Investigating the association between short chain fatty acid antimicrobials and Escherichia coli virulence

Antibiotic resistance in bacteria is often attributed to the excessive use of antibiotics in the agricultural and food processing sectors. Globally, antibiotics are also widely used as growth supplements in livestock, and this practice has led to an increase in multidrug-resistant microbes, raising concerns regarding the use of human-use antibiotics in livestock and food-producing animals. Due to their desirable properties, antibiotic alternatives such as organic acids have recently replaced antibiotics as antimicrobials and preventatives. As a breakdown product of non-digestible carbohydrates, some organic acids, such as propionic acid (PA) and formic acid (FA), are naturally present in the human and animal guts, and they serve crucial roles in regulating the host immune responses. Moreover, a study revealed that long-term exposure of adherent-invasive Escherichia coli (AIEC), a bacterial pathotype linked with Crohn's disease, to PA significantly altered its phenotype, resulting in enhanced adherence and invasion of epithelial cells and increased persistence through biofilm formation. In addition, it remains unclear what makes AIEC pathogenic, but it was proposed that environmental factors such as organic acids have a role in altering AIEC phenotype which could make the strains more pathogenic. Therefore, organic acids and alterations of the AIEC phenotype were investigated. Since AIEC is evolutionarily and phylogenetically related to avian pathogenic Escherichia coli (APEC), and APEC strains are more often exposed to organic acids such as PA and FA due to their widespread use in feeds, the impact of FA and PA on APEC strains was also investigated. In the investigation of organic acids and their association with phenotypic alteration of AIEC and APEC, several methods were used, such as Next-Generation Sequencing and an in vitro fermentation gut model, along with several assays to determine organic acids effects. The results revealed that PA can alter the phenotype of AIEC and increase its virulence in traits such as adhesion, invasion and biofilm formation. In addition, AIEC adapted to PA showed a significant increase in net replication within immune cells when ethanolamine is present, ethanolamine being a carbon source that becomes increasingly available during intestinal inflammation. However, FA has a different effect on AIEC and APEC strains. The gene expression of AIEC adapted to FA revealed that FA has inhibitory effects in contrast to PA. Additionally, an in vitro fermentation gut model indicated that more E. coli can be recovered from the fermentation when PA is present as opposed to FA. In conclusion, organic acids can alter AIEC and APEC phenotypes, and some of these alterations could lead to the emergence of virulent strains of bacteria. The finding that PA increased the virulence of AIEC raises concerns about its long-term effects since it is used as an antibacterial in various food and agricultural sectors. Also, the findings show that FA is a more effective antibacterial for E. coli. However, APEC strains responded differently to the FA, meaning additional research is required to identify specific FA effects on APEC strains

PF-431396 hydrate inhibition of kinase phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn’s disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease and a regulator of intestinal inflammation. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while the inhibitor PF-431396 hydrate, which blocks Pyk2 activation, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD

Inflammation associated ethanolamine facilitates infection by Crohn's disease-linked adherent-invasive Escherichia coli

Background: The predominance of specific bacteria such as adherent-invasive Escherichia coli (AIEC) within the Crohn's disease (CD) intestine remains poorly understood with little evidence uncovered to support a selective pressure underlying their presence. Intestinal ethanolamine is however readily accessible during periods of intestinal inflammation, and enables pathogens to outcompete the host microbiota under such circumstances. Methods: Quantitative RT-PCR (qRT-PCR) to determine expression of genes central to ethanolamine metabolism; transmission electron microscopy to detect presence of bacterial microcompartments (MCPs); in vitro infections of both murine and human macrophage cell lines examining intracellular replication of the AIEC-type strain LF82 and clinical E. coli isolates in the presence of ethanolamine; determination of E. coli ethanolamine utilization (eut) operon transcription in faecal samples from healthy patients, patients with active CD and the same patients in remission following treatment. Results: Growth on the intestinal short chain fatty acid propionic acid (PA) stimulates significantly increased transcription of the eut operon (fold change relative to glucose: >16.9; p-value 4.72; P 15.64; P < .01). Interpretation: Our data indicates a role for ethanolamine metabolism in selecting for AIEC that are consistently overrepresented in the CD intestine. The increased E. coli metabolism of ethanolamine seen in the intestine during active CD, and its decrease during remission, indicates ethanolamine use may be a key factor in shaping the intestinal microbiome in CD patients, particularly during times of inflammation. Fund: This work was funded by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/K008005/1 & BB/P003281/1 to DMW; by a Tenovus Scotland grant to MJO; by Glasgow Children's Hospital Charity, Nestle Health Sciences, Engineering and Physical Sciences Research Council (EPSRC) and Catherine McEwan Foundation grants awarded to KG; and by a Natural Environment Research Council (NERC) fellowship (NE/L011956/1) to UZI. The IBD team at the Royal Hospital for Children, Glasgow are supported by the Catherine McEwan Foundation and Yorkhill IBD fund. RKR and RH are supported by NHS Research Scotland Senior fellowship awards

Intestinal Epithelial Cells Regulate Gut Eotaxin Responses and Severity of Allergy

Intestinal epithelial cells (IECs) are known to regulate allergic sensitization. We addressed the role of the intrinsic IKKβ signaling in IECs in the effector phase of allergy following oral allergen challenge and its impact on the severity of responses is poorly. Upon orally sensitization by co-administration of ovalbumin with cholera toxin as adjuvant, wild-type and mice lacking IKKβ in IECs (IKKβΔIEC mice) developed similar levels of serum IgE and allergen-specific secretory IgA in the gut. However, subsequent allergen challenges in the gut promoted allergic lower responses in KKβΔIEC mice. Analysis of cytokines and chemokines in serum and gut tissues after oral allergen challenge revealed impaired eotaxin responses in IKKβΔIEC mice, which correlated with lower frequencies of eosinophils in the gut lamina propria. We also determined that IECs were a major source of eotaxin and that impaired eotaxin production was due to the lack of IKKβ signaling in IECs. Oral administration of CCL11 to IKKβΔIEC mice during oral allergen challenge enhanced allergic responses to levels in wild-type mice, confirming the role of IEC-derived eotaxin as regulator of the effector phase of allergy following allergen challenge. Our results identified targeting IEC-derived eotaxin as potential strategy to limit the severity of allergic responses to food antigens