Trolox and ascorbic acid reduce direct and indirect oxidative stress in the IPEC-J2 cells, an in vitro model for the porcine gastrointestinal tract

Oxidative stress in the small intestinal epithelium is a major cause of barrier malfunction and failure to regenerate. This study presents a functional in vitro model using the porcine small intestinal epithelial cell line IPEC-J2 to examine the effects of oxidative stress and to estimate the antioxidant and regenerative potential of Trolox, ascorbic acid and glutathione monoethyl ester. Hydrogen peroxide and diethyl maleate affected the tight junction (zona occludens-1) distribution, significantly increased intracellular oxidative stress (CM-H(2)DCFDA) and decreased the monolayer integrity (transepithelial electrical resistance and FD-4 permeability), viability (neutral red) and wound healing capacity (scratch assay). Trolox (2 mM) and 1 mM ascorbic acid pre-treatment significantly reduced intracellular oxidative stress, increased wound healing capacity and reduced FD-4 permeability in oxidatively stressed IPEC-J2 cell monolayers. All antioxidant pre-treatments increased transepithelial electrical resistance and viability only in diethyl maleate-treated cells. Glutathione monoethyl ester (10 mM) pretreatment significantly decreased intracellular oxidative stress and monolayer permeability only in diethyl maleate-treated cells. These data demonstrate that the IPEC-J2 oxidative stress model is a valuable tool to screen antioxidants before validation in piglets

RpoS-independent evolution reveals the importance of attenuated cAMPCRP regulation in high hydrostatic pressure resistance acquisition in E. coli

High hydrostatic pressure (HHP) processing is an attractive non-thermal alternative to food pasteurization. Nevertheless, the large inter- and intra-species variations in HHP resistance among foodborne pathogens and the ease by which they can acquire extreme resistance are an issue of increasing concern. Since RpoS activity has been considered as a central determinant in the HHP resistance of E. coli and its pathovars, this study probed for the potential of an E. coli MG1655 ΔrpoS mutant to acquire HHP resistance by directed evolution. Despite the higher initial HHP sensitivity of the ΔrpoS mutant compared to the wild-type strain, evolved lineages of the former readily managed to restore or even succeed wild-type levels of resistance. A number of these ΔrpoS derivatives were affected in cAMP/CRP regulation, and this could be causally related to their HHP resistance. Subsequent inspection revealed that some of previously isolated HHP-resistant mutants derived from the wild-type strain also incurred a causal decrease in cAMP/CRP regulation. cAMP/CRP attenuated HHP-resistant mutants also exhibited higher resistance to fosfomycin, a preferred treatment for STEC infections. As such, this study reveals attenuation of cAMP/CRP regulation as a relevant and RpoS-independent evolutionary route towards HHP resistance in E. coli that coincides with fosfomycin resistance

Hurdle Technology Approach to Control Listeria monocytogenes Using Rhamnolipid Biosurfactant

This study evaluates the combination of mild heat with a natural surfactant for the inactivation of L. monocytogenes Scott A in low-water-activity (aw) model systems. Glycerol or NaCl was used to reduce the aw to 0.92, and different concentrations of rhamnolipid (RL) biosurfactant were added before heat treatment (60 °C, 5 min). Using glycerol, RL treatment (50–250 µg/mL) reduced bacterial population by less than 0.2 log and heat treatment up to 1.5 log, while the combination of both hurdles reached around 5.0 log reduction. In the NaCl medium, RL treatment displayed higher inactivation than in the glycerol medium at the same aw level and a larger synergistic lethal effect when combined with heat, achieving ≥ 6.0 log reduction at 10–250 µg/mL RL concentrations. The growth inhibition activity of RL was enhanced by the presence of the monovalent salts NaCl and KCl, reducing MIC values from >2500 µg/mL (without salt) to 39 µg/mL (with 7.5% salt). The enhanced antimicrobial activity of RL promoted by the presence of salts was shown to be pH-dependent and more effective under neutral conditions. Overall, results demonstrate that RL can be exploited to design novel strategies based on hurdle approaches aiming to control L. monocytogenes

Artificial rearing influences the morphology, permeability and redox state of the gastrointestinal tract of low and normal birth weight piglets

Background: In this study the physiological implications of artificial rearing were investigated. Low (LBW) and normal birth weight (NBW) piglets were compared as they might react differently to stressors caused by artificial rearing. In total, 42 pairs of LBW and NBW piglets from 16 litters suckled the sow until d19 of age or were artificially reared starting at d3 until d19 of age. Blood and tissue samples that were collected after euthanasia at 0, 3, 5, 8 and 19 d of age. Histology, ELISA, and Ussing chamber analysis were used to study proximal and distal small intestine histo-morphology, proliferation, apoptosis, tight junction protein expression, and permeability. Furthermore, small intestine, liver and systemic redox parameters (GSH, GSSG, GSH-Px and MDA) were investigated using HPLC. Results: LBW and NBW artificially reared piglets weighed respectively 40 and 33% more than LBW and NBW sow-reared piglets at d19 (P < 0.01). Transferring piglets to a nursery at d3 resulted in villus atrophy, increased intestinal FD-4 and HRP permeability and elevated GSSG/GSH ratio in the distal small intestine at d5 (P < 0.05). GSH concentrations in the proximal small intestine remained stable, while they decreased in the liver (P < 0.05). From d5 until d19, villus width and crypt depth increased, whereas PCNA, caspase-3, occludin and claudin-3 protein expressions were reduced. GSH, GSSG and permeability recovered in artificially reared piglets (P < 0.05). Conclusion: The results suggest that artificial rearing altered the morphology, permeability and redox state without compromising piglet performance. The observed effects were not depending on birth weight

Does virulence assessment of Vibrio anguillarum using sea bass (Dicentrarchus labrax) larvae correspond with genotypic and phenotypic characterization?

Background: Vibriosis is one of the most ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as Vibrio (Listonella) anguillarum. Despite a lot of research efforts, the virulence factors and mechanism of V. anguillarum are still insufficiently known, in part because of the lack of standardized virulence assays. Methodology/Principal Findings: We investigated and compared the virulence of 15 V. anguillarum strains obtained from different hosts or non-host niches using a standardized gnotobiotic bioassay with European sea bass (Dicentrarchus labrax L.) larvae as model hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic characteristics, the strains were characterized by random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (rep-PCR) analyses, as well as by phenotypic analyses using Biolog's Phenotype MicroArray (TM) technology and some virulence factor assays. Conclusions/Significance: Virulence testing revealed ten virulent and five avirulent strains. While some relation could be established between serotype, genotype and phenotype, no relation was found between virulence and genotypic or phenotypic characteristics, illustrating the complexity of V. anguillarum virulence. Moreover, the standardized gnotobiotic system used in this study has proven its strength as a model to assess and compare the virulence of different V. anguillarum strains in vivo. In this way, the bioassay contributes to the study of mechanisms underlying virulence in V. anguillarum

Changes of the glutathione redox system during the weaning transition in piglets, in relation to small intestinal morphology and barrier function

Background Weaning is known to result in barrier dysfunction and villus atrophy in the immediate post-weaning phase, and the magnitude of these responses is hypothesized to correlate with changes in the glutathione (GSH) redox system. Therefore, these parameters were simultaneously measured throughout the weaning phase, in piglets differing in birth weight category and weaning age, as these pre-weaning factors are important determinants for the weaning transition. Low birth weight (LBW) and normal birth weight (NBW) littermates were assigned to one of three weaning treatments; i.e. weaning at 3 weeks of age (3w), weaning at 4 weeks of age (4w) and removal from the sow at 3 d of age and fed a milk replacer until weaning at 3 weeks of age (3d3w). For each of these treatments, six LBW and six NBW piglets were euthanized at 0, 2, 5, 12 or 28 d post-weaning piglets, adding up 180 piglets. Results Weaning increased the glutathione peroxidase activity on d 5 post-weaning in plasma, and duodenal and jejunal mucosa. Small intestinal glutathione-S-transferase activity gradually increased until d 12 post-weaning, and this was combined with a progressive rise of mucosal GSH up till d 12 post-weaning. Oxidation of the GSH redox status (GSH/GSSG E-h) was only observed in the small intestinal mucosa of 3d3w weaned piglets at d 5 post-weaning. These piglets also demonstrated increased fluorescein isothiocyanate dextran (FD4) and horseradish peroxidase fluxes in the duodenum and distal jejunum during the experiment, and specifically demonstrated increased FD4 fluxes at d 2 to d 5 post-weaning. On the other hand, profound villus atrophy was observed during the weaning transition for all weaning treatments. Finally, LBW and NBW piglets did not demonstrate notable differences in GSH redox status, small intestinal barrier function and histo-morphology throughout the experiment. Conclusion Although moderate changes in the GSH redox system were observed upon weaning, the GSH redox status remained at a steady state level in 3w and 4w weaned piglets and was therefore not associated with weaning induced villus atrophy. Conversely, 3d3w weaned piglets demonstrated GSH redox imbalance in the small intestinal mucosa, and this co-occurred with a temporal malfunction of their intestinal barrier function

Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach

Kalimantacin is an antimicrobial compound with strong antistaphylococcal activity that is produced by a hybrid trans-acyltransferase polyketide synthase/nonribosomal peptide synthetase system in Pseudomonas fluorescens BCCM_ID9359. We here present a systematic analysis of the substrate specificity of the glycine-incorporating adenylation domain from the kalimantacin biosynthetic assembly line by a targeted mutagenesis approach. The specificity-conferring code was adapted for use in Pseudomonas and mutated adenylation domain active site sequences were introduced in the kalimantacin gene cluster, using a newly adapted ligation independent cloning method. Antimicrobial activity screens and LC-MS analyses revealed that the production of the kalimantacin analogues in the mutated strains was abolished. These results support the idea that further insight in the specificity of downstream domains in nonribosomal peptide synthetases and polyketide synthases is required to efficiently engineer these strains in vivo

The effect of dietary quercetin on the glutathione redox system and small intestinal functionality of weaned piglets

Quercetin has been shown to alleviate mucosal damage and modulate the glutathione (GSH) redox system in the colon of rodents. In the current study, we assessed whether quercetin was able to mitigate small intestinal dysfunction in weaned pigs. Here, 224 weaned piglets were fed a diet containing quercetin at either 0, 100, 300, or 900 mg/kg diet until d14 post-weaning, followed by a common basal diet until d42. Eight animals per treatment were sampled at d5 and d14 post-weaning. In these animals, the small intestinal histomorphology, barrier function, and protein abundance of occludin, caspase-3, and proliferating cell nuclear antigen were assessed. None of these parameters were affected, and neither did quercetin improve performance up to d42 post-weaning. The GSH redox system was evaluated in blood, small intestinal mucosa, and liver. Quercetin did not affect the glutathione peroxidase, glutathione reductase, and glutamate–cysteine ligase activity in these tissues. In contrast, the hepatic glutathione transferase (GST) activity was significantly increased by quercetin supplementation at d5 post-weaning of 100, 300, and 900 mg/kg. Importantly, d5 was characterized by a more oxidized GSH redox status. To conclude, dietary quercetin had little effect on the small intestine, but did upregulate hepatic GST in the occurrence of redox disturbance

In vitro investigation of six antioxidants for pig diets

Oxidative stress in the small intestinal epithelium can lead to barrier malfunction. In this study, the effect of rosmarinic acid (RA), quercetin (Que), gallic acid (GA), lipoic acid (LA), ethoxyquin (ETQ) and Se-methionine (SeMet) pre-treatments using 2 mM Trolox as a control on the viability and the generation of intracellular reactive oxygen species (iROS) of oxidatively (H2O2) stressed intestinal porcine epithelial cells (IPEC-J2) was investigated. A neutral red assay showed that RA (50-400 mu M), Que (12.5-200 mu M), GA (50-400 mu M), ETQ (6.25-100 mu M), and SeMet (125-1000 mu M) pre-treatments but not LA significantly increased the viability of H2O2-stressed IPEC-J2 cells (p < 0.05). A 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H(2)DCFDA) fluorescent probe showed that RA (100-600 mu M), Que (25-800 mu M), ETQ (3.125-100 mu M) and SeMet (500-2000 mu M) pre-treatments significantly reduced iROS in IPEC-J2 monolayers (p < 0.05). Moreover, RA and Que were most effective in reducing iROS. Therefore, the effects of RA and Que on barrier functioning in vitro were examined. RA and Que pre-treatments significantly decreased fluorescein isothiocyanate (FITC)-conjugated dextran-4 (4 kDa) permeability and transepithelial electrical resistance (TEER) of an IPEC-J2 cell monolayer (p < 0.05). These in vitro results of RA and Que hold promise for their use as antioxidants in pig feed