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Background: Halogenated furanones have been found to act as potent quorum sensing inhibitors in several bacterial species. It is believed that dihalofuranones covalently bind to the LuxS enzyme, which is necessary for autoinducer-2 synthesis. In addition to their antimicrobial activity, halogenated furanones also possess anti-cancer, antioxidant, and depigmentation properties. However, traditional routes to these compounds are low-yielding and capricious. Objective: The aim of this study was to investigate higher-yielding preparations of gemdihalofuranones and compare their reactivity using Suzuki chemistry. Methods: Ramirez dibromoolefination of maleic anhydride was optimised using a variety of conditions. A similar route was investigated for the preparation of bromofluorofuranones and dichlorofuranones. The conversion of a dichlorofuranone to the corresponding iodofuranone derivatives using microwave-assisted Finkelstein chemistry was also studied. Lastly, the reactivity of the different dihalofuranones was compared by Pd-mediated coupling with phenylboronic acid. Results: A higher-yielding, concise synthesis of dibromofuranones was developed using a modified Ramirez reaction. Additionally, a telescoped preparation of dichlorofuranone was higher yielding than previous approaches. Bromine- and iodine-substituted dihalofuranones proved more reactive than their chlorine-substituted analogues. Conclusion: Higher yielding routes to bromine-, fluorine-, chlorine- and iodine-containing dihalofuranones were successfully developed. Suzuki couplings of gem-dihalofuranones were found to proceed with high stereoselectivity.

Since their initial isolation from the marine alga Delisea pulchra, bromofuranones have been investigated as potential inhibitors of quorum sensing (QS) in various bacterial strains. QS is an important mechanism by which bacteria co-ordinate their molecular response to the environment. QS is intrinsically linked to bacterial antibiotic resistance. Inspired by nature, chemists have developed a wide variety of synthetic analogs in an effort to elucidate the structure–activity relationships of these compounds, and to ultimately develop novel antimicrobial agents. In this work, we describe advances in this field while paying particular attention to apparent structure–activity relationships. This review is organized according to the main ring systems under investigation, namely furanones, dihydropyrrolones and thiophenones.

Oral delivery of poorly water-soluble drugs (PWSDs), which predominate the development pipeline, poses significant challenges. Weakly basic compounds, such as atazanavir, represent a critical class of PWSDs as even the administration of the crystalline solid may invoke supersaturation during gastric-intestinal transfer. The absorption advantage afforded by this supersaturated state can be offset by inherent metastability and a tendency to revert to the lower energy crystalline state. Therefore, it is important to understand the physiological factors that can affect crystallization to improve in vitro-in vivo predictiveness and to regulate inter-individual responses. The first aim of this study was to elucidate the influence of lyso-phosphatidylcholine (lyso-PC) and sodium oleate on crystallization, as the products of phosphatidylcholine (PC) hydrolysis and the major lipid components of human intestinal fluid (HIF) and updated fasted state simulated intestinal fluid version 3 (FaSSIF-V3). Secondly, as an individual’s bile acid pool is unique, dynamic and related to gut microbiome community structure, it was of interest to investigate the impact of bile acid pool variations on crystallization from supersaturated solutions. To study the effect of PC hydrolysis, media with 2.8 mM sodium glycocholate (GCA) and sodium taurocholate (TCA) (1:1) but varying concentrations of PC, lyso-PC or sodium oleate were prepared. To investigate the influence of inter-individual variations in intestinal bile acid pool size and composition, media simulating the profiles of six healthy Western volunteers were prepared based on previously published data. The crystalline and amorphous solubility of atazanavir, a weakly basic antiretroviral drug, was firstly determined in these media. Nucleation-induction time experiments were then performed at an equivalent extent of supersaturation in each medium (corresponding to the amorphous solubility). At a constant level of GCA/TCA, increasing concentrations of both PC and lyso-PC accelerated crystallization onset; however, this was at least 2-fold more pronounced with lyso-PC at a given molar concentration. The addition of sodium oleate was also observed to induce crystallization. Interestingly, substituting GCA/TCA with the bile salt fraction of other biorelevant media partially circumvented the crystallization-inducing effect of phospholipids and their digests. The presence of dihydroxy bile salts was found to be particularly significant in decelerating the crystallization process. Nucleation-induction times in simulated volunteer pools were found to be dependent upon bile salt concentration, with higher bile salt levels generally prolonging supersaturation. Differences of up to 6-fold were observed. This study demonstrates that the choice of biorelevant medium used to evaluate supersaturating formulations can influence the observed crystallization kinetics. While the presence of lyso-PC and sodium oleate in FaSSIF-V3 medium is more physiologically relevant, further attention should be paid to the bile salt fraction when designing a biorelevant medium for supersaturating formulations. In vivo, inter-individual differences in the amount and types of bile acids and phospholipids present may influence the behaviour of supersaturating formulations.

The microbiota-gut-brain axis encompasses a bidirectional mode of communication between the microorganisms residing in our gut, and our brain function and behavior. The composition of the gut microbiota is subject to diurnal variation and is entrained by host circadian rhythms. In turn, a diverse microbiota is essential for optimal regulation of host circadian pathways. Disruption of the cyclical nature of this microbe-host interaction profoundly influences disease pathology and severity. This review aims to summarize current knowledge on this bidirectional relationship. Indeed, the past few years have revealed promising data regarding the relationship between the microbiota-gut-brain axis and circadian rhythms and how they act in concert to influence disease, but further research needs to be done to examine how they coalesce to modulate severity of, and risk for, certain diseases. Moreover, there is a need for a greater understanding of the molecular mechanisms underlying the close relationship between circadian-microbiome-brain interactions.

We have previously isolated and characterized food-dwelling strains of

Bile acid (BA) signatures are altered in many disease states. BA metabolism is an important microbial function to assist gut colonization and persistence, as well as microbial survival during gastro intestinal (GI) transit and it is an important criteria for potential probiotic bacteria. Microbes that express bile salt hydrolase (BSH), gateway BA modifying enzymes, are considered to have an advantage in the gut. This property is reported as selectively limited to gut-associated microbes. Food-associated microbes have the potential to confer health benefits to the human consumer. Here, we report that food associated Lactobacillus plantarum strains are capable of BA metabolism, they can withstand BA associated stress and propagate, a recognised important characteristic for GIT survival. Furthermore, we report that these food associated Lactobacillus plantarum strains have the selective ability to alter BA signatures in favour of receptor activation that would be beneficial to humans. Indeed, all of the strains examined showed a clear preference to alter human glycol-conjugated BAs, although clear strain-dependent modifications were also evident. This study demonstrates that BA metabolism by food-borne non-pathogenic bacteria is beneficial to both microbe and man and it identifies an evolutionary-conserved characteristic, previously considered unique to gut residents, among food-associated non-pathogenic isolates.

International audience; Listeria monocytogenes is a deadly foodborne pathogen. It is ubiquitous in nature and can be found in the wilderness, in the farm environment, in the food production environment and in food products. Transmission routes are complex and progress from natural environments to contamination of foods and subsequently host infection. The ability of L. monocytogenes to persist in such a large array of habitats relies on gene expression regulation and metabolic features. When contaminated food is consumed, the fate of L. monocytogenes depends on a complex interplay between the composition of the food matrix, host susceptibility as well as strain phylogeny. Indeed, genetic features underlie fitness differences between isolates during the saprophytic lifestyle and during infection.

Aims: The aim of this study was to test the efficacy of new and currently used biocides in the mushroom industry for inactivating L. monocytogenes biofilm. Methods and results: A lab‐scale study was initially carried out to test the efficacy of eleven biocidal products against a cocktail of five L. monocytogenes strains that were grown to three‐day biofilms on stainless steel coupons. Biocidal efficacy was then tested under clean and dirty conditions based on the EN 13697:2015 method. The results for the biocides tested ranged between 1.7‐log to 6‐log reduction of biofilm, with only the efficacy of the sodium hypochlorite‐based biocide being significantly reduced in dirty conditions. A pilot‐scale trial was then carried out on a subset of biocides against L. monocytogenes on concrete floors in a mushroom growing room and it was found that biocide efficacy in lab‐scale did not translate well in pilot‐scale. Conclusions: Biocides that are used in the mushroom industry and potential alternative biocides were determined to be effective against L. monocytogenes biofilm in both lab‐scale and pilot‐scale experiments. Significance and impact of the study: This study has direct impact for the industry as it provides information on the efficacy of currently used biocides and other biocidal products against L. monocytogenes, an added benefit to their primary use.

Background The commercial production of Agaricus bisporus is a three stage process: 1) production of compost, also called “substrate”; 2) production of casing soil; and 3) production of the mushrooms. Hygiene practices are undertaken at each stage: pasteurization of the substrate, hygiene practices applied during the production of casing soil, postharvest steam cookout, and disinfection at the mushroom production facilities. However, despite these measures, foodborne pathogens, including Listeria monocytogenes, are reported in the mushroom production environment. In this work, the presence of L. monocytogenes was evaluated before and after the application of hygiene practices at each stage of mushroom production with swabs, samples of substrate, casing, and spent mushroom growing substrates. Results L. monocytogenes was not detected in any casing or substrate sample by enumeration according to BS EN ISO 11290‐2:1998. Analysis of the substrate showed that L. monocytogenes was absent in 10 Phase II samples following pasteurization, but was then present in 40% of 10 Phase III samples. At the casing production facility, 31% of 59 samples were positive. Hygiene improvements were applied, and after four sampling occasions, 22% of 37 samples were positive, but no statistically significant difference was observed (p > .05). At mushroom production facilities, the steam cookout process inactivated L. monocytogenes in the spent growth substrate, but 13% of 15 floor swabs at Company 1 and 19% of 16 floor swabs at Company 2, taken after disinfection, were positive. Conclusion These results showed the possibility of L. monocytogenes recontamination of Phase III substrate, cross‐contamination at the casing production stage and possible survival after postharvest hygiene practices at the mushroom growing facilities. This information will support the development of targeted measures to minimize L. monocytogenes in the mushroom industry., During the mushroom production, the current hygiene practices are not completely efficient in Listeria monocytogenes removal. Here, we investigate the effectiveness of hygiene practices in critical points throughout the mushroom production chain.

Pharmacokinetic research at the host-microbe interface has been primarily directed toward effects on drug metabolism, with fewer investigations considering the absorption process. We previously demonstrated that the transcriptional expression of genes encoding intestinal transporters involved in lipid translocation are altered in germ-free and conventionalized mice possessing distinct bile acid signatures. It was consequently hypothesized that microbial bile acid metabolism, which is the deconjugation and dehydroxylation of the bile acid steroid nucleus by gut bacteria, may impact upon drug transporter expression and/or activity and potentially alter drug disposition. Using a panel of three human intestinal cell lines (Caco-2, T84, and HT-29) that differ in basal transporter expression level, bile acid conjugation-, and hydroxylation-status was shown to influence the transcription of genes encoding several major influx and efflux transporter proteins. We further investigated if these effects on transporter mRNA would translate to altered drug disposition and activity. The results demonstrated that the conjugation and hydroxylation status of the bile acid steroid nucleus can influence the cellular response to multidrug resistance (MDR) substrates, a finding that did not directly correlate with directionality of gene or protein expression. In particular, we noted that the cytotoxicity of cyclosporine A was significantly augmented in the presence of the unconjugated bile acids deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) in P-gp positive cell lines, as compared to their taurine/glycine-conjugated counterparts, implicating P-gp in the molecular response. Overall this work identifies a novel mechanism by which gut microbial metabolites may influence drug accumulation and suggests a potential role for the microbial bile acid-deconjugating enzyme bile salt hydrolase (BSH) in ameliorating multidrug resistance through the generation of bile acid species with the capacity to access and inhibit P-gp ATPase. The physicochemical property of nonionization is suggested to underpin the preferential ability of unconjugated bile acids to attenuate the efflux of P-gp substrates and to sensitize tumorigenic cells to cytotoxic therapeutics in vitro. This work provides new impetus to investigate whether perturbation of the gut microbiota, and thereby the bile acid component of the intestinal metabolome, could alter drug pharmacokinetics in vivo. These findings may additionally contribute to the development of less toxic P-gp modulators, which could overcome MDR.

Once regarded obscure and underappreciated, the gut microbiota (the microbial communities colonizing the gastrointestinal tract) is gaining recognition as an influencer of many aspects of human health. Also increasingly apparent is the breadth of interindividual variation in these co-evolved microbial-gut associations, presenting novel quests to explore implications for disease and therapeutic response. In this respect, the unearthing of the drug-metabolizing capacity of the microbiota has provided impetus for the integration of microbiological and pharmacological research. This review considers a potential mechanism, 'microbial bile acid metabolism', by which the intricate interplay between the host and gut bacteria may influence drug pharmacokinetics. Bile salts traditionally regarded as biological surfactants, synthesized by the host and biotransformed by gut bacteria, are now also recognized as signalling molecules that affect diverse physiological processes. Accumulating data indicate that bile salts are not equivalent with respect to their physicochemical properties, micellar solubilization capacities for poorly water-soluble drugs, crystallization inhibition tendencies nor potencies for bile acid receptor activation. Herein, the origin, physicochemical properties, physiological functions, plasticity and pharmaceutical significance of the human bile acid pool are discussed. Microbial dependant differences in the composition of the human bile acid pool, simulated intestinal media and commonly used preclinical species is highlighted to better understand in vivo performance predictiveness. While the precise impact of an altered gut microbiome, and consequently bile acid pool, in the biopharmaceutical setting remains largely elusive, the objective of this article is to aid knowledge acquisition through a detailed review of the literature.

peer-reviewed As the new technology of whole genome sequencing (WGS) has been shown to have greater discriminatory power in differentiating strains than the much-used pulsed-field gel electrophoresis (PFGE), there is currently a transition from using PFGE to WGS for disease outbreak investigation. Therefore, there is a need for comparison of bacterial isolates using both PFGE and WGS. In this study, two pairs of L. monocytogenes strains with geographically diverse sources of isolation but which had indistinguishable or closely related PFGE profiles, were subjected to WGS analysis. Comparative analysis of their genomes showed that one pair of strains which had closely related PFGE profiles in fact differed significantly from one another in terms of their antibiotic and heavy metal stress resistance determinants, and mobile genetic elements. Therefore, this research demonstrated the ability of WGS analysis to differentiate very closely related strains and that WGS analysis represents the most effective tool available for subtyping L. monocytogenes isolates.

Bile acids are synthesized from cholesterol in the liver and released into the intestine to aid the digestion of dietary lipids. The host enzymes that contribute to bile acid synthesis in the liver and the regulatory pathways that influence the composition of the total bile acid pool in the host have been well established. In addition, the gut microbiota provides unique contributions to the diversity of bile acids in the bile acid pool. Gut microbial enzymes contribute significantly to bile acid metabolism through deconjugation and dehydroxylation reactions to generate unconjugated bile acids and secondary bile acids. These microbial enzymes (which include bile salt hydrolase (BSH) and bile acid-inducible (BAI) enzymes) are essential for bile acid homeostasis in the host and represent a vital contribution of the gut microbiome to host health. Perturbation of the gut microbiota in disease states may therefore significantly influence bile acid signatures in the host, especially in the context of gastrointestinal or systemic disease. Given that bile acids are ligands for host cell receptors (including the FXR, TGR5 and Vitamin D Receptor) alterations to microbial enzymes and associated changes to bile acid signatures have significant consequences for the host. In this review we examine the contribution of microbial enzymes to the process of bile acid metabolism in the host and discuss the implications for microbe-host signalling in the context of C. difficile infection, inflammatory bowel disease and other disease states.

Background & Aims: Options for the prevention of short-bowel syndromeâassociated liver disease (SBS-ALDs) are limited and often ineffective. The farnesoid X receptor (FXR) is a newly emerging pharmaceutical target and FXR agonists have been shown to ameliorate cholestasis and metabolic disorders. The aim of this study was to assess the efficacy of obeticholic acid (OCA) treatment in preventing SBS-ALDs. Methods: Piglets underwent 75% small-bowel resection (SBS) or sham surgery (sham) and were assigned to either a daily dose of OCA (2.4 mg/kg/day) or were untreated. Clinical measures included weight gain and stool studies. Histologic features were assessed. Ultraperformance liquid chromatography tandem mass spectrometry was used to determine bile acid composition in end point bile and portal serum samples. Gene expression of key FXR targets was assessed in intestinal and hepatic tissues via quantitative polymerase chain reaction. Results: OCA-treated SBS piglets showed decreased stool fat and altered liver histology when compared with nontreated SBS piglets. OCA prevented SBS-associated taurine depletion, however, further analysis of bile and portal serum samples indicated that OCA did not prevent SBS-associated alterations in bile acid composition. The expression of FXR target genes involved in bile acid transport and synthesis increased within the liver of SBS piglets after OCA administration whereas, paradoxically, intestinal expression of FXR target genes were decreased by OCA administration. Conclusions: Administration of OCA in SBS reduced fat malabsorption and altered bile acid composition, but did not prevent the development of SBS-ALDs. We postulate that extensive small resection impacts the ability of the remnant intestine to respond to FXR activation. Keywords: Short-Bowel Syndrome, Liver Disease, Intestinal FailureâAssociated Liver Disease, Obeticholic Acid, Bile Acids, Farnesoid X Receptor

Statins are the most widely prescribed medications worldwide for the treatment of hypercholesterolemia. They inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R), an enzyme involved in cholesterol synthesis in higher organisms and in isoprenoid biosynthesis in some bacteria. We hypothesized that statins may influence the microbial community in the gut through either direct inhibition or indirect mechanisms involving alterations to host responses. We therefore examined the impact of rosuvastatin (RSV) on the community structure of the murine gastrointestinal microbiota. RSV was orally administered to mice and the effects on the gut microbiota, host bile acid profiles, and markers of inflammation were analyzed. RSV significantly influenced the microbial community in both the cecum and feces, causing a significant decrease in α-diversity in the cecum and resulting in a reduction of several physiologically relevant bacterial groups. RSV treatment of mice significantly affected bile acid metabolism and impacted expression of inflammatory markers known to influence microbial community structure (including RegIIIγ and Camp) in the gut. This study suggests that a commonly used statin (RSV) leads to an altered gut microbial composition in normal mice with attendant impacts on local gene expression profiles, a finding that should prompt further studies to investigate the implications of statins for gut microbiota stability and health in humans. NEW & NOTEWORTHY This work demonstrates that rosuvastatin administration in mice affects the gastrointestinal microbiota, influences bile acid metabolism, and alters transcription of genes encoding factors involved in gut homeostasis and immunity in the gastrointestinal tract.

In recent years, the gut microbiome has gained increasing appreciation as a determinant of the health status of the human host. Bile salts that are secreted into the intestine may be biotransformed by enzymes produced by the gut bacteria. To date, bile acid research at the host-microbe interface has primarily been directed toward effects on host metabolism. The aim of this work was to investigate the effect of changes in gut microbial bile acid metabolism on the solubilization capacity of bile salt micelles and consequently intraluminal drug solubility. First, the impact of bile acid metabolism, mediated in vivo by the microbial enzymes bile salt hydrolase (BSH) and 7α-dehydroxylase, on drug solubility was assessed by comparing the solubilization capacity of (a) conjugated vs deconjugated and (b) primary vs secondary bile salts. A series of poorly water-soluble drugs (PWSDs) were selected as model solutes on the basis of an increased tendency to associate with bile micelles. Subsequently, PWSD solubility and dissolution was evaluated in conventional biorelevant simulated intestinal fluid containing host-derived bile acids, as well as in media modified to contain microbial bile acid metabolites. The findings suggest that deconjugation of the bile acid steroidal core, as dictated by BSH activity, influences micellar solubilization capacity for some PWSDs; however, these differences appear to be relatively minor. In contrast, the extent of bile acid hydroxylation, regulated by microbial 7α-dehydroxylase, was found to significantly affect the solubilization capacity of bile salt micelles for all nine drugs studied (p0.05). Subsequent investigations in biorelevant media containing either the trihydroxy bile salt sodium taurocholate (TCA) or the dihydroxy bile salt sodium taurodeoxycholate (TDCA) revealed altered drug solubility and dissolution. Observed differences in biorelevant media appeared to be both drug- and amphiphile (bile salt/lecithin) concentration-dependent. Our studies herein indicate that bile acid modifications occurring at the host-microbe interface could lead to alterations in the capacity of intestinal bile salt micelles to solubilize drugs, providing impetus to consider the gut microbiota in the drug absorption process. In the clinical setting, disruption of the gut microbial ecosystem, through disease or antibiotic treatment, could transform the bile acid pool with potential implications for drug absorption and bioavailability.

The symbiosis between the gut microbiota and the host has been identified as an integral part of normal human physiology and physiological development. Research in germ-free or gnotobiotic animals has demonstrated the importance of this symbiosis in immune, vascular, hepatic, respiratory and metabolic systems. Disruption of the microbiota can also contribute to disease, and the microbiota has been implicated in numerous intestinal and extra-intestinal pathologies including colorectal cancer. Interactions between host and microbiota can occur either directly or indirectly, via microbial-derived metabolites. In this chapter, we focus on two major products of microbial metabolism, short-chain fatty acids and bile acids, and their role in colorectal cancer. Short-chain fatty acids are the products of microbial fermentation of complex carbohydrates and confer protection against cancer risk, while bile acids are compounds which are endogenous to the host, but undergo microbial modification in the large intestine leading to alterations in their bioactivity. Lastly, we discuss the ability of microbial modulation to mediate cancer risk and the potential to harness this ability as a prophylactic or therapeutic treatment in colorectal cancer.

Background The commercial production of Agaricus bisporus is a three stage process: 1) production of compost, also called “substrate”; 2) production of casing soil; and 3) production of the mushrooms. Hygiene practices are undertaken at each stage: pasteurization of the substrate, hygiene practices applied during the production of casing soil, postharvest steam cookout, and disinfection at the mushroom production facilities. However, despite these measures, foodborne pathogens, including Listeria monocytogenes, are reported in the mushroom production environment. In this work, the presence of L. monocytogenes was evaluated before and after the application of hygiene practices at each stage of mushroom production with swabs, samples of substrate, casing, and spent mushroom growing substrates. Results L. monocytogenes was not detected in any casing or substrate sample by enumeration according to BS EN ISO 11290‐2:1998. Analysis of the substrate showed that L. monocytogenes was absent in 10 Phase II samples following pasteurization, but was then present in 40% of 10 Phase III samples. At the casing production facility, 31% of 59 samples were positive. Hygiene improvements were applied, and after four sampling occasions, 22% of 37 samples were positive, but no statistically significant difference was observed (p > .05). At mushroom production facilities, the steam cookout process inactivated L. monocytogenes in the spent growth substrate, but 13% of 15 floor swabs at Company 1 and 19% of 16 floor swabs at Company 2, taken after disinfection, were positive. Conclusion These results showed the possibility of L. monocytogenes recontamination of Phase III substrate, cross‐contamination at the casing production stage and possible survival after postharvest hygiene practices at the mushroom growing facilities. This information will support the development of targeted measures to minimize L. monocytogenes in the mushroom industry.

Listeria monocytogenes is an important food-borne pathogen that is ubiquitous in the environment. It is able to utilize a variety of carbon sources, to produce biofilms on food-processing surfaces and to survive food preservation–associated stresses. In this study, we investigated the effect of three common carbon sources, namely glucose, glycerol and lactose, on growth and activation of the general stress response Sigma factor, SigB, and corresponding phenotypes including stress resistance. A fluorescent reporter coupled to the promoter of lmo2230, a highly SigB-dependent gene, was used to determine SigB activation via quantitative fluorescence spectroscopy. This approach, combined with Western blotting and fluorescence microscopy, showed the highest SigB activation in lactose grown cells and lowest in glucose grown cells. In line with this observation, lactose grown cells showed the highest resistance to lethal heat and acid stress, the highest biofilm formation, and had the highest adhesion/invasion capacity in Caco-2-derived C2Bbe1 cell lines. Our data suggest that lactose utilisation triggers a strong SigB dependent stress response and this may have implications for the resistance of L. monocytogenes along the food chain.

Background Lactobacillus mucosae DPC 6426 has previously demonstrated potentially cardio-protective properties, in the form of dyslipidaemia and hypercholesterolemia correction in an apolipoprotein-E deficient mouse model. This study aims to characterise the manner in which this microbe may modulate host bile pool composition and immune response, in the context of cardiovascular disease. Lactobacillus mucosae DPC 6426 was assessed for bile salt hydrolase activity and specificity. The microbe was compared against several other enteric strains of the same species, as well as a confirmed bile salt hydrolase-active strain, Lactobacillus reuteri APC 2587. Results Quantitative bile salt hydrolase assays revealed that enzymatic extracts from Lactobacillus reuteri APC 2587 and Lactobacillus mucosae DPC 6426 demonstrate the greatest activity in vitro. Bile acid profiling of porcine and murine bile following incubation with Lactobacillus mucosae DPC 6426 confirmed a preference for hydrolysis of glyco-conjugated bile acids. In addition, the purified exopolysaccharide and secretome of Lactobacillus mucosae DPC 6426 were investigated for immunomodulatory capabilities using RAW264.7 macrophages. Gene expression data revealed that both fractions stimulated increases in interleukin-6 and interleukin-10 gene transcription in the murine macrophages, while the entire secretome was necessary to increase CD206 transcription. Moreover, the exopolysaccharide elicited a dose-dependent increase in nitric oxide and interleukin-10 production from RAW264.7 macrophages, concurrent with increased tumour necrosis factor-α secretion at all doses. Conclusions This study indicates that Lactobacillus mucosae DPC 6426 modulates both bile pool composition and immune system tone in a manner which may contribute significantly to the previously identified cardio-protective phenotype. Electronic supplementary material The online version of this article (10.1186/s12866-019-1403-0) contains supplementary material, which is available to authorized users.

The gastrointestinal microbiota plays a central role in the host metabolism of bile acids through deconjugation and dehydroxylation reactions, which generate unconjugated free bile acids and secondary bile acids respectively. These microbially generated bile acids are particularly potent signalling molecules that interact with host bile acid receptors (including the farnesoid X receptor, vitamin D receptor and TGR5 receptor) to trigger cellular responses that play essential roles in host lipid metabolism, electrolyte transport and immune regulation. Perturbations of microbial populations in the gut can therefore profoundly alter bile acid profiles in the host to impact upon the digestive and signalling properties of bile acids in the human superorganism. A number of recent studies have clearly demonstrated the occurrence of microbial disturbances allied to alterations in host bile acid profiles that occur across a range of disease states. Intestinal diseases including irritable bowel syndrome, inflammatory bowel disease (IBD), short bowel syndrome and Clostridium difficile infection all exhibit concurrent alterations in the composition of the gut microbiota and changes to host bile acid profiles. Similarly, extraintestinal diseases and syndromes such as asthma and obesity may be linked to aberrant bile acid profiles in the host. Here, we focus upon recent studies that highlight the links between alterations to gut microbial communities and altered bile acid profiles across a range of diseases from asthma to IBD.

Background and Aim Short bowel syndrome (SBS) is primarily characterised by malabsorption and malnutrition resulting from loss of intestinal absorptive area following massive small bowel resection (SBR). Bile acids and the gut microbiota are functionally-linked within the gut-liver axis, however SBS-associated disturbances within the gut-liver axis remain largely unexplored. The aim of this study was to characterize the evolution of bile acid alterations within the gut-liver axis at both short- and long-term time points and to relate these changes to alterations in colonic bacterial composition. Methods Four week-old piglets were assigned to 75% small bowel resection (SBR), sham operation or non-operation control (NOC) groups. High throughput sequencing was employed to determine bacterial abundance in colonic content and ultra-performance liquid chromatography used to determine the bile acid concentration of gall bladder, portal serum and faecal samples. Results Bile acid complexity and relative abundance are altered in the SBS piglet model at two weeks post-SBR, and these changes persisted at six weeks post-SBR. Our examination of the microbial profile revealed an early and persistent loss in bacteria belonging to the Clostridiales order. Conclusions This study provides evidence of an early and persistent disturbance of the bile acid profile throughout the entero-hepatic circulation with an increase in the proportion of primary bile acids and a decrease in secondary bile acids following small bowel resection. These changes were associated with a loss of bacteria belonging to the Clostridiales order consistent with a disturbance in the bile-microbial axis following SBR.

Statins are widely prescribed cholesterol-lowering medications and act through inhibition of the human enzyme 3-methylglutaryl coenzyme A reductase (HMG-R) which produces mevalonate (MVAL), a key substrate for cholesterol biosynthesis. Some important microbial species also express an isoform of HMG-R; however, the nature of the interaction between statins and bacteria is currently unclear and studies would benefit from protocols to quantify MVAL in complex microbial environments. The objective of this study was to develop a protocol for the analytical quantification of MVAL in bacterial systems and to utilise this approach to analyse the effects of Rosuvastatin (RSV) on bacterial MVAL formation. To determine the effective concentration range of RSV, we examined the dose-dependent inhibition of growth in the HMG-R(+) bacterial pathogens Listeria monocytogenes, Staphylococcus aureus and Enterococcus faecium at various concentrations of pure RSV. Growth inhibition generally correlated with a reduction in bacterial MVAL levels, particularly in culture supernatants at high RSV concentrations, as determined using our ultra-performance liquid chromatography mass spectrometry protocol. This work therefore outlines a refined protocol for the analysis of MVAL in microbial cultures and provides evidence for statin-mediated inhibition of bacterial HMG-R. Furthermore, we show that MVAL is readily transported and secreted from bacterial cells into the growth media.

Proteus mirabilis forms dense crystalline biofilms on catheter surfaces that occlude urine flow, leading to serious clinical complications in long-term catheterized patients, but there are presently no truly effective approaches to control catheter blockage by this organism. This study evaluated the potential for bacteriophage therapy to control P. mirabilis infection and prevent catheter blockage. Representative in vitro models of the catheterized urinary tract, simulating a complete closed drainage system as used in clinical practice, were employed to evaluate the performance of phage therapy in preventing blockage. Models mimicking either an established infection or early colonization of the catheterized urinary tract were treated with a single dose of a 3-phage cocktail, and the impact on time taken for catheters to block, as well as levels of crystalline biofilm formation, was measured. In models of established infection, phage treatment significantly increased time taken for catheters to block (∼3-fold) compared to untreated controls. However, in models simulating early-stage infection, phage treatment eradicated P. mirabilis and prevented blockage entirely. Analysis of catheters from models of established infection 10 h after phage application demonstrated that phage significantly reduced crystalline biofilm formation but did not significantly reduce the level of planktonic cells in the residual bladder urine. Taken together, these results show that bacteriophage constitute a promising strategy for the prevention of catheter blockage but that methods to deliver phage in sufficient numbers and within a key therapeutic window (early infection) will also be important to the successful application of phage to this problem.

Bile acids have emerged as important signaling molecules in the host, as they interact either locally or systemically with specific cellular receptors, in particular the farnesoid X receptor (FXR) and TGR5. These signaling functions influence systemic lipid and cholesterol metabolism, energy metabolism, immune homeostasis, and intestinal electrolyte balance. Through defined enzymatic activities, the gut microbiota can significantly modify the signaling properties of bile acids and therefore can have an impact upon host health. Alterations to the gut microbiota that influence bile acid metabolism are associated with metabolic disease, obesity, diarrhea, inflammatory bowel disease (IBD), Clostridium difficile infection, colorectal cancer, and hepatocellular carcinoma. Here, we examine the regulation of this gut-microbiota-liver axis in the context of bile acid metabolism and indicate how this pathway represents an important target for the development of new nutraceutical (diet and/or probiotics) and targeted pharmaceutical interventions.

peer-reviewed Mushroom growth substrates from different commercial producers of mushrooms (Agaricus bisporus) were screened for the presence of bacteria with potential for use as biocontrol agents for controlling Listeria monocytogenes in the mushroom production environment. Eight anti-listerial strains were isolated from different sources and all were identified using 16s rRNA gene sequencing as Lactococcus lactis subsp. lactis. Whole-genome sequencing of the Lc. lactis isolates indicated that strains from different sites and substrate types were highly similar. Colony MALDI-TOF mass spectrometry found that these strains were Nisin Z producers but inhibitory activity was highly influenced by the incubation conditions and was strain dependant. The biofilm forming ability of these strains was tested using a crystal violet assay and all were found to be strong biofilm formers. Growth of Lc. lactis subsp. lactis using mixed-biofilm conditions with L. monocytogenes on stainless steel resulted in a 4-log reduction of L. monocytogenes cell numbers. Additional sampling of mushroom producers showed that these anti-listerial Lc. lactis strains are commonly present in the mushroom production environment. Lc. lactis has a generally regarded as safe (GRAS) status and therefore has potential for use as an environmentally benign solution to control L. monocytogenes in order to prevent product contamination and to enhance consumer confidence in the mushroom industry.

peer-reviewed Background A westernized diet comprising a high caloric intake from animal fats is known to influence the development of pathological inflammatory conditions. However, there has been relatively little focus upon the implications of such diets for the progression of infectious disease. Here, we investigated the influence of a high-fat (HF) diet upon parameters that influence Listeria monocytogenes infection in mice. Results We determined that short-term administration of a HF diet increases the number of goblet cells, a known binding site for the pathogen, in the gut and also induces profound changes to the microbiota and promotes a pro-inflammatory gene expression profile in the host. Host physiological changes were concordant with significantly increased susceptibility to oral L. monocytogenes infection in mice fed a HF diet relative to low fat (LF)- or chow-fed animals. Prior to Listeria infection, short-term consumption of HF diet elevated levels of Firmicutes including Coprococcus, Butyricicoccus, Turicibacter and Clostridium XIVa species. During active infection with L. monocytogenes, microbiota changes were further exaggerated but host inflammatory responses were significantly downregulated relative to Listeria-infected LF- or chow-fed groups, suggestive of a profound tempering of the host response influenced by infection in the context of a HF diet. The effects of diet were seen beyond the gut, as a HF diet also increased the sensitivity of mice to systemic infection and altered gene expression profiles in the liver. Conclusions We adopted a systems approach to identify the effects of HF diet upon L. monocytogenes infection through analysis of host responses and microbiota changes (both pre- and post-infection). Overall, the results indicate that short-term consumption of a westernized diet has the capacity to significantly alter host susceptibility to L. monocytogenes infection concomitant with changes to the host physiological landscape. The findings suggest that diet should be a consideration when developing models that reflect human infectious disease. This research was funded by the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement No. 641984, through funding of the List_MAPS consortium. We also acknowledge funding and support from Science Foundation Ireland (SFI) in the form of a center grant (APC Microbiome Ireland grant SFI/12/RC/2273).

Faecal microbiota transplant (FMT) effectively treats recurrentUsing stool collected from patients and donors pre-FMT/post-FMT for rCDI, we performed 16S rRNA gene sequencing, ultra performance liquid chromatography mass spectrometry (UPLC-MS) bile acid profiling, BSH activity measurement, and qPCR ofFrom metataxonomics, pre-FMT stool demonstrated a reduced proportion of BSH-producing bacterial species compared with donors/post-FMT. Pre-FMT stool was enriched in taurocholic acid (TCA, a potentRestoration of gut BSH functionality contributes to the efficacy of FMT in treating rCDI.

Listeria monocytogenes is a major human foodborne pathogen that is prevalent in the natural environment and has a high case fatality rate. Whole genome sequencing (WGS) analysis has emerged as a valuable methodology for the classification of L. monocytogenes isolates and the identification of virulence islands that may influence infectivity. In this study, WGS was used to provide an insight into 25 L. monocytogenes isolates from cases of clinical infection in Ireland between 2013 and 2015. Clinical strains were either lineage I (14 isolates) or lineage II (11 isolates), with 12 clonal complexes (CC) represented, of which CC1 (6) and CC101 (4) were the most common. Single nucleotide polymorphism (SNP) analysis demonstrated that clinical isolates from mother-infant pairs (one isolate from the mother and one from the infant) were highly related (3 SNP differences in each) and also identified close similarities between isolates from otherwise distinct cases (1 SNP difference). Clinical strains were positive for common virulence-associated loci and 13 isolates harbour the LIPI-3 locus. Pulsed-field gel electrophoresis (PFGE) was used to compare strains to a database of 1300 Irish food and food processing environment isolates and determined that 64% of clinical pulsotypes were previously encountered in the food or food processing environment. Five of the matching food and food processing environment isolates were sequenced and results demonstrated a correlation between pulsotype and genotype. Overall, the work provides insights into the nature of L. monocytogenes strains currently causing clinical disease in Ireland and indicates that similar isolates can be found in the food or food processing environment.

Autism spectrum disorder (ASD) is one of the most prevalent neurodevelopmental conditions worldwide. There is growing awareness that ASD is highly comorbid with gastrointestinal distress and altered intestinal microbiome, and that host-microbiome interactions may contribute to the disease symptoms. However, the paucity of knowledge on gut-brain axis signaling in autism constitutes an obstacle to the development of precision microbiota-based therapeutics in ASD. To this end, we explored the interactions between intestinal microbiota, gut physiology and social behavior in a BTBR T+Itpr3tf/J mouse model of ASD. Here we show that a reduction in the relative abundance of very particular bacterial taxa in the BTBR gut – namely, bile-metabolizing Bifidobacterium and Blautia species, - is associated with deficient bile acid and tryptophan metabolism in the intestine, marked gastrointestinal dysfunction, as well as impaired social interactions in BTBR mice. Together these data support the concept of targeted manipulation of the gut microbiota for reversing gastrointestinal and behavioral symptomatology in ASD, and offer specific plausible targets in this endeavor., Highlights • BTBR mice display a reduced abundance of bile-metabolizing Bifidobacterium and Blautia bacterial species in the intestine. • BTBR mice demonstrate deficient bacterial metabolism of bile moieties in the gut. • Changes in the gut microbiota are associated with marked gastrointestinal distress and reduced sociability in BTBR mice. There is growing awareness that the gut microbiome may contribute to gastrointestinal and behavioral symptomatology of autism spectrum disorder (ASD). However, the exact mechanisms by which intestinal bacteria can affect gut-brain axis signaling in autism are as yet poorly understood. Here we explore interactions between intestinal microbiota, gut physiology and behavior in a mouse model of ASD. We show that a reduction in the abundance of particular intestinal bacteria in “autistic” mice is associated with gastrointestinal distress and social behavior deficits. This work supports the concept of targeting the gut microbiota for reversing gastrointestinal symptoms in ASD, and identifies specific plausible targets for microbiota-based interventions.

Listeria monocytogenes is a Gram-positive opportunistic pathogen that is the causative agent of listeriosis. Here, we report the draft genome sequences of 25 L. monocytogenes strains isolated from patients with clinical listeriosis in the Republic of Ireland between 2013 and 2015.

Controlled, reductionist approaches are required in order to obtain a more complete understanding of the functional capabilities of the gut microbiota. We recently identified microbial bile salt hydrolase (BSH) activity as a gut microbial activity that has the capacity to profoundly alter both local (gastrointestinal) and systemic (hepatic) host functions. Using both germ free and conventionally-raised mouse models we demonstrated that gastrointestinal expression of BSH results in local bile acid deconjugation with concomitant alterations in lipid and cholesterol metabolism, signaling functions and weight gain. Key mediators of cholesterol homeostasis (Abcg5/8), gut homeostasis (RegIIIγ) and circadian rhythm (Dbp) were influenced by elevated BSH in our study. In this addendum we discuss the implications of this work for the rational development of probiotics with the potential to modulate host weight gain.

The concept of biological containment was developed as a strategy to prevent environmental dissemination of engineered live vaccine or drug delivery vehicles. A mutation in the gene encoding thymidylate synthase (thyA), a key enzyme in the pyrimidine biosynthetic pathway, has previously been shown to limit growth of L. lactis vectors under restrictive conditions. We hypothesized that further mutations in the pyrimidine biosynthetic pathway might enhance the stability and safety of live L. lactis vectors. We show that a double mutation in the genes encoding ThyA and CTP synthase (PyrG) in L. lactis confers double auxotrophy for both thymidine and cytidine. However, the combination of two mutations failed to enhance the biological containment phenotype of the engineered strain. In the absence of thymine/thymidine, the thyA mutant exhibited a strong bactericidal phenotype. However, creation of the double mutant caused the loss of this phenotype, though survival in the mouse GI tract was enhanced. The implications for biological containment of live L. lactis based delivery vectors are discussed.

Listeria monocytogenes is a Gram-positive intracellular pathogen that is responsible for listeriosis, a potentially fatal, food-borne illness. Due to its cytoplasmic location during infection, this pathogen can mediate a long-lasting cellular immune response, which makes attenuated strains strong candidates for vaccine development. Recently, our group identified and characterized frvA (Fur-regulated virulence factor A), and deletion of this gene resulted in disruption of iron homeostasis and a strong attenuation in virulence. Despite significant attenuation in the mouse infection model, the frvA mutant was capable of intracellular growth in antigen-presenting cells. Indeed, mice immunized with L. monocytogenes ΔfrvA were able to effectively stimulate specific CD8(+) T cells to the listerial epitopes LLO(91-99) and P60(217-225) at levels comparable with L. monocytogenes strain EGDe. Most notably, mice immunized with ΔfrvA then subsequently challenged with the wild-type strain were completely protected from listerial infection. On the basis of these results, we advocate the use of ΔfrvA as a live attenuated listerial vaccine, and propose that this mutant may serve as a platform for the development of a future vaccine delivery vehicle.