etiasa groups. It was concluded that the topical administration of 5-ASA can effectively increase the concentration of 5-ASA in the colonic tissue, decrease the expression of proinflammatory cytokines, alleviate the colonic pathological damage and improve the symptoms of TNBS-induced acute UC in rats.

Fatty Acid Synthase Inhibitor C75 Ameliorates Experimental Colitis

Abnormalities of lipid metabolism through overexpression of fatty acid synthase (FASN), which catalyzes the formation of long-chain fatty acids, are associated with the development of inflammatory bowel disease (IBD). C75 is a synthetic α-methylene-γ-butyrolactone compound that inhibits FASN activity. We hypothesized that C75 treatment could effectively reduce the severity of experimental colitis. Male C57BL/6 mice were fed 4% dextran sodium sulfate (DSS) for 7 d. C75 (5 mg/kg body weight) or dimethyl sulfoxide (DMSO) (vehicle) was administered intraperitoneally from d 2 to 6. Clinical parameters were monitored daily. Mice were euthanized on d 8 for histological evaluation and measurements of colon length, chemokine, cytokine and inflammatory mediator expression. C75 significantly reduced body weight loss from 23% to 15% on d 8, compared with the vehicle group. The fecal bleeding, diarrhea and colon histological damage scores in the C75-treated group were significantly lower than scores in the vehicle animals. Colon shortening was significantly improved after C75 treatment. C75 protected colon tissues from DSS-induced apoptosis by inhibiting caspase-3 activity. Macrophage inflammatory protein 2, keratinocyte-derived chemokine, myeloperoxidase activity and proinflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1β and IL-6) in the colon were significantly downregulated in the C75-treated group, compared with the vehicle group. Treatment with C75 in colitis mice inhibited the elevation of FASN, cyclooxygenase-2 and inducible nitric oxide synthase expression as well as IκB degradation in colon tissues. C75 administration alleviates the severity of colon damage and inhibits the activation of inflammatory pathways in DSS-induced colitis. Thus, inhibition of FASN may represent an attractive therapeutic potential for treating IBD.

Comparison of intestinal contents from different regions of the equine gastrointestinal tract as inocula for use in an in vitro gas production technique

The aim of the experiment reported here was to compare equine faeces with inocula obtained from equine caecum, ventral colon and dorsal colon for use in the in vitro gas production technique. Freeze-dried grass (FDG), high-temperature dried grass (HDG) and unmolassed sugar beet pulp (BP) were incubated with either caecal fluid (C), dosal colonic fluid (D), ventral colonic fluid (V) or faeces (F) as the source of inoculum. Substrate/inocula combinations were fermented using an in vitro gas production (GP) technique. Mathematical analysis of cumulative gas production curves revealed an interaction (P<0.001) between inocula and substrate for all of the in vitro parameters measured. However, similar extents of gas production were seen for inocula obtained from caecal fluid and faeces across all substrates, with lower values obtained from bottles incubated with colonic fluid. Thus, results indicate that faeces are a suitable alternative to caecal fluid; however, it would appear that they are less effective in modelling fermentation in the colon. Consequently, further work is required to determine the use of faeces as an alternative to caecal and colonic fluid and to further investigate inocula/substrate interactions.

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin

Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8±5.2ng/ml in proximal small intestine to 54.3±10.3ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.

Risk factors of fecal toxigenic or non-toxigenic Clostridium difficile colonization: impact of Toll-like receptor polymorphisms and prior antibiotic exposure.

BackgroundThis study is to investigate the significance and risk factors of fecal toxigenic (tCdC) or non-toxigenic Clostridium difficile colonization (ntCdC) among hospitalized patients.MethodsAdults admitted to medical wards in a district hospital between January 2011 and June 2012 were enrolled, and those with a history of colectomy, C. difficile fecal colonization or infection or receipt of either metronidazole or oral vancomycin within 3 months, were excluded. Stools collected within 48 hours after admission and every week during hospitalization were cultured for C. difficile.FindingsAmong the 441 enrolled patients, 84 (20.0%) had CdC at initial screening, including 58 (13.2%) with tCdC and 26 (6.8%) with ntCdC. Among patients with initial negative fecal screening for CdC, it took an average of 70.6 days or 66.5 days to develop tCdC or ntCdC during the study period. Finally 78 (17.7%) had tCdC and 34 (7.7%) had ntCdC. During the follow-up period, the patients with tCdC had a higher risk of CDAD (11/79, 14.1%) than those without CdC (3/328, 0.9%) and those with ntCdC (0/34, 0%) (P<0.001). In multivariate analysis, the TLR4 rs1927914 polymorphism (GG genotype) (odds ratio [OR] 4.4, 95% confidence interval [CI] 1.6–11.8, P = 0.003) and recent cefepime therapy (OR 5.3, 95% CI 2.1–13.2, P<0.001) were independently associated with tCdC, whereas recent cefuroxime (OR 11.7, 95% CI 2.3–60.2, P = 0.003) and glycopeptide therapy (OR 10.9, CI: 2.1–57.2, P = 0.005) associated with ntCdC.ConclusionThe incidence of CDAD is highest in patients with tCdC and lowest in patients with ntCdC, and the TLR4 rs1927914 polymorphism GG genotype and recent cefepime therapy were independently associated with tCdC.

Fecal Colonization with ESBL-Producing

Gram-negative gut bacteria — Escherichia coli — constitute the reservoir from which urinary tract infections (UTIs) originate. The prevalence of

Travel-associated faecal colonization with ESBL-producing Enterobacteriaceae: incidence and risk factors

To study the acquisition of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) among the faecal flora during travel, with a focus on risk factors, antibiotic susceptibility and ESBL-encoding genes. An observational prospective multicentre cohort study of individuals attending vaccination clinics in south-east Sweden was performed, in which the submission of faecal samples and questionnaires before and after travelling outside Scandinavia was requested. Faecal samples were screened for ESBL-PE by culturing on ChromID ESBL and an in-house method. ESBL-PE was confirmed by phenotypic and genotypic methods. Susceptibility testing was performed with the Etest. Individuals who acquired ESBL-PE during travel (travel-associated carriers) were compared with non-carriers regarding risk factors, and unadjusted and adjusted ORs after manual stepwise elimination were calculated using logistic regression. Of 262 enrolled individuals, 2.4% were colonized before travel. Among 226 evaluable participants, ESBL-PE was detected in the post-travel samples from 68 (30%) travellers. The most important risk factor in the final model was the geographic area visited: Indian subcontinent (OR 24.8, P < 0.001), Asia (OR 8.63, P < 0.001) and Africa north of the equator (OR 4.94, P = 0.002). Age and gastrointestinal symptoms also affected the risk significantly. Multiresistance was seen in 77 (66%) of the ESBL-PE isolates, predominantly a combination of reduced susceptibility to third-generation cephalosporins, trimethoprim/sulfamethoxazole and aminoglycosides. The most common species and ESBL-encoding gene were Escherichia coli (90%) and CTX-M (73%), respectively. Acquisition of multiresistant ESBL-PE among the faecal flora during international travel is common. The geographical area visited has the highest impact on ESBL-PE acquisition.

Abstract 4426: Metabolism and tissue distribution of curcumin in mouse GI tract.

Abstract Purpose: Curcuminoids have shown pronounced preventive activities on gastrointestinal (GI) cancers in preclinical settings and have now advanced to clinical evaluation. However, their tissue distribution and metabolism in the GI tissues remain largely unknown. The objective of this study is to establish an LC-MS/MS method to profile curcumin (CUR), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC) and CUR Metabolites: CUR O-glucuronide (COG), CUR O-sulfate (COS), and tetrahydrocurcumin (THC) in plasma, and to study their disposition and kinetics in the GI tissues and lumens. Methods: The following ion transitions at m/z: 367.4/149.1, 337.3/216.9, 307.5/186.8, 543.7/216.9, 447.4/216.9, and 371.2/235.1, and 301.5/163.9 were used for monitoring CUR, DMC, BDMC, COG, COS, THC and hesperetin (internal standard), respectively, on an API mass spectrometer. Nano-emulsion CUR (50 mg/kg) was orally administered to CD2F1 mice. Plasma, stomach tissues (ST) and lumens (SL), intestine tissue (IT) and lumens (IL), and colon tissue (CT) and feces (F) were collected up to 6 h after the dose and analyzed using an LC-MS/MS method. Results: CUR and its metabolites were simultaneously quantified in mouse plasma, GI tissue homogenates, and GI lumens using the LC-MS/MS method with a linear range of 2 to 1000 ng/mL. COG was found to be the most abundant circulating CUR metabolite with a peak concentration of 3.99 μM in plasma at 30 min, which is approximately 8 and 36 fold higher than that of CUR and COS, respectively. In contrast, CUR was the most abundant transient species in ST, IT and CT, and reached pharmacological effective peak concentrations (μM) of 118, 55, and 142 in ST, IT and CT at 20, 60 and 30 min, respectively. The concentrations of CUR were 8261, 923 and 9.74 μM in SL, IL and F at 30, 60 and 30 min, respectively. COG and COS were two main metabolites of CUR with relative lower peak concentrations of 6.46 and 8.79 μM in IT and 30.4 and 80.1 μM in IL at 60 min, respectively, while COG and COS concentrations were much lower in ST, CT and F, and they were not detectable in SL. Notably, THC was the main metabolite of CUR with peak concentrations of 24 and 1.89 μM in ST and CT at 10 and 360 min, respectively, while no THC was detected in SL, IT, IL and F. In addition, DMC, BDMC, and THC, and their glucuronide and sulfate adducts were also profiled using these ion transition channels. Conclusions: The LC-MS/MS method provides a critical analytic method to study the metabolism, tissue distribution, and pharmacokinetics (PK) of CUR in mice. These results demonstrated that orally ingested CUR could deliver pharmacological effective level of CUR to GI tissues and lumens, which is completely different from that in plasma. Therefore, further investigation of curcuminoids as a preventive or therapeutic agent for GI cancers is warranted. Supported by R21CA135478 (Liu), CA159077-01 (Liu &amp; Xu) and Biomedical Mass Spectrometric Laboratory (Chan &amp; Liu). Citation Format: Yu Cao, Ming Chiu, Kenneth K. Chan, Ronald Xu, Zhongfa Liu. Metabolism and tissue distribution of curcumin in mouse GI tract. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4426. doi:10.1158/1538-7445.AM2013-4426

Effect of essential oil compound on shedding and colonization of Salmonella enterica serovar Heidelberg in broilers

The objectives of this study were to determine the effect of an essential oil blend (EO; carvacrol, thymol, eucalyptol, lemon) administered in drinking water on the performance, mortality, water consumption, pH of crop and ceca, and Salmonella enterica serovar Heidelberg fecal shedding and colonization in broiler birds following Salmonella Heidelberg challenge and feed withdrawal. Chicks were randomly assigned to water treatments containing 0.05, 0.025, or 0.0125% EO or untreated controls. Treatments were administered in drinking water on 0 to 7 and 35 to 42 d. One-half of the chicks were challenged with Salmonella Heidelberg and placed in pens with unchallenged chicks on d 1. Performance, mortality, water consumption, and pH were determined during the 42-d study. Prevalence of Salmonella Heidelberg was determined on drag swabs (0, 14, and 42 d) and in the ceca and crops (42 d). The 0.05% EO administered in drinking water significantly (P < 0.05) reduced Salmonella Heidelberg colonization in crops of challenged birds, significantly lowered the feed conversion ratio, and increased weight gain compared with controls. The 0.025% and 0.015% EO in drinking water significantly lowered the feed conversion ratio and increased weight gain compared with controls, but did not significantly reduce Salmonella Heidelberg colonization in the crops. The EO in drinking water did not significantly reduce Salmonella Heidelberg colonization in ceca or fecal shedding in broilers. The EO used in the study may control Salmonella Heidelberg contamination in crops of broilers when administered in drinking water and therefore may reduce the potential for cross-contamination of the carcass when the birds are processed.

Dissemination of plasmid-mediated fosfomycin resistance fosA3 among multidrug-resistant Escherichia coli from livestock and other animals

To investigate plasmid-mediated fosfomycin resistance related to fosA3 in Escherichia coli isolates collected from different animals in Hong Kong, China, 2008-2010. In total, 2106 faecal specimens from 210 cattle, 214 pigs, 460 chickens, 398 stray cats, 368 stray dogs and 456 wild rodents were cultured. The faecal colonization rates of fosfomycin-resistant E. coli were as follows: 11.2% in pigs, 8.6% in cattle, 7.3% in chickens, 2.4% in dogs, 0.8% in cats and 1.5% in rodents. The cultures yielded 1693 isolates of which 831 were extended-spectrum β-lactamases (ESBL) producers. Fosfomycin-resistant isolates were more likely than fosfomycin-susceptible isolates to be producers of ESBL and to have resistance to chloramphenicol, ciprofloxacin, cotrimoxazole, gentamicin and tetracycline. Of the 101 fosfomycin-resistant isolates, 97 (96.0%) isolates were fosA3 positive and 94 (93.1%) were bla(CTX) (-M) positive. PCR mapping showed that the fosA3-containing regions were flanked by IS26, both upstream and downstream in 81 (83.5%) isolates, and by an upstream bla(CTX-M-14) -containing transposon-like structure (ΔISEcp1-bla(CTX-M-14) -ΔIS903 or ISEcp1-IS10 -bla(CTX-M-14) -ΔIS903) and a downstream IS26 in 14 (14.4%) isolates. For the remaining two isolates, fosA3 was flanked by a downstream IS26 but the upstream part cannot be defined. In a random subset of 18 isolates, fosA3 was carried on transferable plasmids with sizes of 50-200 kb and the following replicons: F2:A-B- (n = 3), F16:A1:B- (n = 2), F24:A-B- (n = 1), N (n = 1), B/O (n = 1) and untypeable (n = 3). This study demonstrates the emergence of fosA3-mediated fosfomycin resistance among multidrug-resistant E. coli isolates from various animals. IS26 transposon-like structures might be the main vehicles for dissemination of fosA3.

Risk factors for acquisition of CTX-M-15 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae during an outbreak in a neonatal intensive care unit in Norway

A CTX-M-15 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae was responsible for an outbreak in the neonatal intensive care unit (NICU) at Stavanger University Hospital, Norway over a 5-month period (November 2008-April 2009). The risk factors for acquiring ESBL-producing K. pneumoniae during the outbreak were examined in this study. Faecal or rectal cultures were obtained from infants hospitalized in the NICU during the outbreak period and examined for ESBL-producing K. pneumoniae. Data were retrospectively retrieved from the medical records, including sex, gestational age, birth weight, indwelling central vascular catheter, continuous positive airway pressure (CPAP), mechanical ventilation, parenteral nutrition, antibiotic treatment, mode of delivery (vaginal vs caesarean), length of hospital stay, and mortality. A total of 216 infants were hospitalized in the NICU during the outbreak period, of whom 212 were screened; 51 (24%) scored positive for faecal colonization with ESBL-producing K. pneumoniae. One infant acquired a clinical infection. Forty-four colonized infants and 55 non-colonized infants were included in the risk analysis. Colonized infants had a lower birth weight, lower gestational age, and a longer hospital stay compared to non-colonized infants. By logistic regression, prematurity (gestational age <37 weeks) and treatment with antibiotics were independent risk factors for acquiring ESBL-producing K. pneumoniae in the final model. Prematurity and treatment with antibiotics were independent risk factors for colonization during this NICU outbreak with ESBL-producing K. pneumoniae.

Risk factors for KPC-producing Klebsiella pneumoniae faecal colonization of ICU patients

Risk factors for KPC-producing Klebsiella pneumoniae faecal colonization of ICU patients

Isolation of Campylobacter from Feral Swine (Sus scrofa) on the Ranch Associated with the 2006 Escherichia coli O157:H7 Spinach Outbreak Investigation in California

We report the isolation of Campylobacter species from the same population of feral swine that was investigated in San Benito County, California, during the 2006 spinach-related Escherichia coli O157:H7 outbreak. This is the first survey of Campylobacter in a free-ranging feral swine population in the United States. Campylobacter species were cultured from buccal and rectal-anal swabs, colonic faeces and tonsils using a combination of selective enrichment and antibiotic-free membrane filtration methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS, Bruker Daltonics, Inc., Billerica, MA, USA) was used to identify species followed by confirmatory multiplex PCR or 16S rRNA sequencing. Genetic relatedness of Campylobacter jejuni and Campylobacter coli strains was determined by multilocus sequence typing (MLST) and porA allele sequencing. Altogether, 12 (40%) of 30 feral swine gastrointestinal and oral cavity specimens were positive, and six species were isolated: Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalsis, Campylobacter jejuni, Campylobacter lanienae and Campylobacter sputorum. Campylobacter jejuni subtypes were closely related to MLST sequence type 21 (ST-21) and had identical porA sequences. Campylobacter coli subtypes were unrelated to isolates in the pubMLST/porA database. This feral swine population lived in close association with a 'grassfed' beef cattle herd adjacent to spinach and other leafy green row crop fields. The findings underscore the importance of protecting raw vegetable crops from faecal contamination by wild or feral animals. The study also illustrates a potential risk of Campylobacter exposure for hunters during handling and processing of wild swine meat.

Colonization of a Marker and Field Strain of Salmonella Enteritidis and a Marker Strain of Salmonella Typhimurium in Vancomycin-Pretreated and Nonpretreated Laying Hens

This study was conducted to evaluate the influence of a vancomycin pretreatment on the ability of marker (nalidixic-acid resistant) Salmonella Enteritidis (SE(M)), field Salmonella Enteritidis (SE(E)), and marker Salmonella Typhimurium (ST(M)) strains to colonize within the intestinal and reproductive tracts and translocate to other organs of leghorn laying hens. In each of three trials, caged laying hens (76, 26, and 33 wk ofage) were divided into six groups designated to receive SE(M), SE(F), or ST(M), and half were pretreated with vancomycin (n = 11-12 hens). Vancomycin-treated hens received 10 mg vancomycin in saline/kilogram body weight orally for 5 days to inhibit Gram-positive bacteria within the intestines. On Day 6, all hens were concurrently challenged by oral, intravaginal, and intracolonal routes with Salmonella and placed into separate floor chambers by Salmonella strain. Two weeks postinoculation, all hens were euthanatized and the ceca, spleen, liver/gall bladder (LGB), upper (URT), and lower (LRT) reproductive tracts, and ovarian follicles were aseptically collected, and analyzed for Salmonella. Results did not differ for the three hen's ages and were therefore combined. The vancomycin pretreatment also had no significant effect on the colonization ability of SE(M), SE(F) or ST(M), and therefore results were combined within Salmonella strain. The marker strain of Salmonella Enteritidis was recovered from 21% of ceca, 4% of LGB, 9% of LRT, and 17% of the fecal samples. The field strain of Salmonella Enteritidis was recovered from 88% of ceca, 96% of spleen, 92% of LGB, 30% of LRT, 4% of URT, 13% of follicle, and 42% of the fecal samples. The marker strain of Salmonella Typhimurium was recovered from 100% of ceca, 74% of spleen, 91% of LGB, 30% of LRT, 9% of URT, 9% of follicle, and 100% of the fecal samples. Among ceca, spleen, LGB, and fecal samples, SE(F) and ST(M) colonization was significantly greater than SE(M) colonization. Overall prevalence of Salmonella in the reproductive tracts of challenged hens was relatively low, ranging from 4% to 30%.

A combination of calcium phosphate and probiotics beneficially influences intestinal lactobacilli and cholesterol metabolism in humans

The study focuses on the influence of a probiotic supplement alone and in combination with a calcium supplement on faecal lactobacilli colonisation and beneficial health effects such as a lowering of blood cholesterol. Thirty-two men and women participated in the double-blind, placebo-controlled, cross-over study. All participants consumed a probiotic drink containing 10(10)CFU/d Lactobacillus paracasei (LPC37) for four weeks. In addition, one group consumed bread enriched with pentacalcium hydroxy-triphosphate (CaP; 1g Ca/d) and the other group had bread without CaP. After a two-week washout and a two-week placebo period, the intervention was switched for further four weeks. After intervention with LPC37+CaP, total cholesterol and LDL-cholesterol concentration in plasma decreased significantly compared to LPC37 and placebo. The faecal concentration of L.paracasei and that of all lactobacilli increased significantly after LPC37+CaP and LPC37 compared to placebo. Moreover, secondary bile acids in faeces increased significantly after LPC37+CaP intervention compared to placebo. CaP modulates the colonisation of LPC37 in the human gut under combinatory supplementation of CaP and LPC37. The combined supplementation also decreases plasma LDL-cholesterol and the LDL/HDL ratio in healthy, moderately hypercholesterolemic men and women, which could be also due to the CaP supplementation. NCT01033461.

Hypertonic Stress Induces VEGF Production in Human Colon Cancer Cell Line Caco-2: Inhibitory Role of Autocrine PGE2

Vascular Endothelial Growth Factor (VEGF) is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PG)E2 are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE2 generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE2 generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE2 production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE2 on VEGF production, Caco-2 cells were treated with cPLA2 (ATK) and COX-2 (NS-398) inhibitors, that completely block PGE2 generation. The Caco-2 cells were also treated with a non selective PGE2 receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE2 or selective EP2 receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE2 appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl2 was decreased by inhibition of concomitant PGE2 generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE2 plays an inhibitory role on VEGF production by Caco-2 cells exposed to hyperosmotic stress through EP2 activation.

Chronic stress-induced alterations in mouse colonic 5-HT and defecation responses are strain dependent

Mood disorders and chronic stress are frequently associated with gastrointestinal (GI) symptoms including diarrhoea or constipation. Locally produced serotonin [5-hydroxytryptamine (5-HT)] regulates GI motility and is a key factor in the pathophysiology of stress-associated GI disorders. We aimed to establish whether chronic stress can differentially affect faecal output and colon 5-HT concentration in two inbred mouse strains: BALB/c and C57BL/6 which differ in their ability to cope with stress. Adult male BALB/c and C57BL/6 mice were restrained for 2 h daily for 10 days. Defecation was monitored during each stress session. Twenty-four hours after the last session of stress, plasma corticosterone concentration was higher than control in both strains, indicative of a physiological effect of chronic stress; however, stress-induced diarrhoea was more persistent in C57BL/6 mice. Basal concentration of colon 5-HT was higher in C57BL/6 mice, and stress elicited an increase in colon 5-HT only in this strain. Finally, naïve BALB/c mice had a higher sensitivity (incidence of diarrhoea) to 5-HT (0.33 mg/kg, i.p.) than C57BL/6 mice. Our results suggest that differential defecation responses to stress may be associated with colon 5-HT concentration, which may in turn reflect the individual sensitivity to 5-HT. In addition, C57BL/6 mice emerge as a relevant model for studying GI alterations induced by chronic stress.

Bovine Intestinal Bacteria Inactivate and Degrade Ceftiofur and Ceftriaxone with Multiple β-Lactamases

The veterinary cephalosporin drug ceftiofur is rapidly degraded in the bovine intestinal tract. A cylinder-plate assay was used to detect microbiologically active ceftiofur, and high-performance liquid chromatography-mass spectrometry analysis was used to quantify the amount of ceftiofur remaining after incubation with bovine intestinal anaerobic bacteria, which were isolated from colon contents or feces from 8 cattle. Ninety-six percent of the isolates were able to inactivate ceftiofur to some degree, and 54% actually degraded the drug. None of 9 fungal isolates inactivated or degraded ceftiofur. Facultative and obligate anaerobic bacterial species that inactivated or degraded ceftiofur were identified with Vitek and Biolog systems, respectively. A subset of ceftiofur degraders also degraded the chemically similar drug ceftriaxone. Most of the species of bacteria that degraded ceftiofur belonged to the genera Bacillus and Bacteroides. PCR analysis of bacterial DNA detected specific β-lactamase genes. Bacillus cereus and B. mycoides isolates produced extended-spectrum β-lactamases and metallo-β-lactamases. Seven isolates of Bacteroides spp. produced multiple β-lactamases, including possibly CepA, and metallo-β-lactamases. Isolates of Eubacterium biforme, Bifidobacterium breve, and several Clostridium spp. also produced ceftiofur-degrading β-lactamases. An agar gel overlay technique on isoelectric focusing separations of bacterial lysates showed that β-lactamase enzymes were sufficient to degrade ceftiofur. These results suggest that ceftiofur is inactivated nonenzymatically and degraded enzymatically by multiple β-lactamases from bacteria in the large intestines of cattle.

High-density fecal Enterococcus faecium colonization in hospitalized patients is associated with the presence of the polyclonal subcluster CC17

Enterococcus faecium belonging to the polyclonal subcluster CC17, with a typical ampicillin-resistant E. faecium (AREfm) phenotype, have become prevalent among nosocomial infections around the world. High-density intestinal AREfm colonization could be one of the factors contributing to the successful spread of these pathogens. We aimed to quantify the enterococcal intestinal colonization densities in stool samples from AREfm-colonized and non-colonized patients using fluorescent in situ hybridization (FISH). Stool samples were collected from AREfm-colonized (n = 8) and non-colonized (n = 8) patients. The relative number of Enterococcus faecalis and E. faecium was determined by FISH using specific 16S rRNA probes, while the total amount of bacterial cells was counted by staining the sample with 4',6-diamidino-2-phenylindole (DAPI). The median bacterial cell numbers in fecal samples, counted by DAPI staining, were 7.7 × 10(9) and 4.8 × 10(9) cells/g for AREfm-colonized and non-colonized patients, respectively (p = 0.34). The E. faecium densities in AREfm-colonized patients, accounting for 0.5-7% of all fecal bacterial cells, exceeded E. faecalis levels by over ten-fold. E. faecium was not detected in non-colonized patients. This study demonstrated high E. faecium cell densities in stool samples from patients colonized with AREfm. Increased cell densities may contribute to host-to-host transmission and environmental contamination, facilitating the spread of AREfm in the hospital setting.

Çocuklarda Genişletilmiş Spektrumlu Beta Laktamaz Üreten Enterobacteriaceae Fekal Taşıyıcılığı Prevalansı ve Risk Faktörleri

Objective: The isolation of extended-spectrum β-lactamase (ESBL) -producing bacteria has increased worldwide. Fecal colonization with ESBLproducing isolates is considered a prerequisite for infection. Material and Methods: We prospectively evaluated the prevalence and risk factors for fecal carriage of ESBL-producing E. coli and Klebsiella spp. in hospitalized and ambulatory children. A total of 464 fecal samples from pediatric patients (270 hospitalized and 194 ambulatory) were collected from a tertiary care center in Turkey during a non-outbreak period. All stool samples were evaluated for ESBL production by the combination disc test and for imipenem susceptibility by the CLSI agar dilution method. Results: Sixty-six (24% of total) hospitalized children (25 with Escherichia coli, 39 with Klebsiella pneumoniae and 2 with Klebsiella oxytoca) and 14 (7.2% of total) ambulatory children (10 with E. coli, 3 with K. pneumoniae and 1 with K. oxytoca) had a positive fecal sample for ESBL-producing microorganisms. All isolates were susceptible to imipenem; however, the MIC values of strains isolated from hospitalized patients were 3-4 fold higher in dilution than were those of strains isolated from ambulatory children. Univariate analyses showed that intensive care unit stay, urinary catheterization, indwelling catheterization, surgical interventions and prior 3rd-generation cephalosporin usage were associated with ESBL positivity for the hospitalized patients. Recent 2nd- or 3rd-generation cephalosporin use was found to be