Oral tryptophan activates duodenal aryl hydrocarbon receptor in healthy subjects: a crossover randomized controlled trial.

Background and Aims: Tryptophan is an essential amino acid transformed by host and gut microbial enzymes into metabolites that regulate mucosal homeostasis through Aryl hydrocarbon receptor (AhR) activation. Alteration of tryptophan metabolism has been associated with chronic inflammation, however whether tryptophan supplementation affects the metabolite repertoire and AhR activation under physiologic conditions in humans, is unknown. Methods: We performed a randomized, double blind, placebo-controlled, crossover study in 20 healthy volunteers. Subjects on a low tryptophan background diet were randomly assigned to a 3-week L-tryptophan supplementation (3 g/day) or placebo, and after a 2-week washout switched to opposite interventions. We assessed gastrointestinal and psychological symptoms by validated questionnaires, AhR activation by cell reporter assay, tryptophan metabolites by liquid chromatography and high-resolution mass spectrometry, cytokine production in isolated monocytes by ELISA and microbiota profile by 16S rRNA Illumina technique. Results: Oral tryptophan supplementation was well tolerated, with no changes in gastrointestinal or psychological scores. Compared with placebo, tryptophan increased AhR activation capacity by duodenal contents, but not by feces. This was paralleled by higher urinary and plasma kynurenine metabolites and indoles. Tryptophan had a modest impact on fecal microbiome profiles, and no significant effect on cytokine production. Conclusions: At the doses used in this study, oral tryptophan supplementation in humans induces microbial indole and host kynurenine metabolic pathways in the small intestine, known to be immunomodulatory. The results should prompt tryptophan intervention strategies in inflammatory conditions of the small intestine where the AhR pathway is impaired.

Identification of plasminogen-binding sites in Streptococcus suis enolase that contribute to bacterial translocation across the blood-brain barrier.

Streptococcus suis is an emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must cross the blood-brain barrier (BBB) comprising blood vessels that vascularize the central nervous system (CNS). The BBB is highly selective due to interactions with other cell types in the brain and the composition of the extracellular matrix (ECM). Purified streptococcal surface enolase, an essential enzyme participating in glycolysis, can bind human plasminogen (Plg) and plasmin (Pln). Plg has been proposed to increase bacterial traversal across the BBB via conversion to Pln, a protease which cleaves host proteins in the ECM and monocyte chemoattractant protein 1 (MCP1) to disrupt tight junctions. The essentiality of enolase has made it challenging to unequivocally demonstrate its role in binding Plg/Pln on the bacterial surface and confirm its predicted role in facilitating translocation of the BBB. Here, we report on the CRISPR/Cas9 engineering of S. suis enolase mutants eno261, eno252/253/255, eno252/261, and eno434/435 possessing amino acid substitutions at in silico predicted binding sites for Plg. As expected, amino acid substitutions in the predicted Plg binding sites reduced Plg and Pln binding to S. suis but did not affect bacterial growth in vitro compared to the wild-type strain. The binding of Plg to wild-type S. suis enhanced translocation across the human cerebral microvascular endothelial cell line hCMEC/D3 but not for the eno mutant strains tested. To our knowledge, this is the first study where predicted Plg-binding sites of enolase have been mutated to show altered Plg and Pln binding to the surface of S. suis and attenuation of translocation across an endothelial cell monolayer in vitro.

Transcriptomic analysis of intestinal organoids, derived from pigs divergent in feed efficiency, and their response to Escherichia coli.

BACKGROUND: There is increasing interest in using intestinal organoids to study complex traits like feed efficiency (FE) and host-microbe interactions. The aim of this study was to investigate differences in the molecular phenotype of organoids derived from pigs divergent for FE as well as their responses to challenge with adherent and invasive Escherichia coli (E. coli). RESULTS: Colon and ileum tissue from low and high FE pigs was used to generate 3D organoids and two dimensional (2D) monolayers of organoid cells for E. coli challenge. Genome-wide gene expression was used to investigate molecular differences between pigs that were phenotypically divergent for FE and to study the difference in gene expression after challenge with E. coli. We showed, (1) minor differences in gene expression of colon organoids from pigs with low and high FE phenotypes, (2) that an E. coli challenge results in a strong innate immune gene response in both colon and ileum organoids, (3) that the immune response seems to be less pronounced in the colon organoids of high FE pigs and (4) a slightly stronger immune response was observed in ileum than in colon organoids. CONCLUSIONS: These findings demonstrate the potential for using organoids to gain insights into complex biological mechanisms such as FE.

Cross-talk between QseBC and PmrAB two-component systems is crucial for regulation of motility and colistin resistance in Enteropathogenic Escherichia coli.

The quorum sensing two-component system (TCS) QseBC has been linked to virulence, motility and metabolism regulation in multiple Gram-negative pathogens, including Enterohaemorrhagic Escherichia coli (EHEC), Uropathogenic E. coli (UPEC) and Salmonella enterica. In EHEC, the sensor histidine kinase (HK) QseC detects the quorum sensing signalling molecule AI-3 and also acts as an adrenergic sensor binding host epinephrine and norepinephrine. Downstream changes in gene expression are mediated by phosphorylation of its cognate response regulator (RR) QseB, and 'cross-talks' with non-cognate regulators KdpE and QseF to activate motility and virulence. In UPEC, cross-talk between QseBC and TCS PmrAB is crucial in the regulation and phosphorylation of QseB RR that acts as a repressor of multiple pathways, including motility. Here, we investigated QseBC regulation of motility in the atypical Enteropathogenic E. coli (EPEC) strain O125ac:H6, causative agent of persistent diarrhoea in children, and its possible cross-talk with the KdpDE and PmrAB TCS. We showed that in EPEC QseB acts as a repressor of genes involved in motility, virulence and stress response, and in absence of QseC HK, QseB is likely activated by the non-cognate PmrB HK, similarly to UPEC. We show that in absence of QseC, phosphorylated QseB activates its own expression, and is responsible for the low motility phenotypes seen in a QseC deletion mutant. Furthermore, we showed that KdpD HK regulates motility in an independent manner to QseBC and through a third unidentified party different to its own response regulator KdpE. We showed that PmrAB has a role in iron adaptation independent to QseBC. Finally, we showed that QseB is the responsible for activation of colistin and polymyxin B resistance genes while PmrA RR acts by preventing QseB activation of these resistance genes.

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

Resistome expansion in disease-associated human gut microbiomes.

BACKGROUND: The resistome, the collection of antibiotic resistance genes (ARGs) in a microbiome, is increasingly recognised as relevant to the development of clinically relevant antibiotic resistance. Many metagenomic studies have reported resistome differences between groups, often in connection with disease and/or antibiotic treatment. However, the consistency of resistome associations with antibiotic- and non-antibiotic-treated diseases has not been established. In this study, we re-analysed human gut microbiome data from 26 case-control studies to assess the link between disease and the resistome. RESULTS: The human gut resistome is highly variable between individuals both within and between studies, but may also vary significantly between case and control groups even in the absence of large taxonomic differences. We found that for diseases commonly treated with antibiotics, namely cystic fibrosis and diarrhoea, patient microbiomes had significantly elevated ARG abundances compared to controls. Disease-associated resistome expansion was found even when ARG abundance was high in controls, suggesting ongoing and additive ARG acquisition in disease-associated strains. We also found a trend for increased ARG abundance in cases from some studies on diseases that are not treated with antibiotics, such as colorectal cancer. CONCLUSIONS: Diseases commonly treated with antibiotics are associated with expanded gut resistomes, suggesting that historical exposure to antibiotics has exerted considerable selective pressure for ARG acquisition in disease-associated strains. Video Abstract.

Impact of High-Fiber or High-Protein Diet on the Capacity of Human Gut Microbiota To Produce Tryptophan Catabolites.

This study investigated the effect of high-fiber-low-protein (HF) and high-protein-low-fiber (HP) diets on microbial catabolism of tryptophan in the proximal colon (PC) and distal colon(DC) compartments of the Simulator of the Human Intestinal Microbial Ecosystem. The microbiota in PC and DC was dominated by Bacteroidetes and Firmicutes, in which Bacteroidetes were more abundant in DC (∼60% versus 50%) and Firmicutes were more abundant in PC (∼40% versus 25%). Most of the tryptophan catabolites were determined at a higher concentration in PC samples than in DC samples, but the overall concentration of tryptophan catabolites was over 10-fold higher in DC samples than that in PC samples. Interestingly, indole-3-propionic acid and oxindole were only identified in DC samples. A two-week dietary intervention by the HF diet enriched the abundance of Firmicutes in PC, whereas the HP diet enriched the abundance of Proteobacteria. Compared to the HP diet, the HF diet favored the microbial production of indole-3-acetic acid, indole-3-lactic acid, indole-3-aldehyde, and indole-3-propionic acid in both PC and DC compartments. To conclude, these findings increase the understanding of the effect of diets on the microbial production of tryptophan catabolites in the colon.

Agaricus subrufescens fermented rye affects the development of intestinal microbiota, local intestinal and innate immunity in suckling-to-nursery pigs.

BACKGROUND: Agaricus subrufescens is considered as one of the most important culinary-medicinal mushrooms around the world. It has been widely suggested to be used for the development of functional food ingredients to promote human health ascribed to the various properties (e.g., anti-inflammatory, antioxidant, and immunomodulatory activities). In this context, the interest in A. subrufescens based feed ingredients as alternatives for antibiotics has also been fuelled during an era of reduced/banned antibiotics use. This study aimed to investigate the effects of a fermented feed additive -rye overgrown with mycelium (ROM) of A. subrufescens-on pig intestinal microbiota, mucosal gene expression and local and systemic immunity during early life. Piglets received ROM or a tap water placebo (Ctrl) perorally every other day from day 2 after birth until 2 weeks post-weaning. Eight animals per treatment were euthanized and dissected on days 27, 44 and 70. RESULTS: The results showed ROM piglets had a lower inter-individual variation of faecal microbiota composition before weaning and a lower relative abundance of proteobacterial genera in jejunum (Undibacterium and Solobacterium) and caecum (Intestinibacter and Succinivibrionaceae_UCG_001) on day 70, as compared to Ctrl piglets. ROM supplementation also influenced gut mucosal gene expression in both ileum and caecum on day 44. In ileum, ROM pigs showed increased expression of TJP1/ZO1 but decreased expression of CLDN3, CLDN5 and MUC2 than Ctrl pigs. Genes involved in TLR signalling (e.g., TICAM2, IRAK4 and LY96) were more expressed but MYD88 and TOLLIP were less expressed in ROM pigs than Ctrl animals. NOS2 and HIF1A involved in redox signalling were either decreased or increased in ROM pigs, respectively. In caecum, differentially expressed genes between two groups were mainly shown as increased expression (e.g., MUC2, PDGFRB, TOLLIP, TNFAIP3 and MYD88) in ROM pigs. Moreover, ROM animals showed higher NK cell activation in blood and enhanced IL-10 production in ex vivo stimulated MLN cells before weaning. CONCLUSIONS: Collectively, these results suggest that ROM supplementation in early life modulates gut microbiota and (local) immune system development. Consequently, ROM supplementation may contribute to improving health of pigs during the weaning transition period and reducing antibiotics use.

Transcriptomics in serum and culture medium reveal shared and differential gene regulation in pathogenic and commensal Streptococcus suis.

Streptococcus suis colonizes the upper respiratory tract of healthy pigs at high abundance but can also cause opportunistic respiratory and systemic disease. Disease-associated S. suis reference strains are well studied, but less is known about commensal lineages. It is not known what mechanisms enable some S. suis lineages to cause disease while others persist as commensal colonizers, or to what extent gene expression in disease-associated and commensal lineages diverge. In this study we compared the transcriptomes of 21 S. suis strains grown in active porcine serum and Todd-Hewitt yeast broth. These strains included both commensal and pathogenic strains, including several strains of sequence type (ST) 1, which is responsible for most cases of human disease and is considered to be the most pathogenic S. suis lineage. We sampled the strains during their exponential growth phase and mapped RNA sequencing reads to the corresponding strain genomes. We found that the transcriptomes of pathogenic and commensal strains with large genomic divergence were unexpectedly conserved when grown in active porcine serum, but that regulation and expression of key pathways varied. Notably, we observed strong variation of expression across media of genes involved in capsule production in pathogens, and of the agmatine deiminase system in commensals. ST1 strains displayed large differences in gene expression between the two media compared to strains from other clades. Their capacity to regulate gene expression across different environmental conditions may be key to their success as zoonotic pathogens.

Effect of whole foods on the microbial production of tryptophan-derived aryl hydrocarbon receptor agonists in growing pigs.

Effects of whole foods on the microbial production of tryptophan-derived aryl hydrocarbon receptor (AhR) ligands in the intestine were investigated in a pig model. Ileal digesta and faeces of pigs after feeding of eighteen different foods were analyzed. Indole, indole-3-propionic acid, indole-3-acetic acid, indole-3-lactic acid, kynurenine, tryptamine, and indole-3-aldehyde were identified in ileal digesta, which were also identified in faeces but at higher concentrations except indole-3-lactic acid, together with skatole, oxindole, serotonin, and indoleacrylic acid. The panel of tryptophan catabolites in ileal digesta and faeces varied across different foods. Eggs induced the highest overall concentration of catabolites in ileal digesta dominated by indole. Amaranth induced the highest overall concentration of catabolites in faeces dominated by skatole. Using a reporter cell line, we observed many faecal samples but not ileal samples retained AhR activity. Collectively, these findings contribute to food selection targeting AhR ligands production from dietary tryptophan in the intestine.

Distinct effects of fiber and colon segment on microbiota-derived indoles and short-chain fatty acids.

Effects of pectin, inulin, and their combination on the production of microbiota-derived indoles and short-chain fatty acids (SCFAs) from different colon segments were investigated in a batch system inoculated with microbiota from proximal colon (PC) and distal colon (DC) compartments of the Simulator of Human Intestinal Microbial Ecosystem. Bacteria from DC compartment had a higher abundance of Firmicutes and a stronger capacity to produce indoles and SCFAs than bacteria from PC compartment. Fiber supplementation significantly increased the production of SCFAs, indole-3-propionic acid, and indole-3-lactic acid, but decreased the production of oxindole, tryptamine, and serotonin. Pectin specifically promoted the production of indole-3-acetic acid and indole-3-aldehyde. Interestingly, supplementation of pectin or inulin increased the relative abundance of Bacteroidetes whereas supplementation of a mixture of two fibers decreased it. Overall, these results suggest that fiber supplementation and colon segment affect the composition of gut microbiota and the microbial catabolism of tryptophan.

Environmental and maternal factors shaping tonsillar microbiota development in piglets.

BACKGROUND: The palatine tonsils are part of the mucosal immune system and stimulate immune responses through M cell uptake sampling of antigens and bacteria in the tonsillar crypts. Little is known about the development of the tonsillar microbiota and the factors determining the establishment and proliferation of disease-associated bacteria such as Streptococcus suis. In this study, we assessed tonsillar microbiota development in piglets during the first 5 weeks of life and identified the relative importance of maternal and environmental farm parameters influencing the tonsillar microbiota at different ages. Additionally, we studied the effect sow vaccination with a bacterin against S. suis on microbiota development and S. suis colonisation in their offspring. RESULTS: Amplicon sequencing of the 16S rRNA gene V3-V4 region revealed that a diverse tonsillar microbiota is established shortly after birth, which then gradually changes during the first 5 weeks of life without a large impact of weaning on composition or diversity. We found a strong litter effect, with siblings sharing a more similar microbiota compared to non-sibling piglets. Co-housing in rooms, within which litters were housed in separate pens, also had a large impact on microbiota composition. Sow parity and prepartum S. suis bacterin vaccination of sows had weaker but significant associations with microbiota composition, impacting on the abundance of Streptococcus species before and after weaning. Sex and birthweight had limited impact on the tonsillar microbiota, and none of the measured factors had consistent associations with microbiota diversity. CONCLUSIONS: The piglet tonsillar microbiota is established shortly after birth. While microbiota development is associated with both environmental and maternal parameters, weaning has limited impact on microbiota composition. Intramuscular vaccination of sows pre-partum had a significant effect on the tonsillar microbiota composition of their piglets. These findings provide new insights into the mechanisms shaping the tonsillar microbiota.

Comparative genomics of Rothia species reveals diversity in novel biosynthetic gene clusters and ecological adaptation to different eukaryotic hosts and host niches.

Rothia species are understudied members of the phylum Actinobacteria and prevalent colonizers of the human and animal upper respiratory tract and oral cavity. The oral cavity, including the palatine tonsils, is colonized by a complex microbial community, which compete for resources, actively suppress competitors and influence host physiology. We analysed genomic data from 43 new porcine Rothia isolates, together with 112 publicly available draft genome sequences of Rothia isolates from humans, animals and the environment. In all Rothia genomes, we identified biosynthetic gene clusters predicted to produce antibiotic non-ribosomal peptides, iron scavenging siderophores and other secondary metabolites that modulate microbe-microbe and potentially microbe-host interactions. In vitro overlay inhibition assays corroborated the hypothesis that specific strains produce natural antibiotics. Rothia genomes encode a large number of carbohydrate-active enzymes (CAZy), with varying CAZy activities among the species found in different hosts, host niches and environments. These findings reveal competition mechanisms and metabolic specializations linked to ecological adaptation of Rothia species in different hosts.

Adaptive laboratory evolution and independent component analysis disentangle complex vancomycin adaptation trajectories.

Human infections with methicillin-resistant Staphylococcus aureus (MRSA) are commonly treated with vancomycin, and strains with decreased susceptibility, designated as vancomycin-intermediate S. aureus (VISA), are associated with treatment failure. Here, we profiled the phenotypic, mutational, and transcriptional landscape of 10 VISA strains adapted by laboratory evolution from one common MRSA ancestor, the USA300 strain JE2. Using functional and independent component analysis, we found that: 1) despite the common genetic background and environmental conditions, the mutational landscape diverged between evolved strains and included mutations previously associated with vancomycin resistance (in vraT, graS, vraFG, walKR, and rpoBCD) as well as novel adaptive mutations (SAUSA300_RS04225, ssaA, pitAR, and sagB); 2) the first wave of mutations affected transcriptional regulators and the second affected genes involved in membrane biosynthesis; 3) expression profiles were predominantly strain-specific except for sceD and lukG, which were the only two genes significantly differentially expressed in all clones; 4) three independent virulence systems (φSa3, SaeR, and T7SS) featured as the most transcriptionally perturbed gene sets across clones; 5) there was a striking variation in oxacillin susceptibility across the evolved lineages (from a 10-fold increase to a 63-fold decrease) that also arose in clinical MRSA isolates exposed to vancomycin and correlated with susceptibility to teichoic acid inhibitors; and 6) constitutive expression of the VraR regulon explained cross-susceptibility, while mutations in walK were associated with cross-resistance. Our results show that adaptation to vancomycin involves a surprising breadth of mutational and transcriptional pathways that affect antibiotic susceptibility and possibly the clinical outcome of infections.

Babies, Bugs, and Barriers: Dietary Modulation of Intestinal Barrier Function in Early Life.

The intestinal barrier is essential in early life to prevent infection, inflammation, and food allergies. It consists of microbiota, a mucus layer, an epithelial layer, and the immune system. Microbial metabolites, the mucus, antimicrobial peptides, and secretory immunoglobulin A (sIgA) protect the intestinal mucosa against infection. The complex interplay between these functionalities of the intestinal barrier is crucial in early life by supporting homeostasis, development of the intestinal immune system, and long-term gut health. Exclusive breastfeeding is highly recommended during the first 6 months. When breastfeeding is not possible, milk-based infant formulas are a safe alternative. Breast milk contains many bioactive components that help to establish the intestinal microbiota and influence the development of the intestinal epithelium and the immune system. Importantly, breastfeeding lowers the risk for intestinal and respiratory tract infections. Here we review all aspects of intestinal barrier function and the nutritional components that impact its functionality in early life, such asmicronutrients, bioactive milk proteins, milk lipids, and human milk oligosaccharides. These components are present in breast milk and can be added to milk-based infant formulas to support gut health and immunity.

Effects of E scherichia coli Nissle 1917 on the Porcine Gut Microbiota, Intestinal Epithelium and Immune System in Early Life.

Early in life and particularly around weaning, piglets are susceptible to infections because of abrupt social, environmental, and dietary changes. Dietary interventions with probiotic bacteria have gained popularity because of the increased awareness of the direct link between diet and health. In this study, piglets received the probiotic strain Escherichia coli Nissle 1917 (EcN) or a control treatment perorally from day 2 after birth until 2 weeks post-weaning. To investigate spatio-temporal effects of EcN on the gut microbiota composition, intestinal epithelial gene expression and immune system, feces, digesta, blood, scraping material and mesenteric lymph node tissue were collected at different time points. In addition, oral vaccinations against Salmonella enterica serovar Typhimurium were administered on days 21 and 45 of the study to assess the immunocompetence. EcN-treated pigs showed a reduced diversity of taxa within the phylum Proteobacteria and a lower relative abundance of taxa within the genus Treponema during the pre-weaning period. Moreover, EcN induced T cell proliferation and Natural Killer cell activation in blood and enhanced IL-10 production in ex vivo stimulated mesenteric lymph node cells, the latter pointing toward a more regulatory or anti-inflammatory state of the local gut-associated immune system. These outcomes were primarily observed pre-weaning. No significant differences were observed between the treatment groups with regards to body weight, epithelial gene expression, and immune response upon vaccination. Differences observed during the post-weaning period between the treatment groups were modest. Overall, this study demonstrates that the pre-weaning period offers a 'window of opportunity' to modulate the porcine gut microbiota and immune system through dietary interventions such as EcN supplementation.

Tryptophan Supplementation Increases the Production of Microbial-Derived AhR Agonists in an In Vitro Simulator of Intestinal Microbial Ecosystem.

The aryl hydrocarbon receptor (AhR) plays an important role in intestinal homeostasis, and some microbial metabolites of tryptophan are known AhR agonists. In this study, we assessed the impact of tryptophan supplementation on the formation of tryptophan metabolites, AhR activation, and microbiota composition in the simulator of the human intestinal microbial ecosystem (SHIME). AhR activation, microbial composition, and tryptophan metabolites were compared during high tryptophan supplementation (4 g/L tryptophan), control, and wash-out periods. During tryptophan supplementation, the concentration of several tryptophan metabolites was increased compared to the control and wash-out period, but AhR activation by fermenter supernatant was significantly decreased. This was due to the higher levels of tryptophan, which was found to be an antagonist of AhR signaling. Tryptophan supplementation induced most microbial changes in the transverse colon including increased relative abundance of lactobacillus. We conclude that tryptophan supplementation leads to increased formation of AhR agonists in the colon.

Lichenysin Production by Bacillus licheniformis Food Isolates and Toxicity to Human Cells.

Bacillus licheniformis can cause foodborne intoxication due to the production of the surfactant lichenysin. The aim of this study was to measure the production of lichenysin by food isolates of B. licheniformis in LB medium and skimmed milk and its cytotoxicity for intestinal cells. Out of 11 B. licheniformis isolates tested, most showed robust growth in high salt (1M NaCl), 4% ethanol, at 37 or 55°C, and aerobic and anaerobic conditions. All strains produced lichenysin (in varying amounts), but not all strains were hemolytic. Production of this stable compound by selected strains (high producers B4094 and B4123, and type strain DSM13 T ) was subsequently determined using LB medium and milk, at 37 and 55°C. Lichenysin production in LB broth and milk was not detected at cell densities < 5 log10 CFU/ml. The highest concentrations were found in the stationary phase of growth. Total production of lichenysin was 4-20 times lower in milk than in LB broth (maximum 36 µg/ml), and ∼10 times lower in the biomass obtained from milk agar than LB agar. Under all conditions tested, strain B4094 consistently yielded the highest amounts. Besides strain variation and medium composition, temperature also had an effect on lichenysin production, with twofold lower amounts of lichenysin produced at 55°C than at 37°C. All three strains produced lichenysin A with varying acyl chain lengths (C11-C18). The relative abundance of the C14 variant was highest in milk and the C15 variant highest in LB. The concentration of lichenysin needed to reduce cell viability by 50% (IC50) was 16.6 µg/ml for Caco-2 human intestinal epithelial cells and 16.8 µg/ml for pig ileum organoids. Taken together, the presence of low levels (<5 log10 CFU/ml) of B. licheniformis in foods is unlikely to pose a foodborne hazard related to lichenysin production. However, depending on the strain present, the composition, and storage condition of the food, a risk of foodborne intoxication may arise if growth to high levels is supported and such product is ingested.

Collection of human and environmental data on pesticide use in Europe and Argentina: Field study protocol for the SPRINT project.

Current farm systems rely on the use of Plant Protection Products (PPP) to secure high productivity and control threats to the quality of the crops. However, PPP use may have considerable impacts on human health and the environment. A study protocol is presented aiming to determine the occurrence and levels of PPP residues in plants (crops), animals (livestock), humans and other non-target species (ecosystem representatives) for exposure modelling and impact assessment. To achieve this, we designed a cross-sectional study to compare conventional and organic farm systems across Europe. Environmental and biological samples were/are being/will be collected during the 2021 growing season, at 10 case study sites in Europe covering a range of climate zones and crops. An additional study site in Argentina will inform the impact of PPP use on growing soybean which is an important European protein-source in animal feed. We will study the impact of PPP mixtures using an integrated risk assessment methodology. The fate of PPP in environmental media (soil, water and air) and in the homes of farmers will be monitored. This will be complemented by biomonitoring to estimate PPP uptake by humans and farm animals (cow, goat, sheep and chicken), and by collection of samples from non-target species (earthworms, fish, aquatic and terrestrial macroinvertebrates, bats, and farm cats). We will use data on PPP residues in environmental and biological matrices to estimate exposures by modelling. These exposure estimates together with health and toxicity data will be used to predict the impact of PPP use on environment, plant, animal and human health. The outcome of this study will then be integrated with socio-economic information leading to an overall assessment used to identify transition pathways towards more sustainable plant protection and inform decision makers, practitioners and other stakeholders regarding farming practices and land use policy.

Adjuvant Effect of Orally Applied Preparations Containing Non-Digestible Polysaccharides on Influenza Vaccination in Healthy Seniors: A Double-Blind, Randomised, Controlled Pilot Trial.

Senior individuals can suffer from immunosenescence and novel strategies to bolster the immune response could contribute to healthy ageing. In this double-blind, randomised, controlled pilot trial, we investigated the ability of non-digestible polysaccharide (NPS) preparations to enhance the immune response in a human vaccination model. In total, 239 subjects (aged 50-79 years) were randomised to consume one of five different NPS (yeast ß-glucan (YBG), shiitake ß-glucan (SBG), oat ß-glucan (OBG), arabinoxylan (AX), bacterial exopolysaccharide (EPS)) or control (CTRL) product daily for five weeks. After two weeks of intervention, subjects were vaccinated with seasonal influenza vaccine. The post-vaccination increases in haemagglutination inhibition antibody titres and seroprotection rate against the influenza strains were non-significantly enhanced in the NPS intervention groups compared to CTRL. Specifically, a trend towards a higher mean log2 fold increase was observed in the AX group (uncorrected p = 0.074) combined with a trend for an increased seroprotection rate, AX group (48.7%) compared to CTRL (25.6%) (uncorrected p = 0.057), for the influenza A H1N1 strain. Subjects consuming AX also had a reduced incidence of common colds compared to CTRL (1 vs. 8; p = 0.029 in Fisher exact test). No adverse effects of NPS consumption were reported. The findings of this pilot study warrant further research to study AX as an oral adjuvant to support vaccine efficacy.