Construction of the ETECFinder database for the characterization of enterotoxigenic Escherichia coli (ETEC) and revision of the VirulenceFinder web tool at the CGE website.

The identification of pathogens is essential for effective surveillance and outbreak detection, which lately has been facilitated by the decreasing cost of whole-genome sequencing (WGS). However, extracting relevant virulence genes from WGS data remains a challenge. In this study, we developed a web-based tool to predict virulence-associated genes in enterotoxigenic Escherichia coli (ETEC), which is a major concern for human and animal health. The database includes genes encoding the heat-labile toxin (LT) (eltA and eltB), heat-stable toxin (ST) (est), colonization factors CS1 through 30, F4, F5, F6, F17, F18, and F41, as well as toxigenic invasion and adherence loci (tia, tibAC, etpBAC, eatA, yghJ, and tleA). To construct the database, we revised the existing ETEC nomenclature and used the VirulenceFinder webtool at the CGE website [VirulenceFinder 2.0 (dtu.dk)]. The database was tested on 1,083 preassembled ETEC genomes, two BioProjects (PRJNA421191 with 305 and PRJNA416134 with 134 sequences), and the ETEC reference genome H10407. In total, 455 new virulence gene alleles were added, 50 alleles were replaced or renamed, and two were removed. Overall, our tool has the potential to greatly facilitate ETEC identification and improve the accuracy of WGS analysis. It can also help identify potential new virulence genes in ETEC. The revised nomenclature and expanded gene repertoire provide a better understanding of the genetic diversity of ETEC. Additionally, the user-friendly interface makes it accessible to users with limited bioinformatics experience. IMPORTANCE: Detecting colonization factors in enterotoxigenic Escherichia coli (ETEC) is challenging due to their large number, heterogeneity, and lack of standardized tests. Therefore, it is important to include these ETEC-related genes in a more comprehensive VirulenceFinder database in order to obtain a more complete coverage of the virulence gene repertoire of pathogenic types of E. coli. ETEC vaccines are of great importance due to the severity of the infections, primarily in children. A tool such as this could assist in the surveillance of ETEC in order to determine the prevalence of relevant types in different parts of the world, allowing vaccine developers to target the most prevalent types and, thus, a more effective vaccine.

Carrier state of enterotoxigenic Escherichia coli virulence markers in pigs: Effects on gut microbiota modulation and immune markers transcription.

Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in pigs at early age, leading to high mortality rates and significant economic losses in the swine industry. ETEC effect on gut microbiota and immune system is mostly studied in diarrheic model under controlled laboratory conditions, however its impact on asymptomatic carriers remains unknown. Thus, we investigated whether ETEC can modulate gut microbiota or regulate the transcription of immune markers in asymptomatic pigs in farm environment. Stool samples from newborn piglets, nursery and growing pigs, and sows were screened for ETEC markers, then submitted to 16S-rDNA sequencing to explore gut microbiota composition in carriers (ETEC+) and non-carriers (ETEC-) animals. We observed a reduced α-diversity in ETEC+ animals (p < 0.05), while bacterial compositions were mostly driven by ageing (p > 0.05). Prevotella marked ETEC-carrier group, while Rikenellaceae RC9 gut group was a marker for a healthy gut microbiota, suggesting that they might be biomarker candidates for surveillance and supplementation purposes. Furthermore, we observed transcription regulation of il6 and tff2 genes in ETEC+ in newborn and nursery stages, respectively. Our findings indicate that ETEC presence modulate gut microbiota and the immune response in asymptomatic pigs; nevertheless, further studies using a probabilistic design must be performed to assess the effect of ETEC presence on gut imbalance in pigs despite the age bias.

The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion.

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32-0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6.

Heat-stable enterotoxin inhibits intestinal stem cell expansion to disrupt the intestinal integrity by downregulating the Wnt/ß-catenin pathway.

Enterotoxigenic Escherichia coli causes severe infectious diarrhea with high morbidity and mortality in newborn and weanling pigs mainly through the production of heat-stable enterotoxins (STs). However, the precise regulatory mechanisms involved in ST-induced intestinal epithelium injury remain unclear. Consequently, we conducted the experiments in vivo (mice), ex vivo (mouse and porcine enteroids), and in vitro (MODE-K and IPEC-J2 cells) to explore the effect of STp (one type of STa) on the integrity of the intestinal epithelium. The results showed that acute STp exposure led to small intestinal edema, disrupted intestinal integrity, induced crypt cell expansion into spheroids, and downregulated Wnt/ß-catenin activity in the mice. Following a similar trend, the enteroid-budding efficiency and the expression of Active ß-catenin, ß-catenin, Lgr5, PCNA, and KRT20 were significantly decreased after STp treatment, as determined ex vivo. In addition, STp inhibited cell proliferation, induced cell apoptosis, destroyed cell barriers, and reduced Wnt/ß-catenin activity by downregulating its membrane receptor Frizzled7 (FZD7). In contrast, Wnt/ß-catenin reactivation protected the IPEC-J2 cells from STp-induced injury. Taking these findings together, we conclude that STp inhibits intestinal stem cell expansion to disrupt the integrity of the intestinal mucosa through the downregulation of the Wnt/ß-catenin signaling pathway.

Establishment and Validation of Pathogenic CS17+ and CS19+ Enterotoxigenic Escherichia coli Challenge Models in the New World Primate Aotus nancymaae.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrheal illness in the military, travelers, and children living in low- to middle-income countries. Increased antibiotic resistance, the absence of a licensed vaccine, and the lack of broadly practical therapeutics perpetuate the significant health and financial burden resulting from ETEC infection. A critical step in the evaluation of vaccines and therapeutics is preclinical screening in a relevant animal disease model that closely replicates human disease. We previously developed a diarrheal model of class 5a colonization factor (CF) CFA/I-expressing ETEC in the New World owl monkey species Aotus nancymaae using ETEC strain H10407. In order to broaden the use of the model, we report here on the development of A. nancymaae models of ETEC expressing the class 5b CFs CS17 and CS19 with strains LSN03-016011/A and WS0115A, respectively. For both models, we observed diarrheal attack rates of ≥80% after oral inoculation with 5 × 1011 CFU of bacteria. These models will aid in assessing the efficacy of future ETEC vaccine candidates and therapeutics.

Protective Effects of Selenium Nanoparticle-Enriched Lactococcus lactis NZ9000 against Enterotoxigenic Escherichia coli K88-Induced Intestinal Barrier Damage in Mice.

Composite microecological agents have received widespread attention due to their advantageous properties, including safety, multiple effects, and low cost. This study was conducted to evaluate the protective effects of selenium (Se) nanoparticle (SeNP)-enriched Lactococcus lactis NZ9000 (L. lactis NZ9000-SeNPs) against enterotoxigenic Escherichia coli (ETEC) K88-induced intestinal barrier damage in C57BL/6 mice. The oral administration of L. lactis NZ9000-SeNPs significantly increased the villus height and the number of goblet cells in the ileum; reduced the levels of serum and ileal interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ); and increased the activities of thioredoxin reductase (TrxR) and glutathione peroxidase (GSH-Px) compared with the ETEC K88-infected group not treated with L. lactis NZ9000-SeNPs. In addition, L. lactis NZ9000-SeNPs significantly attenuated the reduction of the expression levels of occludin and claudin-1, dysbiosis of the gut microbiome, and activation of the Toll-like receptor (TLR)/nuclear factor kappa B (NF-κB)-mediated signaling pathway induced by ETEC K88. These findings suggested that L. lactis NZ9000-SeNPs may be a promising and safe Se supplement for food or feed additives. IMPORTANCE The beneficial effects of microecological agents have been widely proven. Se, which is a nutritionally essential trace element for humans and animals, is incorporated into selenoproteins that have a wide range of pleiotropic effects, ranging from antioxidant to anti-inflammatory effects. However, sodium selenite, a common addition form of Se in feed and food, has disadvantages such as strong toxicity and low bioavailability. We investigated the protective effects of L. lactis NZ9000-SeNPs against ETEC K88-induced intestinal barrier injury in C57BL/6 mice. Our results show that L. lactis NZ9000-SeNPs effectively alleviate ETEC K88-induced intestinal barrier dysfunction. This study highlights the importance of developing a promising and safe Se supplement for the substitution of sodium selenite applied in food, feed, and biomedicine.

Icariin and its phosphorylated derivatives alleviate intestinal epithelial barrier disruption caused by enterotoxigenic Escherichia coli through modulate p38 MAPK in vivo and in vitro.

The natural product icariin (ICA) and its phosphorylated derivatives (pICA) have been shown to have outstanding anti-inflammatory and antioxidant properties. This study was to explore the protective effects of ICA and pICA on the intestinal epithelium of enterotoxigenic Escherichia coli (ETEC)-induced piglet diarrhea and its underlying mechanisms in vivo and in vitro. ETEC K88 increased pro-inflammatory cytokine expression, activated oxidative stress and inhibited antioxidant enzyme activity, induced phosphorylated p38 MAPK gene and protein expression, disrupted intestinal barrier function, and led to diarrhea in piglets. Pretreatment with ICA and pICA effectively alleviated ETEC-induced intestinal barrier dysfunction in vivo and in vitro. Pretreatment with p38 MAPK inhibitor (SB203580) significantly rescued the IPEC-J2 cells barrier function damaged by ETEC challenge. However, pretreatment with p38 MAPK activator (anisomycin) did not alleviated the IPEC-J2 cells barrier function damaged by ETEC challenge. Our data demonstrated that ICA and pICA regulate the inflammatory response and oxidative stress of intestinal epithelial cells by inhibiting the expression of p38 MAPK, thereby alleviating ETEC K88-induced disruption of intestinal barrier function and intestinal permeability. These findings provide new insights into the prevention and treatment of intestinal barrier dysfunction induced by ETEC K88.

A new multidrug-resistant enterotoxigenic Escherichia coli pulsed-field gel electrophoresis cluster associated with enrofloxacin non-susceptibility in diseased pigs.

AIMS: To describe the temporal trends in Escherichia coli pathotypes and antimicrobial resistance detected in isolates from diseased-pig cases submitted to the EcL from 2008 to 2016, in Quebec, Canada, and to investigate the presence of spatiotemporal and phylogenetic clusters. METHODS AND RESULTS: Detection of 12 genes coding for virulence factors in pathogenic E. coli in pigs by PCR and antimicrobial resistance standard disc diffusion assay were performed. Demographic and clinical data were entered in the Animal Pathogenic and Zoonotic E. coli (APZEC) database. ETEC:F4 was the most prevalent pathovirotype among the 3773 cases submitted. The LT:STb:F4 virotype was predominant until 2014, then was overtaken by the LT:STb:STa:F4 virotype. More than 90% of the ETEC:F4 isolates were multidrug resistant. A spatiotemporal cluster of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin was detected between 4/2015 and 9/2016. Pulsed-field gel electrophoresis analysis of 137 ETEC:F4 isolates revealed the presence of a cluster composed mainly of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin. CONCLUSIONS: The APZEC database was useful to highlight temporal trends in E. coli pathotypes. A high-risk ETEC:F4 clone might disseminate in the pig population in Quebec since 2015. SIGNIFICANCE AND IMPACT OF THE STUDY: Surveillance is crucial to identify new clones and develop control strategies.

Lactobacillus plantarum inhibited the inflammatory response induced by enterotoxigenic Escherichia coli K88 via modulating MAPK and NF-κB signalling in intestinal porcine epithelial cells.

AIMS: To investigate the effects of Lactobacillus plantarum on inflammatory responses induced by ETEC K88 and explore the underlying molecular mechanisms. METHODS AND RESULTS: Intestinal porcine cells (IPEC-1) were incubated with 0 or 1 × 108  CFU per well L. plantarum for 4 h, and then these cells were challenged with 0 or 1 × 108  CFU per well ETEC K88 for 2 h. The results showed that pre-treatment of IPEC-1 cells with L. plantarum prevented the increases in the transcript abundance of interleukin-1α (IL-1α), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α) (P < 0·05) caused by ETEC K88. Additionally, L. plantarum inhibited the reduction in peroxisome proliferator-activated receptor-γ (PPAR-γ) expression caused by ETEC K88 (P < 0·05). Moreover, L. plantarum pre-treatment downregulated the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular regulated protein kinases 1 and 2 (ERK1/2) and p38 and the nuclear concentration of nuclear factor kappa B p65 (NF-κB p65) (P < 0·05) compared with ETEC K88 group. Silencing experiment further supported that the protective effect of L. plantarum P might mediated by suppression of ETEC-provoked activation of MAPK and NF-κB signalling pathways. CONCLUSIONS: Lactobacillus plantarum inhibited the inflammatory response induced by ETEC K88 in IPEC-1 cells via modulating MAPK and NF-κB signalling. SIGNIFICANCE AND IMPACT OF THE STUDY: This study elucidated the underlying mechanism in which probiotics protect against intestinal inflammation caused by ETEC K88.

Effects of IQW and IRW on Inflammation and Gut Microbiota in ETEC-Induced Diarrhea.

METHODS: The mice were randomly distributed into four groups: (a) control (CTRL) group, (b) ETEC group, (c) IQW-ETEC group, and (d) IRW-ETEC group. Villus length and crypt depth were measured after hematoxylin and eosin staining. The inflammatory reaction was analyzed via inflammatory cytokines (i.e., TNF-α, IL-1ß, IL-6, and IL-10) using the enzyme-linked immunosorbent assay (ELISA). The microbiota in the colon was sequenced using 16S ribosomal RNA. RESULTS: The villus length decreased, the crypt depth decreased, and the expression of inflammatory cytokines (i.e., TNF-α, IL-1ß, IL-6, and IL-10) increased due to ETEC. In the IRW-ETEC and IQW-ETEC groups, the Shannon index decreased (P < 0.05). IQW and IRW increased the abundance of Firmicutes, Proteobacteria, Clostridiales, Lachnospiraceae, and Alloprevotella; contrastingly, it decreased the abundance of Epsilonproteobacteria, Erysipelotrichales, Prevotellaceae, and Flavobacteriaceae compared to the ETEC group (P <0.05). CONCLUSION: This study ascertained that the addition of IQW and IRW could alleviate jejunal inflammation and increase microbiota community diversity.

Evaluation of the probiotic and functional potential of Lactobacillus agilis 32 isolated from pig manure.

Escherichia coli is a symbiotic bacterium in humans and animals and an important pathogen of humans and animals. Prevention and suppression of E. coli infection is of great concern. In this study, we isolated a strain of Lactobacillus agilis 32 from pig manure and evaluated its biological characteristics, and found that its bacterial survival rate was 25% after 4 h of treatment at pH 2, and under the condition of 0·5% bile concentration, its survival rate exceeds 30%. In addition, L. agilis 32 has a cell surface hydrophobicity of 77·8%, and exhibits 67·1% auto-aggregation and 63·2% aggregation with Enterotoxigenic E. coli 10 (ETEC 10). FITC fluorescence labelling showed that the fluorescence intensity of cecum was significantly higher than that of duodenum, jejunum or colon (P < 0·05), but no significant difference from ileum. Lactobacillus agilis 32 bacterial culture and CFS showed average inhibition zone diameters of 14·2 and 15·4 mm respectively. Lactobacillus agilis 32 CFS treatment can significantly reduce the pathogenicity of ETEC 10. These results show that L. agilis 32 is an active and potential probiotic, and it has a good antibacterial effect on ETEC10, which provides basic research for probiotics to prevent and treat intestinal diarrhoea pathogen infection.

Interplay of a secreted protein with type IVb pilus for efficient enterotoxigenic Escherichia coli colonization.

Initial attachment and subsequent colonization of the intestinal epithelium comprise critical events allowing enteric pathogens to survive and express their pathogenesis. In enterotoxigenic Escherichia coli (ETEC), these are mediated by a long proteinaceous fiber termed type IVb pilus (T4bP). We have reported that the colonization factor antigen/III (CFA/III), an operon-encoded T4bP of ETEC, possesses a minor pilin, CofB, that carries an H-type lectin domain at its tip. Although CofB is critical for pilus assembly by forming a trimeric initiator complex, its importance for bacterial attachment remains undefined. Here, we show that T4bP is not sufficient for bacterial attachment, which also requires a secreted protein CofJ, encoded within the same CFA/III operon. The crystal structure of CofB complexed with a peptide encompassing the binding region of CofJ showed that CofJ interacts with CofB by anchoring its flexible N-terminal extension to be embedded deeply into the expected carbohydrate recognition site of the CofB H-type lectin domain. By combining this structure and physicochemical data in solution, we built a plausible model of the CofJ-CFA/III pilus complex, which suggested that CofJ acts as a molecular bridge by binding both T4bP and the host cell membrane. The Fab fragments of a polyclonal antibody against CofJ significantly inhibited bacterial attachment by preventing the adherence of secreted CofJ proteins. These findings signify the interplay between T4bP and a secreted protein for attaching to and colonizing the host cell surface, potentially constituting a therapeutic target against ETEC infection.

Enterotoxigenic E. coli virulence gene regulation in human infections.

Enterotoxigenic Escherichia coli (ETEC) is a global diarrheal pathogen that utilizes adhesins and secreted enterotoxins to cause disease in mammalian hosts. Decades of research on virulence factor regulation in ETEC has revealed a variety of environmental factors that influence gene expression, including bile, pH, bicarbonate, osmolarity, and glucose. However, other hallmarks of the intestinal tract, such as low oxygen availability, have not been examined. Further, determining how ETEC integrates these signals in the complex host environment is challenging. To address this, we characterized ETEC's response to the human host using samples from a controlled human infection model. We found ETEC senses environmental oxygen to globally influence virulence factor expression via the oxygen-sensitive transcriptional regulator fumarate and nitrate reduction (FNR) regulator. In vitro anaerobic growth replicates the in vivo virulence factor expression profile, and deletion of fnr in ETEC strain H10407 results in a significant increase in expression of all classical virulence factors, including the colonization factor antigen I (CFA/I) adhesin operon and both heat-stable and heat-labile enterotoxins. These data depict a model of ETEC infection where FNR activity can globally influence virulence gene expression, and therefore proximity to the oxygenated zone bordering intestinal epithelial cells likely influences ETEC virulence gene expression in vivo. Outside of the host, ETEC biofilms are associated with seasonal ETEC epidemics, and we find FNR is a regulator of biofilm production. Together these data suggest FNR-dependent oxygen sensing in ETEC has implications for human infection inside and outside of the host.

Spatial and temporal modulation of enterotoxigenic E. coli H10407 pathogenesis and interplay with microbiota in human gut models.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) substantially contributes to the burden of diarrheal illnesses in developing countries. With the use of complementary in vitro models of the human digestive environment, TNO gastrointestinal model (TIM-1), and Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME), we provided the first detailed report on the spatial-temporal modulation of ETEC H10407 survival, virulence, and its interplay with gut microbiota. These systems integrate the main physicochemical parameters of the human upper digestion (TIM-1) and simulate the ileum vs ascending colon microbial communities and luminal vs mucosal microenvironments, captured from six fecal donors (M-SHIME). RESULTS: A loss of ETEC viability was noticed upon gastric digestion, while a growth renewal was found at the end of jejunal and ileal digestion. The remarkable ETEC mucosal attachment helped to maintain luminal concentrations above 6 log10 mL-1 in the ileum and ascending colon up to 5 days post-infection. Seven ETEC virulence genes were monitored. Most of them were switched on in the stomach and switched off in the TIM-1 ileal effluents and in a late post-infectious stage in the M-SHIME ascending colon. No heat-labile enterotoxin production was measured in the stomach in contrast to the ileum and ascending colon. Using 16S rRNA gene-based amplicon sequencing, ETEC infection modulated the microbial community structure of the ileum mucus and ascending colon lumen. CONCLUSIONS: This study provides a better understanding of the interplay between ETEC and gastrointestinal cues and may serve to complete knowledge on ETEC pathogenesis and inspire novel prophylactic strategies for diarrheal diseases.

Antibacterial and Antibiofilm Activities of Novel Antimicrobial Peptides against Multidrug-Resistant Enterotoxigenic Escherichia Coli.

Post-weaning diarrhea due to enterotoxigenic Escherichia coli (ETEC) is a common disease of piglets and causes great economic loss for the swine industry. Over the past few decades, decreasing effectiveness of conventional antibiotics has caused serious problems because of the growing emergence of multidrug-resistant (MDR) pathogens. Various studies have indicated that antimicrobial peptides (AMPs) have potential to serve as an alternative to antibiotics owing to rapid killing action and highly selective toxicity. Our previous studies have shown that AMP GW-Q4 and its derivatives possess effective antibacterial activities against the Gram-negative bacteria. Hence, in the current study, we evaluated the antibacterial efficacy of GW-Q4 and its derivatives against MDR ETEC and their minimal inhibition concentration (MIC) values were determined to be around 2~32 µg/mL. Among them, AMP Q4-15a-1 with the second lowest MIC (4 µg/mL) and the highest minimal hemolysis concentration (MHC, 256 µg/mL), thus showing the greatest selectivity (MHC/MIC = 64) was selected for further investigations. Moreover, Q4-15a-1 showed dose-dependent bactericidal activity against MDR ETEC in time-kill curve assays. According to the cellular localization and membrane integrity analyses using confocal microscopy, Q4-15a-1 can rapidly interact with the bacterial surface, disrupt the membrane and enter cytosol in less than 30 min. Minimum biofilm eradication concentration (MBEC) of Q4-15a-1 is 4× MIC (16 µg/mL), indicating that Q4-15a-1 is effective against MDR ETEC biofilm. Besides, we established an MDR ETEC infection model with intestinal porcine epithelial cell-1 (IPEC-1). In this infection model, 32 µg/mL Q4-15a-1 can completely inhibit ETEC adhesion onto IPEC-1. Overall, these results suggested that Q4-15a-1 may be a promising antibacterial candidate for treatment of weaned piglets infected by MDR ETEC.

Porcine and Bovine Forms of Lactoferrin Inhibit Growth of Porcine Enterotoxigenic Escherichia coli and Degrade Its Virulence Factors.

Postweaning diarrhea (PWD) is an economically important, multifactorial disease affecting pigs within the first 2 weeks after weaning. The most common agent associated with PWD is enterotoxigenic Escherichia coli (ETEC). Currently, antibiotics are used to control PWD, and this has most likely contributed to an increased prevalence of antibiotic-resistant strains. This puts pressure on veterinarians and farmers to decrease or even abandon the use of antibiotics, but these measures need to be supported by alternative strategies for controlling these infections. Naturally derived molecules, such as lactoferrin, could be potential candidates due to their antibacterial or immune-modulating activities. Here, we analyzed the ability of bovine lactoferrin (bLF), porcine lactoferrin (pLF), and ovotransferrin (ovoTF) to inhibit ETEC growth, degrade ETEC virulence factors, and inhibit adherence of these pathogens to porcine intestinal epithelial cells. Our results revealed that bLF and pLF, but not ovoTF, inhibit the growth of ETEC. Furthermore, bLF and pLF can degrade several virulence factors produced by ETEC strains, more specifically F4 fimbriae, F18 fimbriae, and flagellin. On the other hand, ovoTF degrades F18 fimbriae and flagellin but not F4 fimbriae. An in vitro adhesion assay showed that bLF, ovoTF, and pLF can decrease the number of bacteria adherent to epithelial cells. Our findings demonstrate that lactoferrin can directly affect porcine ETEC strains, which could allow lactoferrin to serve as an alternative to antimicrobials for the prevention of ETEC infections in piglets.IMPORTANCE Currently, postweaning F4+ and F18+Escherichia coli infections in piglets are controlled by the use of antibiotics and zinc oxide, but the use of these antimicrobial agents most likely contributes to an increase in antibiotic resistance. Our work demonstrates that bovine and porcine lactoferrin can inhibit the growth of porcine enterotoxigenic E. coli strains. In addition, we also show that lactoferrin can reduce the adherence of these strains to small intestinal epithelial cells, even at a concentration that does not inhibit bacterial growth. This research could allow us to develop lactoferrin as an alternative strategy to prevent enterotoxigenic E. coli (ETEC) infections in piglets.

A tale of two bacterial enteropathogens and one multivalent vaccine.

Shigella and enterotoxigenic Escherichia coli (ETEC) are among the top four enteric pathogens that cause diarrheal illness in young children in developing countries and are major etiologic agents of travellers' diarrhoea. A single vaccine that could target both of these pathogens would have significant public health impact. In this review, we highlight the many pivotal contributions of Phillippe Sansonetti to the identification of molecular mechanisms of pathogenesis of Shigella that paved the way for the development of rationally designed, novel vaccines candidates. The CVD developed a series of live attenuated Shigella vaccine strains based on the most prevalent serotypes associated with disease. Shigella vaccine strains were engineered to express critical ETEC antigens to form a broadly protective Shigella-ETEC multivalent vaccine.

Lasso Peptide Microcin J25 Effectively Enhances Gut Barrier Function and Modulates Inflammatory Response in an Enterotoxigenic Escherichia coli-Challenged Mouse Model.

Bacterial resistance leads to severe public health and safety issues worldwide. Alternatives to antibiotics are currently needed. A promising lasso peptide, microcin J25 (MccJ25), is considered to be the best potential substitute for antibiotics to treat pathogen infection, including enterotoxigenic Escherichia coli (ETEC). This study evaluated the efficacy of MccJ25 in the prevention of ETEC infection. Forty-five female BALB/c mice of clean grade (aged seven weeks, approximately 16.15 g) were randomly divided into three experimental groups as follows: (i) control group (uninfected); (ii) ETEC infection group; (iii) MccJ25 + ETEC group. Fifteen mice per group in five cages, three mice/cage. MccJ25 conferred effective protection against ETEC-induced body weight loss, decrease in rectal temperature and increase in diarrhea scores in mice. Moreover, in ETEC-challenged mice model, MccJ25 significantly improved intestinal morphology, decreased intestinal histopathological scores and attenuated intestinal inflammation by decreasing proinflammatory cytokines and intestinal permeability, including reducing serum diamine oxidase and D-lactate levels. MccJ25 enhanced epithelial barrier function by increasing occludin expression in the colon and claudin-1 expression in the jejunum, ultimately improving intestinal health of host. MccJ25 was further found to alleviate gut inflammatory responses by decreasing inflammatory cytokine production and expression via the activation of the mitogen-activated protein kinase and nuclear factor κB signaling pathways. Taken together, the results indicated that MccJ25 protects against ETEC-induced intestinal injury and intestinal inflammatory responses, suggesting the potential application of MccJ25 as an excellent antimicrobial or anti-inflammation agent against pathogen infections.

Antidiarrheal potential of lactobacillus strains isolated from pharmaceutical formulations for the treatment of pediatric diarrhea.

The consumption of probiotics in the prevention and treatment of diarrhea have been clinically justified, comprehensive studied and explored in many products around the world. In Pakistan, recommendation of probiotic formulations is being emerged to control the increased mortality and morbidity from diarrhea under 5 years of age children. The objective of the study was to evaluate the antimicrobial potential of isolated Lactobacillus strains against diarrheagenic Escherichia coli. Twelve strains were isolated from different probiotic pharmaceutical formulations available in Pakistan. Physiological and biochemical characteristics of isolates were analyzed. Selective media was used for the growth of probiotic isolates and E. coli. Agar spot and well diffusion methods were employed to evaluate the antimicrobial activity of isolates and measured as a zone of inhibition (mm). Changes in cell morphology was observed by Scanning Electron Microscopy. Statistical analysis was adopted with a level of significance p<0.05. L. reuteri (28 mm) and L.plantarum (26 mm) showed significant inhibitory actions against E. coli due to increased organic acids and bacteriocins formations. Rest of isolates exhibited mild to moderate activity with an average inhibition (20 mm). L. sporogenes demonstrated weak antagonistic behavior. Use of multiple strains of Lactobacillus along with L. reuteri or L.plantarum as a therapeutic agent or in nutritional supplements could be a novel approach for the prevention and treatment of pediatric diarrhea.

Changes in the Ileal, but Not Fecal, Microbiome in Response to Increased Dietary Protein Level and Enterotoxigenic Escherichia coli Exposure in Pigs.

The relationship between porcine gut microbiota composition and health is an important area of research, especially due to the need to find alternatives to antimicrobial use to manage disease in livestock production systems. Previous work has indicated that lower crude dietary protein levels can reduce the impacts of postweaning colibacillosis, which is a porcine diarrheal disease caused by enterotoxigenic Escherichia coli (ETEC). Here, to explore the complex interactions between the gut microbiota, protein nutrition, and ETEC exposure, the microbial compositions of both ileal digesta and feces were analyzed with or without ETEC exposure from pigs fed a low- or high-protein diet. Since ETEC colonization is mostly localized to the ileum, changes in the small intestinal microbiota were expected in response to ETEC exposure. This was supported by the study findings, which identified significant microbiota changes in ileal samples but not in fecal samples. Both increased dietary protein and ETEC exposure impacted on ileal microbiota alpha diversity (richness and diversity indices) and beta diversity (structure, stability, and relative taxon abundances) at certain sampling points, although the combination of a high-protein diet and ETEC exposure had the most profound impact on ileal microbiota composition. An understanding of how infection and nutrition lead to microbiota changes is likely to be required if dietary strategies are to be developed for the management of enteric diseases.IMPORTANCE Gut bacterial communities have been shown to play a key role in pig health and development and are strongly influenced by host diet, but studies highlighting the complex interactions between nutrition, gut infections and the microbiome tend to focus on bacterial populations in the feces and not other important gut locations. We found that alteration of dietary protein level and exposure to a pathogenic microorganism, enterotoxigenic Escherichia coli (ETEC), changed bacterial populations in the distal small intestine (i.e., the ileum). We found that the most profound changes occurred in pigs fed a high-protein diet in combination with exposure to ETEC, showing a clear interaction between dietary composition and exposure to a key pathogen. These changes were not observed in the fecal samples, revealing the importance of studying biologically pertinent sites in the gut, and so the data will help to inform the development of alternative management strategies for enteric disorders.