PUFAs add fuel to Crohn's disease-associated AIEC-induced enteritis by exacerbating intestinal epithelial lipid peroxidation.

Polyunsaturated fatty acids (PUFAs) have been shown to exacerbate Crohn's disease (CD) by promoting lipid peroxidation (LPO) of intestinal epithelial cells (IECs). Dysbiosis of the gut microbiota may play a crucial role in this process. CD patients often exhibit an increased abundance of Escherichia coli (E. coli) in the gut, and the colonization of adherent-invasive E. coli (AIEC) is implicated in the initiation of intestinal inflammation in CD. However, the impact of AIEC on LPO remains unclear. In this study, we observed that AIEC colonization in the terminal ileum of CD patients was associated with decreased levels of glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH) in the intestinal epithelium, along with elevated levels of 4-Hydroxynonenal (4-HNE). In vitro experiments demonstrated that AIEC infection reduced the levels of GPX4 and FTH, increased LPO, and induced ferroptosis in IECs. Furthermore, arachidonic acid (AA) and docosahexaenoic acid (DHA) supplementation in AIEC-infected IECs significantly aggravated LPO and ferroptosis. However, overexpression of GPX4 rescued AIEC-induced LPO and ferroptosis in IECs. Our results further confirmed that AIEC with AA supplementation, associated with excessive LPO and cell death in IECs, worsened colitis in the DSS mouse model and induced enteritis in the antibiotic cocktail pre-treatment mouse model in vivo. Moreover, treatment with ferrostatin-1, a ferroptosis inhibitor, alleviated AIEC with AA supplementation-induced enteritis in mice, accompanied by reduced LPO and cell death in IECs. Our findings suggest that AIEC, in combination with PUFA supplementation, can induce and exacerbate intestinal inflammation, primarily through increased LPO and ferroptosis in IECs.

Leucine Reconstitutes Phagocytosis-Induced Cell Death in E. coli-Infected Neonatal Monocytes-Effects on Energy Metabolism and mTOR Signaling.

MΦ differentiate from circulating monocytes (Mo). The reduced ability of neonatal Mo to undergo apoptosis after E. coli infection (phagocytosis-induced cell death (PICD)) could contribute to sustained inflammatory processes. The objective of our study was to investigate whether immune metabolism in Mo can be modified to gain access to pro-apoptotic signaling. To this end, we supplemented Mo from neonates and from adults with the branched amino acid leucine. In neonatal Mo, we observed increased energy production via oxidative phosphorylation (Oxphos) after E. coli infection via Seahorse assay. Leucine did not change phagocytic properties. In neonatal Mo, we detected temporal activation of the AKT and mTOR pathways, accompanied with subsequent activation of downstream targets S6 Kinase (S6K) and S6. FACS analyses showed that once mTOR activation was terminated, the level of anti-apoptotic BCL-2 family proteins (BCL-2; BCL-XL) decreased. Release of cytochrome C and cleavage of caspase-3 indicated involvement of the intrinsic apoptotic pathway. Concomitantly, the PICD of neonatal Mo was initiated, as detected by hypodiploid DNA. This process was sensitive to rapamycin and metformin, suggesting a functional link between AKT, mTOR and the control of intrinsic apoptotic signaling. These features were unique to neonatal Mo and could not be observed in adult Mo. Supplementation with leucine therefore could be beneficial to reduce sustained inflammation in septic neonates.

Polysaccharides extract from Vaccaria segetalis seeds inhibits kidney infection by regulating cathelicidin expression.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the Chinese Pharmacopoeia, the seeds of Vaccaria segetalis, a traditional medicinal herb, can be used for treating urinary diseases. The polysaccharides extract from V. segetalis seeds (VSP) has been shown to prevent urinary tract infections (UTIs). AIM OF THE STUDY: Investigate the effects of VSP on treating kidney infection induced by uropathogenic Escherichia coli (UPEC) and the underlying mechanisms. MATERIALS AND METHODS: Both in vivo and in vitro infection models were established with the UPEC strain CFT073. After oral administration of VSP, the levels of bacterial load, cathelicidin (CRAMP), Toll-like receptors (TLRs) in the kidney were evaluated. The expression of cathelicidin (LL-37) in human renal cell carcinoma cell line (A498) was tested after the treatment of VSP. RESULTS: In the kidneys of infection models, high-titer bacteria was detected. In the kidney of rat model, the expression of CRAMP was down-regulated, no significant change was observed in the levels of TLRs. After oral administration of VSP, the bacterial load was significantly decreased in rat and mouse models, and the levels of CRAMP and TLRs were significantly up-regulated in rat model. In vitro, the expression of LL-37 was significantly inhibited by CFT073. VSP up-regulated the expression of LL-37 in A498 cells. CONCLUSIONS: The up-regulation of cathelicidin expression may contribute to the therapeutic effects of VSP on kidney infection.

Magnesium Sensing Regulates Intestinal Colonization of Enterohemorrhagic Escherichia coli O157:H7.

The large intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 detects host cues to regulate virulence gene expression during colonization and infection. However, virulence regulatory mechanisms of EHEC O157:H7 in the human large intestine are not fully understood. Herein, we identified a virulence-regulating pathway where the PhoQ/PhoP two-component regulatory system senses low magnesium levels and signals to the O island 119-encoded Z4267 (LmiA; low magnesium-induced regulator A), directly activating loci of enterocyte effacement genes to promote EHEC O157:H7 adherence in the large intestine. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, feeding mice a magnesium-rich diet significantly reduced EHEC O157:H7 adherence in vivo This LmiA-mediated virulence regulatory pathway is also conserved among several EHEC and enteropathogenic E. coli serotypes; therefore, our findings support the use of magnesium as a dietary supplement and provide greater insights into the dietary cues that can prevent enteric infections.IMPORTANCE Sensing specific gut metabolites is an important strategy for inducing crucial virulence programs by enterohemorrhagic Escherichia coli (EHEC) O157:H7 during colonization and infection. Here, we identified a virulence-regulating pathway wherein the PhoQ/PhoP two-component regulatory system signals to the O island 119-encoded low magnesium-induced regulator A (LmiA), which, in turn, activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence in the low-magnesium conditions of the large intestine. This regulatory pathway is widely present in a range of EHEC and enteropathogenic E. coli (EPEC) serotypes. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, mice fed a magnesium-rich diet showed significantly reduced EHEC O157:H7 adherence in vivo, indicating that magnesium may help in preventing EHEC and EPEC infection in humans.

In Vitro and In Vivo Biological Activity of Berberine Chloride against Uropathogenic E. coli Strains Using Galleria mellonella as a Host Model.

Berberine is an alkaloid of the protoberberine type used in traditional oriental medicine. Its biological activities include documented antibacterial properties against a wide variety of microorganisms; nonetheless, its use against Escherichia coli strains isolated from urinary infections has not yet been widely investigated in vivo. The emergence of antimicrobial resistance requires new therapeutic approaches to ensure the continued effectiveness of antibiotics for the treatment and prevention of urinary infections. Moreover, uropathogenic Escherichia coli (UPEC) has developed several virulence factors and resistance to routine antibiotic therapy. To this end, several in vitro and in vivo tests were conducted to assess the activity of berberine on uropathogenic E. coli strains. Galleria mellonella as an infection model was employed to confirm the in vivo translatability of in vitro data on berberine activity and its influence on adhesion and invasion proprieties of E. coli on human bladder cells. In vitro pre-treatment with berberine was able to decrease the adhesive and invasive UPEC ability. In vivo treatment increased the larvae survival infected with UPEC strains and reduced the number of circulating pathogens in larvae hemolymph. These preliminary findings demonstrated the efficacy and reliability of G. mellonella as in vivo model for pre-clinical studies of natural substances.

Methyl-donor supplementation prevents intestinal colonization by Adherent-Invasive E. coli in a mouse model of Crohn's disease.

Deficiencies in methyl-donor molecules (folate, B12 vitamin), DNA methylation alteration and high prevalence of Adherent-Invasive Escherichia coli (AIEC) are frequently observed in Crohn's disease (CD) patients. AIEC bacteria adhere to the enterocytes through abnormally expressed carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) glycoprotein on host cells. This work aims at studying the relationship between methyl-donor molecules and AIEC-induced intestinal inflammatory response. CEABAC10 mice, a mouse model of CD, were fed a control or Methyl-donor Supplemented diet (MS diet). CEACAM6 promoter was hypermethylated in intestinal epithelial cells from mice fed an MS diet, which was associated with a significant decrease in CEACAM6 expression. Transcriptomic analysis revealed increased expression of anti-microbial peptides, increase in HSP70 gene family expression and a decreased expression of inflammatory marker Calprotectin upon MS diet, associated to a lower ability of AIEC bacteria to colonize gut mucosa. We observed in a cohort of CD patients that serum folate concentration was inversely correlated to Crohn's disease endoscopic index of severity and to fecal inflammatory markers. This study demonstrates that methyl-donor supplementation through the diet induces a specific intestinal micro-environment limiting pathobiont colonization of the gut. Clinicians may wish to consider methyl-donor supplementation for methyl-donor deficient CD patients.

Mitochondrial Signature in Human Monocytes and Resistance to Infection in C. elegans During Fumarate-Induced Innate Immune Training.

Monocytes can develop immunological memory, a functional characteristic widely recognized as innate immune training, to distinguish it from memory in adaptive immune cells. Upon a secondary immune challenge, either homologous or heterologous, trained monocytes/macrophages exhibit a more robust production of pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α, than untrained monocytes. Candida albicans, ß-glucan, and BCG are all inducers of monocyte training and recent metabolic profiling analyses have revealed that training induction is dependent on glycolysis, glutaminolysis, and the cholesterol synthesis pathway, along with fumarate accumulation; interestingly, fumarate itself can induce training. Since fumarate is produced by the tricarboxylic acid (TCA) cycle within mitochondria, we asked whether extra-mitochondrial fumarate has an effect on mitochondrial function. Results showed that the addition of fumarate to monocytes induces mitochondrial Ca2+ uptake, fusion, and increased membrane potential (Δψm), while mitochondrial cristae became closer to each other, suggesting that immediate (from minutes to hours) mitochondrial activation plays a role in the induction phase of innate immune training of monocytes. To establish whether fumarate induces similar mitochondrial changes in vivo in a multicellular organism, effects of fumarate supplementation were tested in the nematode worm Caenorhabditis elegans. This induced mitochondrial fusion in both muscle and intestinal cells and also increased resistance to infection of the pharynx with E. coli. Together, these findings contribute to defining a mitochondrial signature associated with the induction of innate immune training by fumarate treatment, and to the understanding of whole organism infection resistance.

Crude polysaccharides from the seeds of Vaccaria segetalis prevent the urinary tract infection through the stimulation of kidney innate immunity.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Vaccaria segetalis (Neck.) Garcke is used for the treatment of urinary diseases in Traditional Chinese Medicine according to the Chinese Pharmacopoeia. Crude polysaccharides and the aqueous extract from the seeds of V. segetalis (SVCP) were proved to be effective on treating benign prostatic hyperplasia. AIM OF THE STUDY: The aim of this study was to test the effects of SVCP on urinary tract infection (UTI) induced by uropathogenic Escherichia coli (UPEC) strain CFT073 in the rat model and to investigate the underlying mechanisms. MATERIALS AND METHODS: A rat UTI model was established with the infection of UPEC strain CFT073. After oral administration of SVCP, the urinalysis and histological examination were evaluated. The levels of pro-inflammatory cytokines, procalcitonin (PCT) and polymeric Ig receptor (PIGR) were used to test the effects of SVCP on host immunity. The mRNA level of PapG in CFT073 was used to test the influence of SVCP on virulence factor. The effects of SVCP on the inhibition of bacterial adhesion were evaluated with mice UTI model. RESULTS: In the rat UTI model, the levels of bacterial load, white blood cells (WBC) and red blood cells (RBC) in urine and the pathological injury in the bladder were significantly up-regulated, the expression of PIGR in kidney was down-regulated, no significant change was observed on the pro-inflammatory cytokines in urine. After oral administration of SVCP for 3 days, the levels of bacterial load, WBC and RBC in urine were significantly decreased, the pathological injury in the bladder were remarkably inhibited. The expression of IL-6, IL-8 in urine and PIGR in kidney were significantly up-regulated by SVCP (200 mg/kg). SVCP showed no effect on the concentration of PCT in serum. SVCP failed to down-regulate the mRNA level of PapG in CFT073. In the mice UTI model, pre-treatment of SVCP failed to inhibit the intracellular bacterial load in the bladder. CONCLUSIONS: The therapeutic effects of SVCP on treating UTIs might result from the up-regulation of innate immunity in the kidney. SVCP can be used as an alternative therapeutic agent for UTIs.

The antidiarrhoeal evaluation of Psidium guajava L. against enteropathogenic Escherichia coli induced infectious diarrhoea.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Psidium guajava L. (Myrtaceae), commonly used as an edible fruit is traditionally used worldwide in treatment of various gastrointestinal problems including diarrhoea. The leaves of the plant have also been evaluated for antidiarrhoeal activity in various chemical induced diarrhoea models. OBJECTIVE: The main objective of the present study was to evaluate the potency of P. guajava leaves and its major biomarker quercetin against enteropathogenic Escherichia coli (EPEC) induced infectious diarrhoea using preclinical and computational model. MATERIAL AND METHODS: P. guajava alcoholic leaf extract (PGE) was initially standardized using HPLC taking quercetin as a biomarker and was then subjected to antidiarrhoeal evaluation on rats in an EPEC induced diarrhoea rat model. The study included assessment of various behavioral parameters, initially for 6 h and then for up to 24 h of induction which was followed by estimation of stool water content, density of EPEC in stools and blood parameters evaluation. The colonic and small intestinal tissues of the treated animals were subjected to various biochemical estimations, in vivo antioxidant evaluation, estimation of ion concentration, Na+/K+-ATPase activity, assessment of pro-inflammatory cytokines and histopathological studies. Further, the major biomarker off PGE, quercetin was subjected to molecular docking studies with Na+/K+-ATPase and EPEC. RESULTS: The results demonstrated a significant antidiarrhoeal activity of quercetin (50 mg/kg), PGE at 200 and 400 mg/kg, p.o., where quercetin and PFGE at 200 mg/kg, p.o. were found to be more prominent, as confirmed through higher % protection, water content of stools and density of EPEC in stools. PGE and its biomarker quercetin also significantly recovered the WBC, Hb, platelets loss and also revealed a significant restoration of altered antioxidants level, pro-inflammatory cytokines (IL-1ß and TNF-α) expression and had positive influence on Na+/K+-ATPase activity. The docking studies of quercetin with Na+/K+-ATPase showed favourable interactions and residues Glu 327, Ser 775, Asn 776, Glu 779 and Asp 804 of Na+/K+-ATPase were adequately similar to quercetin for donating ligands for binding, while quercetin was also found to terminate the linkage between mammalian cells and EPEC thus, preventing further infection from EPEC. CONCLUSION: Inhibition in intestinal secretion, reduced nitric oxide production and inflammatory expression along with reactivation of Na+/K-ATPase activity could be attributed to the observed antidiarrhoeal potential of PGE against infectious diarrhoea, where quercetin was confirmed to be the main contributing factor.

Isoleucine attenuates infection induced by E. coli challenge through the modulation of intestinal endogenous antimicrobial peptide expression and the inhibition of the increase in plasma endotoxin and IL-6 in weaned pigs.

Enteric infection is a major cause of morbidity and mortality in both humans and animals worldwide. Immunotherapy against intestinal infection is a well-known alternative to the antibiotic strategy. Herein, we demonstrated that isoleucine significantly suppressed the multiplication of E. coli in the presence of IPEC-J2 cells. Isoleucine supplementation enhanced the concentrations of total plasma protein and IgA in pigs compared to the alanine control diet, while inhibiting the increase in plasma endotoxin and IL-6 contents induced by E. coli challenge. A significant interaction between the E. coli challenge and the diet treatment was found in the red blood cell volume. Isoleucine improved the expression of porcine ß-defensin-1 (pBD-1), pBD-2, pBD-3, pBD-114 and pBD-129 in the jejunum and ileum of pigs with or without E. coli challenge. Conclusively, isoleucine attenuated the infection caused by the E. coli challenge possibly through increasing the intestinal ß-defensin expression and inhibiting the increase in plasma endotoxin and IL-6 in weaned pigs.

Effective Oral Combination Treatment for Extended-Spectrum Beta-Lactamase-Producing Escherichia coli.

Background: Extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) is increasing worldwide. The drugs of choice for treatment of ESBLs are parenteral carbapenems. The aim of this study was to evaluate the in vitro and in vivo efficacy of a new combination of oral cephalosporins and amoxicillin/clavulanate in treatment of ESBL-EC. Methods: A total of 150 ESBL-EC samples were collected over 1 year from two referral centers. Synergistic studies of cephalosporins and amoxicillin/clavulanate were performed in vitro using disk approximation and disk replacement methods. Combination treatment was assessed in vivo on 20 ESBL-EC urinary tract infection (UTI) patients. Results: ESBL-EC isolates were confirmed in 150 patients with a mean age of 46.67 years, 75.2% of them being women. Antibiotic susceptibility testing of isolates indicated high resistance rate to oral antibiotics. The frequency of positive synergy and mean distance of synergy between cephalosporins and amoxicillin/clavulanate was significantly higher with cefotaxime and cefixime compared with cefpodoxime, cefdinir, and ceftazidime using disk approximation and disk replacement methods (p < 0.05). Addition of amoxicillin/clavulanate enhanced the susceptibility rate with cefixime from 8.6% to 86.3%, significantly higher than with other cephalosporins (p < 0.0005). Cefixime and amoxicillin/clavulanate synergy was not affected by age, gender, hospital, department, sample type, or bacterial load. Eighteen of 20 ESBL-EC-positive UTI patients had a positive in vitro synergy test and complete clinical and microbiological resolution after completion of cefixime and amoxicillin/clavulanate oral treatment course. Conclusions: Cefixime and amoxicillin/clavulanate combination therapy could be an effective oral outpatient treatment option for ESBL-EC. In vitro synergistic testing is simple and predictive of successful treatment.

Antimicrobial and Immunomodulatory Effect of Gum Arabic on Human and Bovine Granulocytes Against Staphylococcus aureus and Escherichia coli.

Gum arabic (GA) is a traditional herbal medicine from Acacia Senegal (L.) Willdenow trees, which consist of a complex mixture of polysaccharides and glycoproteins. It is used in daily applications for several diseases and is considered to protect against bacterial infections. The detailed mechanisms behind these observations are still unclear. In this study, we investigated the direct antibacterial activity of GA water and ethanol extracts against Staphylococcus (S.) aureus or Escherichia (E.) coli and the immunomodulating properties of those extracts on granulocytes as a first line of defense against bacteria. Firstly, the direct antimicrobial effect of GA was tested on three different S. aureus strains and two E. coli strains. The growth of bacteria was analyzed in the presence of different GA concentrations over time. GA water as well as ethanol extracts showed a significant growth inhibition in a concentration-dependent manner in the case of S. aureus Newman, S. aureus Rd5, and E. coli 25922, but not in the case of S. aureus USA300 and E. coli K1. Transmission electron microscopic analysis confirmed an antibacterial effect of GA on the bacteria. Secondly, the immunomodulatory effect of GA on the antimicrobial activity of bovine or human blood-derived granulocytes was evaluated. Interestingly, water and ethanol extracts enhanced antimicrobial activity of granulocytes by the induction of intracellular ROS production. In line with these data, GA increased the phagocytosis rate of E. coli. No effect was seen on neutrophil extracellular trap (NET) formation that mediates killing of extracellular bacteria such as S. aureus. In conclusion, we show that GA exhibits a direct antibacterial effect against some S. aureus and E. coli strains. Furthermore, GA boosts the antimicrobial activities of granulocytes and increases intracellular ROS production, which may lead to more phagocytosis and intracellular killing. These data might explain the described putative antimicrobial activity of GA used in traditional medicine.

Alanyl-glutamine Protects Against Damage Induced by Enteroaggregative Escherichia coli Strains in Intestinal Cells.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an important pathogen causing enteric infections worldwide. This pathotype is linked to malnutrition in children from developing countries. Alanyl-glutamine (Ala-Gln) is an immune modulator nutrient that acts during intestinal damage and/or inflammation. This study investigated the effect of EAEC infection and Ala-Gln on cell viability, cell death, and inflammation of intestinal epithelium cells (IEC-6). METHODS: Cells were infected with an EAEC prototype 042 strain, an EAEC wild-type strain isolated from a Brazilian malnourished child, and a commensal E coli HS. Gene transcription and protein levels of caspases-3, -8, and -9 and cytokine-induced neutrophil chemoattractant 1 (CINC-1/CXCL1) were evaluated using RT-qPCR, western blot analysis, and ELISA. RESULTS: Infections with both EAEC strains decreased cell viability and induced apoptosis and necrosis after 24 hours. Ala-Gln supplementation increased cell proliferation and reduced cell death in infected cells. Likewise, EAEC strain 042 significantly increased the transcript levels of caspases-3, -8, and -9 when compared to the control group, and Ala-Gln treatment reversed this effect. Furthermore, EAEC induced CXCL1 protein levels, which were also reduced by Ala-Gln supplementation. CONCLUSION: These findings suggest that EAEC infection promotes apoptosis, necrosis, and intestinal inflammation with involvement of caspases. Supplementation of Ala-Gln inhibits cell death, increases cell proliferation, attenuates mediators associated with cell death, and inflammatory pathways in infected cells.

Increased Iron Availability Aggravates the Infection of Escherichia coli O157:H7 in Mice.

Iron plays an important role both in bacterial pathogenicity and in host defense mechanisms, which has frequently been underestimated. The primary purpose of this study was to investigate the influence of iron supplementation on the progression of bacterial infection. We used mice as an experimental model to supplement iron after Escherichia coli (E. coli) O157:H7 infection and found that iron supplementation exacerbated clinical symptoms of bacterial infection by increasing mortality and reducing body weight. Iron supplementation promoted the colonization of bacteria and enhanced inflammatory responses by increasing C-reaction protein level and the phagocytic capacity of PBMCs, as well as upregulating the expression of TNF-α and IL-1ß in E. coli O157:H7-challenged mice. In vitro cell experiment confirmed that an excess of iron would enhance the growth of E. coli O157:H7 and worsen the outcome of bacterial infection. Therefore, it is certainly plausible that iron supplementation in bacterial infection may worsen rather than improve host outcome.

Alginate oligosaccharide alleviates enterotoxigenic Escherichia coli-induced intestinal mucosal disruption in weaned pigs.

Alginate oligosaccharide (AOS) is a non-toxic, non-immunogenic, non-carcinogenic and biodegradable product generated by depolymerisation of alginate, and exhibits various salutary properties. The present study was designed to evaluate whether AOS supplementation could attenuate enterotoxigenic Escherichia coli (ETEC)-induced intestinal mucosal injury in weaned pigs. Twenty-four weaned pigs were randomly assigned to three treatments: (1) non-challenged control; (2) ETEC-challenged control; and (3) ETEC challenge + AOS treatment (100 mg kg-1). On day 12, pigs in the non-challenged group were orally infused with sterilised Luria-Bertani culture while pigs in other groups were orally infused with ETEC (2.6 × 1011 colony-forming units). At 3 days after the challenge, all pigs were orally administered d-xylose at 0.1 g per kg body weight and then euthanised 1 h later to obtain serum and intestinal mucosa samples. Our results showed that ETEC infection both reduced (P < 0.05) the villus height and proportion of epithelial cells in the S phase and elevated (P < 0.05) the percentage of total apoptotic epithelial cells in the jejunum and ileum; these deleterious effects caused by ETEC were alleviated (P < 0.05) by supplemental AOS. Meanwhile, AOS ingestion attenuated (P < 0.05) not only the up-regulated tumour necrosis factor receptor 1 (TNFR1), cysteinyl aspartate-specific protease-3 (caspase-3), -8 and -9 transcriptions, as well as the enhanced caspase activities (caspase-3, -8 and -9), but also the down-regulated cyclin E1 and cyclin-dependent kinase 2 (CDK2) transcriptions in jejunal and ileal mucosae, caused by the ETEC challenge. In conclusion, it is possible that the protective effects of AOS against ETEC-induced intestinal mucosal disruption in weaned pigs are associated with the restrained enterocyte death, by reducing both mitochondria-dependent and TNFR1-dependent apoptosis and the accelerated enterocyte proliferation, via enhancing the cyclin E-CDK2 complex formation.

Self-Assembling Myristoylated Human α-Defensin 5 as a Next-Generation Nanobiotics Potentiates Therapeutic Efficacy in Bacterial Infection.

The increasing prevalence of antibacterial resistance globally underscores the urgent need to the update of antibiotics. Here, we describe a strategy for inducing the self-assembly of a host-defense antimicrobial peptide (AMP) into nanoparticle antibiotics (termed nanobiotics) with significantly improved pharmacological properties. Our strategy involves the myristoylation of human α-defensin 5 (HD5) as a therapeutic target and subsequent self-assembly in aqueous media in the absence of exogenous excipients. Compared with its parent HD5, the C-terminally myristoylated HD5 (HD5-myr)-assembled nanobiotic exhibited significantly enhanced broad-spectrum bactericidal activity in vitro. Mechanistically, it selectively killed Escherichia coli ( E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) through disruption of the cell wall and/or membrane structure. The in vivo results further demonstrated that the HD5-myr nanobiotic protected against skin infection by MRSA and rescued mice from E. coli-induced sepsis by lowering the systemic bacterial burden and alleviating organ damage. The self-assembled HD5-myr nanobiotic also showed negligible hemolytic activity and substantially low toxicity in animals. Our findings validate this design rationale as a simple yet versatile strategy for generating AMP-derived nanobiotics with excellent in vivo tolerability. This advancement will likely have a broad impact on antibiotic discovery and development efforts aimed at combating antibacterial resistance.

Dietary supplementation with Clostridium butyricum helps to improve the intestinal barrier function of weaned piglets challenged with enterotoxigenic Escherichia coli K88.

AIMS: The objective of this study was twofold: (i) to examine the effect of Clostridium butyricum on intestinal barrier function and (ii) to elucidate the mechanisms involved in enhanced intestinal barrier function. METHODS AND RESULTS: Forty-eight weaned piglets were assigned randomly to either a basal diet or a C. butyricum-supplemented diet. On day 15, all pigs were orally challenged with enterotoxigenic Escherichia coli (ETEC) K88 or saline. Clostridium butyricum decreased serum diamine oxidase activity and d-lactic acid concentration, as well as increased intestinal tight junction proteins (ZO-1, claudin-3 and occludin) expression in ETEC K88-infected pigs. Moreover, C. butyricum decreased IL-1ß and IL-18 levels in serum and gut, whereas it increased IL-10 levels. Furthermore, C. butyricum downregulated NLRP3 and caspase-1 expression in ETEC K88-challenged pig gut, but did not affect apoptosis-associated speck-like protein expression. CONCLUSIONS: Clostridium butyricum enhanced intestinal barrier function and inhibited apoptosis-associated speck-like protein-independent NLRP3 inflammasome signalling pathway in weaned piglets after ETEC K88 challenge. SIGNIFICANCE AND IMPACT OF THE STUDY: The novelty of this study lies in the beneficial effects of C. butyricum on intestinal health, likely by improving intestinal barrier function and alleviating inflammatory reactions.

Antiadhesive Activity and Metabolomics Analysis of Rat Urine after Cranberry (Vaccinium macrocarpon Aiton) Administration.

Cranberry (Vaccinium macrocarpon Aiton) is used to treat noncomplicated urinary tract infections (UTIs). A-type procyanidins (PAC-A) are considered the active constituents able to inhibit bacterial adhesion to the urinary epithelium. However, the role of PAC-A in UTIs is debated, because of their poor bioavailability, extensive metabolism, limited knowledge about urinary excretion, and contradictory clinical trials. The effects of 35-day cranberry supplementation (11 mg/kg PAC-A, 4 mg/kg PAC-B) were studied in healthy rats using a ultra performance liquid chromatography-mass spectrometry (UPLC-MS)-based metabolomics approach. Microbial PAC metabolites, such as valeric acid and valerolactone derivatives, were related to cranberry consumption. An increased urinary excretion of glucuronidated metabolites was also observed. In a further experiment, urine samples were collected at 2, 4, 8, and 24 h after cranberry intake and their antiadhesive properties were tested against uropathogenic Escherichia coli. The 8 h samples showed the highest activity. Changes in urinary composition were studied by ultra performance liquid chromatography-time-of-flight (UPLC-QTOF), observing the presence of PAC metabolites. The PAC-A2 levels were measured in all collected samples, and the highest amounts, on the order of ng/mL, were found in the samples collected after 4 h. Results indicate that the antiadhesive activity against uropathogenic bacteria observed after cranberry consumption is ascribable to PAC-A metabolites rather than to a direct PAC-A effect, as the measured PAC-A levels in urine was lower than those reported as active in the literature.

Inhibitory effect of Zanthoxylum bungeanum essential oil (ZBEO) on Escherichia coli and intestinal dysfunction.

The inhibitory effects of Zanthoxylum bungeanum essential oil (ZBEO) on Escherichia coli (E. coli) in vitro and in vivo were investigated, as well as its function of improvement of intestinal health. The results of in vitro studies, such as minimal inhibitory concentration (MIC) analysis, agar disc diffusion test and growth curve analysis of E. coli, showed that ZBEO had an excellent inhibitory effect on the growth of E. coli, which may be related to the loss of the normal shape of the cell membranes and the leakage of intracellular constituents, on the basis of SEM observation and cell constituents' release assay. ZBEO also had an inhibitory effect on enteritis and intestinal dysfunction induced by infection of E. coli in vivo, and histopathological observation indicated that ZBEO could markedly ameliorate the structural destruction of intestinal tissues, which might be related to its inhibitory effect on the gene expression of inflammatory cytokines (TLR2, TLR4, TNFα and IL-8). In conclusion, ZBEO showed an excellent inhibitory effect on E. coli both in vitro and in vivo, suggesting the potential application of ZBEO as a kind of functional component having the effects of improving intestinal function and health.

Comparative pharmacokinetics of eight major bioactive components in normal and bacterial diarrhea mini-pigs after oral administration of Gegen Qinlian Decoction.

Healthy animals are most widely used in current pharmacokinetic(PK) studies. However, neglecting the effects of specific diseases on drug absorption results in the PK parameters of those experiments not accurately reflecting in vivo drug concentration changes during treatment. In this study, an E. coli infective diarrheal minipig model was applied to explore the pharmacokinetics of Gegen Qinlian decoction (GQD). A simple and rapid ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine the concentrations of the eight GQD components in minipig plasma after intragastric administration of GQD. The PK parameters of the main GQD components in normal and model minipigs after oral administration of GQD were compared. There were statistically significant differences (p<0.05) in the pharmacokinetic parameters of Puerarin, Wogonin and Daidzein involving the AUC0-t, Cmax, MRT(0-t), t1/2z between normal and model minipigs. Results showed that bacterial diarrhea had a great impact on the biological availability of the main ingredients in GQD. More importantly, the results obtained suggest that the bacterial diarrheal minipig model can be successfully applied in PK studies and may be used in other PK studies of drugs targeting intestinal disease.