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Mammalian cells utilize glucose as a primary carbon source to produce energy for most cellular functions. However, the bioenergetic homeostasis of cells can be perturbed by environmental alterations, such as changes in oxygen levels which can be associated with bacterial infection. Reduction in oxygen availability leads to a state of hypoxia, inducing numerous cellular responses that aim to combat this stress. Importantly, hypoxia strongly augments cellular glycolysis in most cell types to compensate for the loss of aerobic respiration. Understanding how this host cell metabolic adaptation to hypoxia impacts the course of bacterial infection will identify new anti-microbial targets. This review will highlight developments in our understanding of glycolytic substrate channeling and spatiotemporal enzymatic organization in response to hypoxia, shedding light on the integral role of the hypoxia-inducible factor (HIF) during host-pathogen interactions. Furthermore, the ability of intracellular and extracellular bacteria (pathogens and commensals alike) to modulate host cellular glucose metabolism will be discussed., (© 2024. The Author(s).)

Infection with the protozoan parasite Giardia duodenalis (syn. intestinalis , lamblia ) has been associated with intestinal mucus disruptions and microbiota dysbiosis. The mechanisms remain incompletely understood. Mucus consists primarily of densely glycosylated mucin glycoproteins. Mucin O -glycans influence mucus barrier properties and mucin-microbe interactions and are frequently altered during disease. In this study, we observed time-dependent and regiospecific alterations to intestinal mucin glycosylation patterns and the expression of mucin-associated glycosyltransferase genes during Giardia infection. Glycosylation alterations were observed in Giardia -infected mice in the upper small intestine, the site of parasite colonization, and in the distal colon, where active trophozoites were absent. Alterations occurred as early as day 2 post-infection and persisted in mice after parasite clearance. We also observed small intestinal goblet cell hyperplasia and thinning of the distal colon mucus barrier during early infection, and microbiota alterations and altered production of cecal SCFAs. Giardia -induced alterations to mucin glycosylation were at least in part dependent on microbiota dysbiosis, as transplantation of a dysbiotic mucosal microbiota collected from Giardia -infected mice recapitulated some alterations. This study describes a novel mechanism by which Giardia alters intestinal mucin glycosylation, and implicates the small intestinal microbiota in regulation of mucin glycosylation patterns throughout the gastrointestinal tract.

In recent years, multidrug-resistant Acinetobacter baumannii has emerged globally as a major threat to the healthcare system. It is now listed by the World Health Organization as a priority one for the need of new therapeutic agents. A. baumannii has the capacity to develop robust biofilms on biotic and abiotic surfaces. Biofilm development allows these bacteria to resist various environmental stressors, including antibiotics and lack of nutrients or water, which in turn allows the persistence of A. baumannii in the hospital environment and further outbreaks. Investigation into therapeutic alternatives that will act on both biofilm formation and antimicrobial resistance (AMR) is sorely needed. The aim of the present review is to critically discuss the various mechanisms by which AMR and biofilm formation may be co-regulated in A. baumannii in an attempt to shed light on paths towards novel therapeutic opportunities. After discussing the clinical importance of A. baumannii, this critical review highlights biofilm-formation genes that may be associated with the co-regulation of AMR. Particularly worthy of consideration are genes regulating the quorum sensing system AbaI/AbaR, AbOmpA (OmpA protein), Bap (biofilm-associated protein), the two-component regulatory system BfmRS, the PER-1 β-lactamase, EpsA, and PTK. Finally, this review discusses ongoing experimental therapeutic strategies to fight A. baumannii infections, namely vaccine development, quorum sensing interference, nanoparticles, metal ions, natural products, antimicrobial peptides, and phage therapy. A better understanding of the mechanisms that co-regulate biofilm formation and AMR will help identify new therapeutic targets, as combined approaches may confer synergistic benefits for effective and safer treatments., (© 2023. The Author(s).)

Wastewater-based surveillance (WBS) has been established as a powerful tool that can guide health policy at multiple levels of government. However, this approach has not been well assessed at more granular scales, including large work sites such as University campuses. Between August 2021 and April 2022, we explored the occurrence of SARS-CoV-2 RNA in wastewater using qPCR assays from multiple complimentary sewer catchments and residential buildings spanning the University of Calgary's campus and how this compared to levels from the municipal wastewater treatment plant servicing the campus. Real-time contact tracing data was used to evaluate an association between wastewater SARS-CoV-2 burden and clinically confirmed cases and to assess the potential of WBS as a tool for disease monitoring across worksites. Concentrations of wastewater SARS-CoV-2 N1 and N2 RNA varied significantly across six sampling sites - regardless of several normalization strategies - with certain catchments consistently demonstrating values 1-2 orders higher than the others. Relative to clinical cases identified in specific sewersheds, WBS provided one-week leading indicator. Additionally, our comprehensive monitoring strategy enabled an estimation of the total burden of SARS-CoV-2 for the campus per capita, which was significantly lower than the surrounding community (p≤0.001). Allele-specific qPCR assays confirmed that variants across campus were representative of the community at large, and at no time did emerging variants first debut on campus. This study demonstrates how WBS can be efficiently applied to locate hotspots of disease activity at a very granular scale, and predict disease burden across large, complex worksites., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Extracellular vesicles (EVs) recently emerged as important players in the pathophysiology of parasitic infections. While the protist parasite  Giardia duodenalis can produce EVs, their role in giardiasis remains obscure. Giardia can disrupt gut microbiota biofilms and transform commensal bacteria into invasive pathobionts at sites devoid of colonizing trophozoites via unknown mechanisms. We hypothesized that Giardia EVs could modify gut bacterial behaviour via a novel mode of trans-kingdom communication. Our findings indicate that Giardia EVs exert bacteriostatic effects on Escherichia coli HB101 and Enterobacter cloacae TW1, increasing their swimming motility. Giardia EVs also decreased the biofilm-forming ability of E. coli HB101 but not by E. cloacae TW1, supporting the hypothesis that these effects are, at least in part, bacteria-selective. E. coli HB101 and E. cloacae TW1 exhibited increased adhesion/invasion onto small intestine epithelial cells when exposed to Giardia EVs. EVs labelled with PKH67 revealed colocalization with E. coli HB101 and E. cloacae TW1 bacterial cells. Small RNA sequencing revealed a high abundance of ribosomal RNA (rRNA)- and transfer RNA (tRNA)-derived small RNAs, short-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) within Giardia EVs. Proteomic analysis of EVs uncovered the presence of RNA chaperones and heat shock proteins that can facilitate the thermal stability of EVs and its sRNA cargo, as well as protein-modifying enzymes. In vitro, RNase heat-treatment assays showed that total RNAs in EVs, but not proteins, are responsible for modulating bacterial swimming motility and biofilm formation. G. duodenalis small RNAs of EVs, but not proteins, were responsible for the increased bacterial adhesion to intestinal epithelial cells induced upon exposure to Giardia EVs. Together, the findings indicate that Giardia EVs contain a heat-stable, RNase-sensitive cargo that can trigger the development of pathobiont characteristics in Enterobacteria, depicting a novel trans-kingdom cross-talk in the gut., Competing Interests: All co‐authors have seen and agree with the contents of the manuscript and there is no conflict of interest., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Body tissues are subjected to various oxygenic gradients and fluctuations and hence can become transiently hypoxic. Hypoxia-inducible factor (HIF) is the master transcriptional regulator of the cellular hypoxic response and is capable of modulating cellular metabolism, immune responses, epithelial barrier integrity, and local microbiota. Recent reports have characterized the hypoxic response to various infections. However, little is known about the role of HIF activation in the context of protozoan parasitic infections. Growing evidence suggests that tissue and blood protozoa can activate HIF and subsequent HIF target genes in the host, helping or hindering their pathogenicity. In the gut, enteric protozoa are adapted to steep longitudinal and radial oxygen gradients to complete their life cycle, yet the role of HIF during these protozoan infections remains unclear. This review focuses on the hypoxic response to protozoa and its role in the pathophysiology of parasitic infections. We also discuss how hypoxia modulates host immune responses in the context of protozoan infections.

Mucin O -linked glycans are important mediators of host-microbiota-pathogen interactions in the gastrointestinal tract. The major component of intestinal mucus, the MUC2 mucin, is densely glycosylated, with up to 80% of its weight-to-volume ratio represented by O -linked glycans. Glycosylation of secretory gel-forming mucins has an enormous impact on intestinal barrier function, microbial metabolism, and mucus colonization by both pathogenic and commensal microbes. Mucin O -glycans and glycan-derived sugars may be degraded and used as a nutrient source and may regulate microbial gene expression and virulence. Short-chain fatty acids, produced as a by-product of glycan fermentation, can regulate host immunity and goblet cell activity and are important for host-microbe homeostasis. Mucin glycans may also act as microbial binding sites, influencing intestinal colonization and translocation through the mucus gel barrier. Recent findings indicate that alterations to mucin glycosylation impact the susceptibility of mucins to degradation, resulting in altered barrier function and intestinal permeability. Alterations to mucin glycosylation patterns are frequently observed during intestinal infection and inflammation and have been implicated in microbiota dysbiosis and expansion of pathobionts. Recent work has demonstrated that these alterations can play key roles in disease pathogenesis. The precise mechanisms remain obscure. This review highlights the important roles of O -linked glycans in host-microbe interactions and disease pathogenesis in the context of intestinal infections.

Gut microbiota contain communities of viruses, bacteria, fungi, and Eukarya, and live as biofilms. In health, these biofilms adhere to the intestinal mucus surface without contacting the epithelium. Disruptions to the equilibrium between these biofilms and the host may create invasive pathobionts from these commensal communities and contribute to disease pathogenesis. Environmental factors appear to dominate over genetics in determining the shifts in microbiota populations and function, including when comparing microbiota between low-income and industrialized countries. The observations discussed herein carry enormous potential for the development of novel therapies targeting phenotype in microbiota dysbiosis., (© 2023 Buret and Allain.)

Importance: Infection transmission following endoscopic retrograde cholangiopancreatography (ERCP) can occur due to persistent contamination of duodenoscopes despite high-level disinfection to completely eliminate microorganisms on the instrument., Objective: To determine (1) contamination rates after high-level disinfection and (2) technical performance of duodenoscopes with disposable elevator caps compared with those with standard designs., Design, Setting, and Participants: In this parallel-arm multicenter randomized clinical trial at 2 tertiary ERCP centers in Canada, all patients 18 years and older and undergoing ERCP for any indication were eligible., Intervention: The intervention was use of duodenoscopes with disposable elevator caps compared with duodenoscopes with a standard design., Main Outcomes and Measures: Coprimary outcomes were persistent microbial contamination of the duodenoscope elevator or channel, defined as growth of at least 10 colony-forming units of any organism or any growth of gram-negative bacteria following high-level disinfection (superiority outcome), and technical success of ERCP according to a priori criteria (noninferiority outcome with an a priori noninferiority margin of 7%), assessed by blinded reviewers., Results: From December 2019 to February 2022, 518 patients were enrolled (259 disposable elevator cap duodenoscopes, 259 standard duodenoscopes). Patients had a mean (SD) age of 60.7 (17.0) years and 258 (49.8%) were female. No significant differences were observed between study groups, including in ERCP difficulty. Persistent microbial contamination was detected in 11.2% (24 of 214) of standard duodenoscopes and 3.8% (8 of 208) of disposable elevator cap duodenoscopes (P = .004), corresponding to a relative risk of 0.34 (95% CI, 0.16-0.75) and number needed to treat of 13.6 (95% CI, 8.1-42.7) to avoid persistent contamination. Technical success using the disposable cap scope was noninferior to that of the standard scope (94.6% vs 90.7%, P = .13). There were no differences between study groups in adverse events and other secondary outcomes., Conclusions and Relevance: In this randomized clinical trial, disposable elevator cap duodenoscopes exhibited reduced contamination following high-level disinfection compared with standard scope designs, without affecting the technical performance and safety of ERCP., Trial Registration: ClinicalTrials.gov Identifier: NCT04040504.

Host immune cells and clinical interventions often fail to eradicate biofilm-mediated infections, resulting in chronic inflammation. The role of the biofilm three-dimensional structure in this tolerant phenotype has been studied extensively; however, the impact of small molecules released from biofilm-bacteria in modulating host immune function is less well understood. A model of mixed-species biofilms composed of Fusobacterium necrophorum and Porphyromonas levii was developed to evaluate bovine neutrophil responses to bioactive molecules released from either biofilm or planktonic bacteria. We hypothesized that different soluble extracellular factors (ECFs) would be released from planktonic and biofilm bacteria, resulting in altered neutrophil function. Neutrophils exposed to ECFs from planktonic bacteria showed significantly elevated levels of reactive oxygen species (ROS). In contrast, biofilm components from these same species of bacteria failed to induce such a response. Size-exclusion filtration of ECFs revealed that the bioactive molecule causing neutrophil ROS responses was below 3 kDa. Intensive heat, nuclease, lipase, or protease treatments of the <3 kDa fractions did not alter neutrophil functional responses. Protoporphyrin IX (PPIX) is an important heme precursor and growth requirement for many anaerobes. Porphyromonas species can accumulate environmental PPIX at the cell surface as a strategy to protect the bacteria from oxidative stress and we investigated the direct interaction of bovine neutrophils with PPIX. In the present study, evidence suggests that the accumulation of protoporphyrin in these dual-species biofilm ECFs attenuates neutrophil ROS production and chemotaxis. The diminished neutrophil response to biofilm ECFs via the action of PPIX may represent a biofilm immune-evasion strategy that could assist in explaining the ineffectual host clearance of biofilm-mediated infections involving these bacteria., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Giardia duodenalis cysteine proteases have been identified as key virulence factors and have been implicated in alterations to intestinal goblet cell activity and mucus production during Giardia infection. The present findings demonstrate a novel mechanism by which Giardia cysteine proteases modulate goblet cell activity via cleavage and activation of protease-activated receptor 2. Giardia duodenalis (assemblage A) increased MUC2 mucin gene expression in human colonic epithelial cells in a manner dependent upon both protease-activated receptor 2 activation and Giardia cysteine protease activity. Protease-activated receptor 2 cleavage within the N-terminal activation domain by Giardia proteases was confirmed using a nano-luciferase tagged recombinant protease-activated receptor 2. In keeping with these observations, the synthetic protease-activated receptor 2-activating peptide 2fLIGRLO-amide increased Muc2 gene expression in a time-dependent manner. Calcium chelation and inhibition of the ERK1/2 mitogen activated protein kinase pathway inhibited Muc2 upregulation during Giardia infection, consistent with canonical protease-activated receptor 2 signaling pathways. Giardia cysteine proteases cleaved both recombinant protease-activated receptor 1 and protease-activated receptor 2 within their extracellular activation domains with isolate-dependent efficiency that correlated with the production of cysteine protease activity. Protease-activated receptors represent a novel target for Giardia cysteine proteases, and these findings demonstrate that protease-activated receptor 2 can regulate mucin gene expression in intestinal goblet cells., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Significance: Hydrogen sulfide (H 2 S), an important regulator of physiology and health, helps resolve inflammation and promotes tissue repair in the gastrointestinal tract. Recent Advances: Gut microbiota live as a multispecies biofilm in close interaction with the upper mucus layer lining the epithelium. The relative abundance, spatial organization, and function of these microorganisms affect a broad range of health outcomes. This article provides a state-of-the-art review of our understanding of the cross talk between H 2 S, the gut microbiota, and health. H 2 S can have toxic or therapeutic effects, depending on its concentration and source. When produced at excessive concentrations by local microbiota, H 2 S may cause mucus disruption and inflammation and contribute to development of cancer. In contrast, low levels of endogenous or exogenous H 2 S directly stabilize mucus layers, prevent fragmentation and adherence of the microbiota biofilm to the epithelium, inhibit the release of invasive pathobionts, and help resolve inflammation and tissue injury. Although scarce, research findings suggest that dietary H 2 S obtained from plants or ingestion of the H 2 S precursor, L-cysteine, may also modulate the abundance and function of microbiota. Critical Issues: A critical issue is the lack of understanding of the metagenomic, transcriptomic, and proteomic alterations that characterize the interactions between H 2 S and gut microbiota to shape health outcomes. Future Directions: The ambivalent roles of H 2 S in the gut offer a fertile ground for research on such critical issues. The findings will improve our understanding of how H 2 S modulates the microbiota to affect body function and will help identify novel therapeutic strategies. Antioxid. Redox Signal . 36, 211-219.

Current limitations in the understanding and control of antimicrobial resistance (AMR) in Canada are described through a comprehensive review focusing on: (1) treatment optimization; (2) surveillance of antimicrobial use and AMR; and (3) prevention of transmission of AMR. Without addressing gaps in identified areas, sustained progress in AMR mitigation is unlikely. Expert opinions and perspectives contributed to prioritizing identified gaps. Using Canada as an example, this review emphasizes the importance and necessity of a One Health approach for understanding and mitigating AMR. Specifically, antimicrobial use in human, animal, crop, and environmental sectors cannot be regarded as independent; therefore, a One Health approach is needed in AMR research and understanding, current surveillance efforts, and policy. Discussions regarding addressing described knowledge gaps are separated into four categories: (1) further research; (2) increased capacity/resources; (3) increased prescriber/end-user knowledge; and (4) policy development/enforcement. This review highlights the research and increased capacity and resources to generate new knowledge and implement recommendations needed to address all identified gaps, including economic, social, and environmental considerations. More prescriber/end-user knowledge and policy development/enforcement are needed, but must be informed by realistic recommendations, with input from all relevant stakeholders. For most knowledge gaps, important next steps are uncertain. In conclusion, identified knowledge gaps underlined the need for AMR policy decisions to be considered in a One Health framework, while highlighting critical needs to achieve realistic and meaningful progress., Competing Interests: JC has received peer-reviewed research grants from the Canadian Institutes for Health Research (CIHR) on development of a rapid assay for detection of methicillin-resistant Staphylococcus aureus and Alberta Innovates Health Solutions on use of probiotics as primary prophylaxis for Clostridium difficile colitis. JC has also received financial support from Pfizer for the STRIVE S. aureus vaccine randomized clinical trial in vertebral spinal surgery with instrumentation and CIHR for creation of an AMS app to improve antimicrobial prescribing. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 McCubbin, Anholt, de Jong, Ida, Nóbrega, Kastelic, Conly, Götte, McAllister, Orsel, Lewis, Jackson, Plastow, Wieden, McCoy, Leslie, Robinson, Hardcastle, Hollis, Ashbolt, Checkley, Tyrrell, Buret, Rennert-May, Goddard, Otto and Barkema.)

Exogenous factors that may influence the pathophysiology of Giardia infection remain incompletely understood. We have investigated the role of dietary fat in the pathogenesis of Giardia infection. Male 3 to 4-week-old C57BL/6 mice were fed either a low fat (LF) or a high fat (HF) diet for 12 days and challenged with G. duodenalis. In infected animals, the trophozoite burden was higher in HF + Giardia mice compared to the LF + Giardia group at day 7 post infection. Fatty acids exerted direct pro-growth effects on Giardia trophozoites. Analysis of disease parameters showed that HF + Giardia mice exhibited more mucosal infiltration by inflammatory cells, decreased villus/crypt ratios, goblet cell hyperplasia, mucus disruption, increased gut motility, and elevated fecal water content compared with LF + Giardia. HF diet-dependent exacerbation of Giardia-induced goblet cell hyperplasia was associated with elevated Atoh1 and Muc2 gene expression. Gut microbiota analysis revealed that the HF diet alone induces a taxonomic shift. HF + Giardia mice exhibited microbiota dysbiosis characterized by an increase of Firmicutes and a decrease of Bacteroidetes and significant changes in α- and β-diversity metrics. Taken together, the findings suggest that a HF diet exacerbates the outcome of Giardia infection. The data demonstrate that elevated dietary fat represents an important exogenous factor promoting the pathophysiology of giardiasis., (© 2021. The Author(s).)

Background: Studies on the inhibition of inflammation by infection with helminth parasites have, until recently, overlooked a key determinant of health: the gut microbiota. Infection with helminths evokes changes in the composition of their host's microbiota: one outcome of which is an altered metabolome (e.g., levels of short-chain fatty acids (SCFAs)) in the gut lumen. The functional implications of helminth-evoked changes in the enteric microbiome (composition and metabolites) are poorly understood and are explored with respect to controlling enteric inflammation., Methods: Antibiotic-treated wild-type, germ-free (GF) and free fatty-acid receptor-2 (ffar2) deficient mice were infected with the tapeworm Hymenolepis diminuta, then challenged with DNBS-colitis and disease severity and gut expression of the il-10 receptor-α and SCFA receptors/transporters assessed 3 days later. Gut bacteria composition was assessed by 16 s rRNA sequencing and SCFAs were measured. Other studies assessed the ability of feces or a bacteria-free fecal filtrate from H. diminuta-infected mice to inhibit colitis., Results: Protection against disease by infection with H. diminuta was abrogated by antibiotic treatment and was not observed in GF-mice. Bacterial community profiling revealed an increase in variants belonging to the families Lachnospiraceae and Clostridium cluster XIVa in mice 8 days post-infection with H. diminuta, and the transfer of feces from these mice suppressed DNBS-colitis in GF-mice. Mice treated with a bacteria-free filtrate of feces from H. diminuta-infected mice were protected from DNBS-colitis. Metabolomic analysis revealed increased acetate and butyrate (both or which can reduce colitis) in feces from H. diminuta-infected mice, but not from antibiotic-treated H. diminuta-infected mice. H. diminuta-induced protection against DNBS-colitis was not observed in ffar2 -/- mice. Immunologically, anti-il-10 antibodies inhibited the anti-colitic effect of H. diminuta-infection. Analyses of epithelial cell lines, colonoids, and colon segments uncovered reciprocity between butyrate and il-10 in the induction of the il-10-receptor and butyrate transporters., Conclusion: Having defined a feed-forward signaling loop between il-10 and butyrate following infection with H. diminuta, this study identifies the gut microbiome as a critical component of the anti-colitic effect of this helminth therapy. We suggest that any intention-to-treat with helminth therapy should be based on the characterization of the patient's immunological and microbiological response to the helminth., (© 2021. The Author(s).)

Enteric tuft cells are chemosensory epithelial cells gaining attention in the field of host-parasite interactions. Expressing a repertoire of chemosensing receptors and mediators, these cells have the potential to detect lumen-dwelling helminth and protozoan parasites and coordinate epithelial, immune, and neuronal cell defenses against them. This review highlights the versatility of enteric tuft cells and sub-types thereof, showcasing nuances of tuft cell responses to different parasites, with a focus on helminths reflecting the current state of the field. The role of enteric tuft cells in irritable bowel syndrome, inflammatory bowel disease and intestinal viral infection is assessed in the context of concomitant infection with parasites. Finally, the review presents pertinent questions germane to understanding the enteric tuft cell and its role in enteric parasitic infections. There is much to be done to fully elucidate the response of this intriguing cell type to parasitic-infection and there is negligible data on the biology of the human enteric tuft cell-a glaring gap in knowledge that must be filled.

Background and Aims: Thrombin levels in the colon of Crohn's disease patients have recently been found to be elevated 100-fold compared with healthy controls. Our aim was to determine whether and how dysregulated thrombin activity could contribute to local tissue malfunctions associated with Crohn's disease., Methods: Thrombin activity was studied in tissues from Crohn's disease patients and healthy controls. Intracolonic administration of thrombin to wild-type or protease-activated receptor-deficient mice was used to assess the effects and mechanisms of local thrombin upregulation. Colitis was induced in rats and mice by the intracolonic administration of trinitrobenzene sulphonic acid., Results: Active forms of thrombin were increased in Crohn's disease patient tissues. Elevated thrombin expression and activity were associated with intestinal epithelial cells. Increased thrombin activity and expression were also a feature of experimental colitis in rats. Colonic exposure to doses of active thrombin comparable to what is found in inflammatory bowel disease tissues caused mucosal damage and tissue dysfunctions in mice, through a mechanism involving both protease-activated receptors -1 and -4. Intracolonic administration of the thrombin inhibitor dabigatran, as well as inhibition of protease-activated receptor-1, prevented trinitrobenzene sulphonic acid-induced colitis in rodent models., Conclusions: Our data demonstrated that increased local thrombin activity, as it occurs in the colon of patients with inflammatory bowel disease, causes mucosal damage and inflammation. Colonic thrombin and protease-activated receptor-1 appear as possible mechanisms involved in mucosal damage and loss of function and therefore represent potential therapeutic targets for treating inflammatory bowel disease., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Microorganisms colonize various ecological niches in the human habitat, as they do in nature. Predominant forms of multicellular communities called biofilms colonize human tissue surfaces. The gastrointestinal tract is home to a profusion of microorganisms with intertwined, but not identical, lifestyles: as isolated planktonic cells, as biofilms and in biofilm-dispersed form. It is therefore of major importance in understanding homeostatic and altered host-microorganism interactions to consider not only the planktonic lifestyle, but also biofilms and biofilm-dispersed forms. In this Review, we discuss the natural organization of microorganisms at gastrointestinal surfaces, stratification of microbiota taxonomy, biogeographical localization and trans-kingdom interactions occurring within the biofilm habitat. We also discuss existing models used to study biofilms. We assess the contribution of the host-mucosa biofilm relationship to gut homeostasis and to diseases. In addition, we describe how host factors can shape the organization, structure and composition of mucosal biofilms, and how biofilms themselves are implicated in a variety of homeostatic and pathological processes in the gut. Future studies characterizing biofilm nature, physical properties, composition and intrinsic communication could shed new light on gut physiology and lead to potential novel therapeutic options for gastrointestinal diseases.

Alteration of the intestinal microbiome by enteropathogens is commonly associated with gastrointestinal diseases and disorders and has far-reaching consequences for overall health. Significant advances have been made in understanding the role of microbial dysbiosis during intestinal infections, including infection with the protozoan parasite Giardia duodenalis , one of the most prevalent gut protozoa. Altered species composition and diversity, functional changes in the commensal microbiota, and changes to intestinal bacterial biofilm structure have all been demonstrated during the course of Giardia infection and have been implicated in Giardia pathogenesis. Conversely, the gut microbiota has been found to regulate parasite colonization and establishment and plays a critical role in immune modulation during mono and polymicrobial infections. These disruptions to the commensal microbiome may contribute to a number of acute, chronic, and post-infectious clinical manifestations of giardiasis and may account for variations in disease presentation within and between infected populations. This review discusses recent advances in characterizing Giardia -induced bacterial dysbiosis in the gut and the roles of dysbiosis in Giardia pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors AB., (Copyright © 2021 Fekete, Allain, Siddiq, Sosnowski and Buret.)

Aims: The covalent linking of nonsteroidal anti-inflammatory drugs to a hydrogen sulfide (H 2 S)-releasing moiety has been shown to dramatically reduce gastrointestinal (GI) damage and bleeding, as well as increase anti-inflammatory and analgesic potency. We have tested the hypothesis that an H 2 S-releasing derivative of ketoprofen (ATB-352) would exhibit enhanced efficacy without significant GI damage in a mouse model of allodynia/hyperalgesia. Results: ATB-352 was significantly more potent and effective as an analgesic than ketoprofen and did not elicit GI damage. Pretreatment with an antagonist of the CB1 cannabinoid receptor (AM251) significantly reduced the analgesic effects of ATB-352. The CB1 antagonist exacerbated GI damage when coadministered with ketoprofen, but GI damage was not induced by the combination of ATB-352 and the CB1 antagonist. In vitro , ATB-352 was substantially more potent than ketoprofen as an inhibitor of fatty acid amide hydrolase, consistent with a contribution of endogenous cannabinoids to the analgesic effects of this drug. Blood anandamide levels were significantly depressed by ketoprofen, but remained unchanged after treatment with ATB-352. Innovation: Ketoprofen is a potent analgesic, but its clinical use, even in the short term, is significantly limited by its propensity to cause significant ulceration and bleeding in the GI tract. Covalently linking an H 2 S-releasing moiety to ketoprofen profoundly reduces the GI toxicity of the drug, while boosting analgesic effectiveness. Conclusion: This study demonstrates a marked enhancement of the potency and effectiveness of ATB-352, an H 2 S-releasing derivative of ketoprofen, in part, through the involvement of the endogenous cannabinoid system. This may have significant advantages for the control and management of pain, such as in a postoperative setting.

The tapeworm Hymenolepis diminuta fails to establish in mice. Given the potential for helminth-bacteria interaction in the gut and the influence that commensal bacteria exert on host immunity, we tested if worm expulsion was related to alterations in the gut microbiota. Specific pathogen-free (SPF) mice, treated with broad-spectrum antibiotics, or germ-free wild-type mice were infected with H. diminuta , gut bacterial composition assessed by 16S rRNA gene sequencing, and worm counts, blood eosinophilia, goblet cells, splenic IL-4, -5 and -10, and colonic cytokines/chemokines mRNA were assessed. Effects of a PBS-soluble extract of adult H. diminuta on bacterial growth in vitro was tested. H. diminuta -infected mice displayed increased α and β diversity in colonic mucosa-associated and fecal bacterial communities, characterized by increased Lachnospiraceae and clostridium cluster XIVa. In vitro analysis revealed that the worm extract promoted the growth of anaerobic bacteria on M2GSC agar. H. diminuta- infection was accompanied by increased Th2 immune responses, and colon from infected mice had increased levels of IL-10, IL-25, Muc2, trefoil factor 3, and β2-defensin mRNA. SPF-mice treated with antibiotics, or germ-free mice, expelled H. diminuta with kinetics similar to control SPF mice. In both settings, measurements of Th2-immune responses were not significantly different across the groups. Thus, while infection with H. diminuta results in subtle but distinct changes to the colonic microbiota, we have no evidence to support an essential role for gut bacteria in the expulsion of the worm from the mouse host.

Polymicrobial infections of the gastro-intestinal tract are common in areas with poor sanitation. Disease outcome is the result of complex interactions between the host and pathogens. Such interactions lie at the core of future management strategies of enteric diseases. In developed countries of the world, Giardia duodenalis is a common cause of diarrheal disease. In contrast, giardiasis appears to protect children against diarrhea in countries with poor sanitation, via obscure mechanisms. We hypothesized that Giardia may protect its host from disease induced by a co-infecting pathogen such as attaching and effacing Escherichia coli. This enteropathogen is commonly implicated in pediatric diarrhea in developing countries. The findings indicate that co-infection with Giardia attenuates the severity of disease induced by Citrobacter rodentium, an equivalent of A/E E. coli in mice. Co-infection with Giardia reduced colitis, blood in stools, fecal softening, bacterial invasion, and weight loss; the protective effects were lost when co-infection occurred in Nod-like receptor pyrin-containing 3 knockout mice. In co-infected mice, elevated levels of antimicrobial peptides Murine β defensin 3 and Trefoil Factor 3, and enhanced bacterial killing, were NLRP3-dependent. Inhibition of the NLRP3 inflammasome in human enterocytes blocked the activation of AMPs and bacterial killing. The findings uncover novel NLRP3-dependent modulatory mechanisms during co-infections with Giardia spp. and A/E enteropathogens, and demonstrate how these interactions may regulate the severity of enteric disease., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Background: Endoscopic retrograde cholangio-pancreatography (ERCP) is commonly performed in the management of pancreatic and biliary disease. Duodenoscopes are specialized endoscopes used to perform ERCP, and inherent to their design, a high rate of persistent bacterial contamination exists even after automated reprocessing and disinfection. Consequently, in recent years, ERCP has been associated with infection transmission, leading to several fatal patient outbreaks. Due to increasing fears over widespread future duodenoscope-related outbreaks, regulatory bodies have called for alterations in the design of duodenoscopes. A duodenoscope has recently been developed that employs a disposable cap. This novel design theoretically eliminates the mechanism behind persistent bacterial contamination and infection transmission. However, there are no data demonstrating persistent bacterial contamination rates, technical success rates, or clinical outcomes associated with these duodenoscopes., Methods: A parallel arm randomized controlled trial will be performed for which 520 patients will be recruited. The study population will consist of consecutive patients undergoing ERCP procedures for any indication at a high-volume tertiary care centre in Calgary, Alberta, Canada. Patients will be randomized to an intervention group, that will undergo ERCP with a novel duodenoscope with disposable cap, or to a control group who will undergo ERCP with a traditional duodenoscope. Co-primary outcomes will include persistent bacterial contamination rates (post automated reprocessing) and ERCP technical success rates. Secondary outcomes include clinical success rates, overall and specific early and late adverse event rates, 30-day mortality and healthcare utilization rates, procedure and reprocessing times, and ease of device use., Discussion: The ICECAP trial will answer important questions regarding the use of a novel duodenoscope with disposable cap. Specifically, persistent bacterial contamination, technical performance, and relevant clinical outcomes will be assessed. Given the mortality and morbidity burden associated with ERCP-related infectious outbreaks, the results of this study have the capacity to be impactful at an international level., Trial Registration: This trial was registered on clinicaltrials.gov (NCT04040504) on July 31, 2019.

Background and Purpose: ATB-346 is a hydrogen sulfide (H 2 S)-releasing anti-inflammatory and analgesic drug. Animal studies demonstrated negligible gastrointestinal (GI) damage despite marked inhibition of COX activity and significant analgesic and anti-inflammatory effects. In humans, ATB-346 (250 mg once daily) was found to inhibit COX to the same extent as naproxen (550 mg twice daily)., Experimental Approach: Two hundred forty-four healthy volunteers completed a 2-week, double-blind study, taking either ATB-346 (250 mg once daily) or naproxen (550 mg twice daily), with upper GI ulceration being examined endoscopically., Key Results: Forty-two per cent of the subjects taking naproxen developed at least one ulcer (≥3-mm diameter), while only 3% of the subjects taking ATB-346 developed at least one ulcer. The two drugs produced comparable and substantial (>94%) suppression of COX activity. Subjects in the naproxen group developed more ulcers per subject than ATB-346-treated subjects and a greater incidence of larger ulcers (≥5-mm diameter). The incidence of dyspepsia, abdominal pain, gastro-oesophageal reflux, and nausea was lower with ATB-346 than with naproxen. Subjects treated with ATB-346 had significantly higher plasma levels of H 2 S than those treated with naproxen., Conclusions and Implications: This Phase 2B study provides unequivocal evidence for a marked reduction of GI toxicity of the H 2 S-releasing analgesic/anti-inflammatory drug, ATB-346, as compared to the conventional dose of naproxen that produced equivalent suppression of COX., Linked Articles: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc., (© 2019 The British Pharmacological Society.)

Giardia is an important cause of diarrhoea, and results in post-infectious and extra-intestinal complications. This chapter presents a state-of-the art of our understanding of how this parasite may cause such abnormalities, which appear to develop at least in part in Assemblage-dependent manner. Findings from prospective longitudinal cohort studies indicate that Giardia is one of the four most prevalent enteropathogens in early life, and represents a risk factor for stunting at 2 years of age. This may occur independently of diarrheal disease, in strong support of the pathophysiological significance of the intestinal abnormalities induced by this parasite. These include epithelial malabsorption and maldigestion, increased transit, mucus depletion, and disruptions of the commensal microbiota. Giardia increases epithelial permeability and facilitates the invasion of gut bacteria. Loss of intestinal barrier function is at the core of the acute and post-infectious complications associated with this infection. Recent findings demonstrate that the majority of the pathophysiological responses triggered by this parasite can be recapitulated by the effects of its membrane-bound and secreted cysteine proteases., (© 2020 Elsevier Ltd All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus that grows in macrophages and causes acute pneumonia in pigs. PRRSV causes devastating losses to the porcine industry. However, due to its high antigenic variability and poorly understood immunopathogenesis, there is currently no effective vaccine or treatment to control PRRSV infection. The common occurrence of PRRSV infection with bacterial infections as well as its inflammatory-driven pathobiology raises the question of the value of antibiotics with immunomodulating properties for the treatment of the disease it causes. The macrolide antibiotic Tulathromycin (TUL) has been found to exhibit potent anti-inflammatory and immunomodulating properties in cattle and pigs. The aim of this study was to characterize the anti-viral and immunomodulating properties of TUL in PRRSV-infected porcine macrophages. Our findings indicate that blood monocyte-derived macrophages are readily infected by PRRSV and can be used as an effective cellular model to study PRRSV pathogenesis. TUL did not change intracellular or extracellular viral titers, not did it alter viral receptors (CD163 and CD169) expression on porcine macrophages. In contrast, TUL exhibited potent immunomodulating properties, which therefore occurred in the absence of any direct antiviral effects against PRRSV. TUL had an additive effect with PRRSV on the induction of macrophage apoptosis, and inhibited virus-induced necrosis. TUL significantly attenuated PRRSV-induced macrophage pro-inflammatory signaling (CXCL-8 and mitochondrial ROS production) and prevented PRRSV inhibition of non-opsonized and opsonized phagocytic function. Together, these data demonstrate that TUL inhibits PRRSV-induced inflammatory responses in porcine macrophages and protects against the phagocytic impairment caused by the virus. Research in live pigs is warranted to assess the potential clinical benefits of this antibiotic in the context of virally induced inflammation and tissue injury., Competing Interests: The authors declare that the research was conducted in the absence of any competing interests.

Giardia duodenalis is one of the most prevalent human enteropathogens and a major cause of diarrheal disease worldwide. Cysteine proteases (CPs) have been identified as major virulence factors in protozoan parasites, playing important roles in disease pathogenesis and in parasitic life cycles. G. duodenalis exhibits high proteolytic activity, and CPs play significant roles in giardiasis. Giardia CPs are directly involved in intestinal epithelial junctional complex disruption, intestinal epithelial cell apoptosis, and degradation of host immune factors, including chemokines and immunoglobulins. Giardia CPs have also been implicated in mucus depletion and microbiota dysbiosis induced by the parasite. This review discusses the most recent advances in characterization of Giardia Assemblage A and B CPs, including cathepsin B (catB)-like proteases., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Proteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin.

Here we characterized the murine dextran sulfate sodium (DSS) model of acute colitis. Specifically, we evaluated azithromycin and metronidazole treatment regimens to assess their effects on animal wellbeing, pathologic changes, barrier function, cytokine and chemokine profiles, and neutrophil migration in colon tissue. Azithromycin treatment significantly reduced the severity of colitis, as assessed through body weight change, water consumption, macroscopic lesions, and animal behaviors (activity level, climbing, and grooming), but did not alter food consumption or feeding behavior. Mucosal barrier function (evaluated by using FITC-labeled dextran) was decreased after DSS exposure; azithromycin did not significantly alter barrier function in mice with colitis, whereas metronidazole exacerbated the colitis-related deficit in barrier function. In addition, metronidazole appeared to exacerbate disease as assessed through water consumption and animal behaviors (overall activity, climbing, grooming, and drinking) but had no effect on weight loss, macroscopic lesions, or eating behavior. Pathologic changes were typical for DSS treatment. Antibiotic treatment resulted in reduced levels of proinflammatory cytokines and chemokines and decreased neutrophil adhesion and emigration in DSS-exposed mice. The results highlight the importance of clinical and behavioral assessments in addition to laboratory evaluation as tools to evaluate animal welfare and therapeutic efficacy in disease models. Data from this study suggest that azithromycin may convey some benefits in the mouse DSS colitis model through modulation of the immune response, including neutrophil migration into tissues, whereas metronidazole may exacerbate colitis.

Gut microbiota interacting with an intact mucosal surface are key to the maintenance of homeostasis and health. This review discusses the current state of knowledge of the biofilm mode of growth of these microbiota communities, and how in turn their disruptions may cause disease. Beyond alterations of relative microbial abundance and diversity, the aim of the review is to focus on the disruptions of the microbiota biofilm structure and function, the dispersion of commensal bacteria, and the mechanisms whereby these dispersed commensals may become pathobionts. Recent findings have linked iron acquisition to the expression of virulence factors in gut commensals that have become pathobionts. Causal studies are emerging, and mechanisms common to enteropathogen-induced disruptions, as well as those reported for Inflammatory Bowel Disease and colo-rectal cancer are used as examples to illustrate the great translational potential of such research. These new observations shed new light on our attempts to develop new therapies that are able to protect and restore gut microbiota homeostasis in the many disease conditions that have been linked to microbiota dysbiosis.

Significant alterations of intestinal microbiota and anemia are hallmarks of inflammatory bowel disease (IBD). It is widely accepted that iron is a key nutrient for pathogenic bacteria, but little is known about its impact on microbiota associated with IBD. We used a model device to grow human mucosa-associated microbiota in its physiological anaerobic biofilm phenotype. Compared to microbiota from healthy donors, microbiota from IBD patients generate biofilms ex vivo that were larger in size and cell numbers, contained higher intracellular iron concentrations, and exhibited heightened virulence in a model of human intestinal epithelia in vitro and in the nematode Caenorhabditis elegans. We also describe an unexpected iron-scavenging property for an experimental hydrogen sulfide-releasing derivative of mesalamine. The findings demonstrate that this new drug reduces the virulence of IBD microbiota biofilms through a direct reduction of microbial iron intake and without affecting bacteria survival or species composition within the microbiota. Metabolomic analyses indicate that this drug reduces the intake of purine nucleosides (guanosine), increases the secretion of metabolite markers of purine catabolism (urate and hypoxanthine), and reduces the secretion of uracil (a pyrimidine nucleobase) in complex multispecies human biofilms. These findings demonstrate a new pathogenic mechanism for dysbiotic microbiota in IBD and characterize a novel mode of action for a class of mesalamine derivatives. Together, these observations pave the way towards a new therapeutic strategy for treatment of patients with IBD.

Excessive accumulation of neutrophils and their uncontrolled death by necrosis at the site of inflammation exacerbates inflammatory responses and leads to self-amplifying tissue injury and loss of organ function, as exemplified in a variety of respiratory diseases. In homeostasis, neutrophils are inactivated by apoptosis, and non phlogistically removed by neighboring macrophages in a process known as efferocytosis, which promotes the resolution of inflammation. The present study assessed the potential anti-inflammatory and pro-resolution benefits of tylvalosin, a recently developed broad-spectrum veterinary macrolide derived from tylosin. Recent findings indicate that tylvalosin may modulate inflammation by suppressing NF-κB activation. Neutrophils and monocyte-derived macrophages were isolated from fresh blood samples obtained from 12- to 22-week-old pigs. Leukocytes exposed to vehicle or to tylvalosin (0.1, 1.0, or 10 µg/mL; 0.096-9.6 µM) were assessed at various time points for apoptosis, necrosis, efferocytosis, and changes in the production of cytokines and lipid mediators. The findings indicate that tylvalosin increases porcine neutrophil and macrophage apoptosis in a concentration- and time-dependent manner, without altering levels of necrosis or reactive oxygen species production. Importantly, tylvalosin increased the release of pro-resolving Lipoxin A 4 (LXA 4 ) and Resolvin D1 (RvD 1 ) while inhibiting the production of pro-inflammatory Leukotriene B4 (LTB 4 ) in Ca 2+ ionophore-stimulated porcine neutrophils. Tylvalosin increased neutrophil phospholipase C activity, an enzyme involved in releasing arachidonic acid from membrane stores. Tylvalosin also inhibited pro-inflammatory chemokine (C-X-C motif) ligand 8 (CXCL-8, also known as Interleukin-8) and interleukin-1 alpha (IL-1α) protein secretion in bacterial lipopolysaccharide-stimulated macrophages. Together, these data illustrate that tylvalosin has potent immunomodulatory effects in porcine leukocytes in addition to its antimicrobial properties.

A diverse range of effects of the intestinal microbiota on mucosal defense and injury has become increasingly clear over the past decade. Hydrogen sulfide (H 2 S) has emerged as an important mediator of many physiological functions, including gastrointestinal mucosal defense and repair. Hydrogen sulfide is produced by gastrointestinal tract tissues and by bacteria residing within the gut and can influence the function of a wide range of cells. The microbiota also appears to be an important target of hydrogen sulfide. H 2 S donors can modify the gut microbiota, and the gastrointestinal epithelium is a major site of oxidation of microbial-derived H 2 S. When administered together with nonsteroidal anti-inflammatory drugs, H 2 S can prevent some of the dysbiosis those drugs induce, possibly contributing to the observed prevention of gastrointestinal damage. Exogenous H 2 S can also markedly reduce the severity of experimental colitis and plays important roles in modulating epithelial cell-mucus-bacterial interactions in the intestine, contributing to its ability to promote resolution of inflammation and repair of tissue injury. In this paper we review recent studies examining the roles of H 2 S in mucosal defense, the possibility that H 2 S can damage the gastrointestinal epithelium, and effects of H 2 S on the gut microbiota and on mucus and biofilm interactions in the context of intestinal inflammation.

Giardia duodenalis (syn. G. lamblia, G. intestinalis ) is the protozoan parasite responsible for giardiasis, the most common and widely spread intestinal parasitic disease worldwide, affecting both humans and animals. After cysts ingestion (through either contaminated food or water), Giardia excysts in the upper intestinal tract to release replicating trophozoites that are responsible for the production of symptoms. In the gut, Giardia cohabits with the host's microbiota, and several studies have revealed the importance of this gut ecosystem and/or some probiotic bacteria in providing protection against G. duodenalis infection through mechanisms that remain incompletely understood. Recent findings suggest that Bile-Salt-Hydrolase (BSH)-like activities from the probiotic strain of Lactobacillus johnsonii La1 may contribute to the anti-giardial activity displayed by this strain. Here, we cloned and expressed each of the three bsh genes present in the L. johnsonii La1 genome to study their enzymatic and biological properties. While BSH47 and BSH56 were expressed as recombinant active enzymes, no significant enzymatic activity was detected with BSH12. In vitro assays allowed determining the substrate specificities of both BSH47 and BSH56, which were different. Modeling of these BSHs indicated a strong conservation of their 3-D structures despite low conservation of their primary structures. Both recombinant enzymes were able to mediate anti-giardial biological activity against Giardia trophozoites in vitro . Moreover, BSH47 exerted significant anti-giardial effects when tested in a murine model of giardiasis. These results shed new light on the mechanism, whereby active BSH derived from the probiotic strain Lactobacillus johnsonii La1 may yield anti-giardial effects in vitro and in vivo . These findings pave the way toward novel approaches for the treatment of this widely spread but neglected infectious disease, both in human and in veterinary medicine.

The microbiome influences host processes including nutritional availability, development, immunity, and behavioral responses. Caenorhabditis elegans is a powerful model to study molecular mechanisms of host-microbial interactions. Recent efforts have been made to profile the natural microbiome of C. elegans , laying a foundation for mechanistic studies of host-microbiome interactions in this genetically tractable model system. Studies using single-species microbes, multi-microbial systems, and humanized worm-microbiome interaction studies reveal metabolic and microbial-microbial interactions relevant in higher organisms. This article discusses recent developments in modeling the effects of host-microbiome interactions in C. elegans .

The intestinal mucous layer provides a critical host defense against pathogen exposure and epithelial injury, yet little is known about how enteropathogens may circumvent this physiologic barrier. Giardia duodenalis is a small intestinal parasite responsible for diarrheal disease and chronic postinfectious illness. This study reveals a complex interaction at the surface of epithelial cells, between G. duodenalis and the intestinal mucous layer. Here, we reveal mechanisms whereby G. duodenalis evades and disrupts the first line of host defense by degrading human mucin-2 (MUC2), depleting mucin stores and inducing differential gene expression in the mouse small and large intestines. Human colonic biopsy specimens exposed to G. duodenalis were depleted of mucus, and in vivo mice infected with G. duodenalis had a thinner mucous layer and demonstrated differential Muc2 and Muc5ac mucin gene expression. Infection in Muc2 -/- mice elevated trophozoite colonization in the small intestine and impaired weight gain. In vitro, human LS174T goblet-like cells were depleted of mucus and had elevated levels of MUC2 mRNA expression after G. duodenalis exposure. Importantly, the cysteine protease inhibitor E64 prevented mucous degradation, mucin depletion, and the increase in MUC2 expression. This article describes a novel role for Giardia's cysteine proteases in pathogenesis and how Giardia's disruptions of the mucous barrier facilitate bacterial translocation that may contribute to the onset and propagation of disease., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Biofilms composed of anaerobic bacteria can result in persistent infections and chronic inflammation. Host immune cells have difficulties clearing biofilm-related infections and this can result in tissue damage. Neutrophils are a vital component of the innate immune system and help clear biofilms. The comparative neutrophilic response to biofilms versus planktonic bacteria remains incompletely understood, particularly in the context of mixed infections. The objective of this study was to generate mixed species anaerobic bacterial biofilms composed of two opportunistic pathogens, Fusobacterium necrophorum and Porphyromonas levii, and evaluate neutrophil responses to extracellular fractions from both biofilms and planktonic cell co-cultures of the same bacteria. Purified bovine neutrophils exposed to culture supernatants from mixed species planktonic bacteria showed elevated oxidative activity compared to neutrophils exposed to biofilms composed of the same bacteria. Bacterial lipopolysaccharide plays a significant role in the stimulation of neutrophils; biofilms produced substantially more lipopolysaccharide than planktonic bacteria under these experimental conditions. Removal of lipopolysaccharide significantly reduced neutrophil oxidative response to culture supernatants of planktonic bacteria. Oxidative responses to LPS-removed biofilm supernatants and LPS-removed planktonic cell supernatants were similar. The limited neutrophil response to biofilm bacteria observed in this study supports the reduced ability of the innate immune system to eradicate biofilm-associated infections. Lipopolysaccharide is likely important in neutrophil response; however, the presence of other extracellular, immune modifying molecules in the bacterial media also appears to be important in altering neutrophil function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Our understanding of polymicrobial gastrointestinal infections and their effects on host biology remains incompletely understood. Giardia duodenalis is an ubiquitous intestinal protozoan parasite infecting animals and humans. Concomitant infections with Giardia and other gastrointestinal pathogens commonly occur. In countries with poor sanitation, Giardia infection has been associated with decreased incidence of diarrheal disease and fever, and reduced serum inflammatory markers release, via mechanisms that remain obscure. This study analyzed Giardia spp. co-infections with attaching and effacing (A/E) pathogens, and assessed whether and how the presence of Giardia modulates host responses to A/E enteropathogens, and alters intestinal disease outcome. In mice infected with the A/E pathogen Citrobacter rodentium, co-infection with Giardia muris significantly attenuated weight loss, macro- and microscopic signs of colitis, bacterial colonization and translocation, while concurrently enhancing the production and secretion of antimicrobial peptides (AMPs) mouse β-defensin 3 and trefoil factor 3 (TFF3). Co-infection of human intestinal epithelial cells (Caco-2) monolayers with G. duodenalis trophozoites and enteropathogenic Escherichia coli (EPEC) enhanced the production of the AMPs human β-defensin 2 (HBD-2) and TFF3; this effect was inhibited with treatment of G. duodenalis with cysteine protease inhibitors. Collectively, these results suggest that Giardia infections are capable of reducing enteropathogen-induced colitis while increasing production of host AMPs. Additional studies also demonstrated that Giardia was able to directly inhibit the growth of pathogenic bacteria. These results reveal novel mechanisms whereby Giardia may protect against gastrointestinal disease induced by a co-infecting A/E enteropathogen. Our findings shed new light on how microbial-microbial interactions in the gut may protect a host during concomitant infections.

Giardia duodenalis is a prevalent cause of acute diarrheal disease worldwide. However, recent outbreaks in Italy and Norway have revealed a link between giardiasis and the subsequent development of chronic post-infectious irritable bowel syndrome. While the mechanisms underlying the causation of post-infectious irritable bowel syndrome remain obscure, recent findings suggest that alterations in gut microbiota communities are linked to the pathophysiology of irritable bowel syndrome. In the present study, we use a laboratory biofilm system to culture and enrich mucosal microbiota from human intestinal biopsies. Subsequently, we show that co-culture with Giardia induces disturbances in biofilm species composition and biofilm structure resulting in microbiota communities that are intrinsically dysbiotic - even after the clearance of Giardia. These microbiota abnormalities were mediated in part by secretory-excretory Giardia cysteine proteases. Using in vitro cell culture and germ-free murine infection models, we show that Giardia-induced disruptions of microbiota promote bacterial invasion, resulting in epithelial apoptosis, tight junctional disruption, and bacterial translocation across an intestinal epithelial barrier. Additionally, these dysbiotic microbiota communities resulted in increased activation of the Toll-like receptor 4 signalling pathway, and overproduction of the pro-inflammatory cytokine IL-1beta in humanized germ-free mice. Previous studies that have sought explanations and risk factors for the development of post-infectious irritable bowel syndrome have focused on features of enteropathogens and attributes of the infected host. We propose that polymicrobial interactions involving Giardia and gut microbiota may cause persistent dysbiosis, offering a new interpretation of the reasons why those afflicted with giardiasis are predisposed to gastrointestinal disorders post-infection., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The nasopharyngeal (NP) microbiota plays an important role in bovine health, comprising a rich and diverse microbial community. The nasopharynx is also the niche for potentially pathogenic agents which are associated with bovine respiratory disease (BRD), a serious and costly illness in feedlot cattle. We used 14 beef heifers from a closed and disease-free herd to assess the dynamics of the NP microbiota of cattle that are transported to a feedlot. Cattle were sampled prior to transport to the feedlot (day 0) and at days 2, 7, and 14., Results: The structure of the NP microbiota changed significantly over the course of the study, with the largest shift occurring between day 0 (prior to transport) and day 2 (P < 0.001). Phylogenetic diversity and richness increased following feedlot placement (day 2; P < 0.05). The genera Pasteurella, Bacillus, and Proteus were enriched at day 0, Streptococcus and Acinetobacter at day 2, Bifidobacterium at day 7, and Mycoplasma at day 14. The functional potential of the NP microbiota was assessed using PICRUSt, revealing that replication and repair, as well as translation pathways, were more relatively abundant in day 14 samples. These differences were driven mostly by Mycoplasma. Although eight cattle were culture-positive for the BRD-associated bacterium Pasteurella multocida at one or more sampling times, none were culture-positive for Mannheimia haemolytica or Histophilus somni., Conclusions: This study investigated the effect that feedlot placement has on the NP microbiota of beef cattle over a 14-d period. Within two days of transport to the feedlot, the NP microbiota changed significantly, increasing in both phylogenetic diversity and richness. These results demonstrate that there is an abrupt shift in the NP microbiota of cattle after transportation to a feedlot. This may have importance for understanding why cattle are most susceptible to BRD after feedlot placement.

Understanding how intestinal enteropathogens cause acute and chronic alterations has direct animal and human health perspectives. Significant advances have been made on this field by studies focusing on the dynamic crosstalk between the intestinal protozoan parasite model Giardia duodenalis and the host intestinal mucosa. The concept of intestinal barrier function is of the highest importance in the context of many gastrointestinal diseases such as infectious enteritis, inflammatory bowel disease, and post-infectious gastrointestinal disorders. This crucial function relies on 3 biotic and abiotic components, first the commensal microbiota organized as a biofilm, then an overlaying mucus layer, and finally the tightly structured intestinal epithelium. Herein we review multiple strategies used by Giardia parasite to circumvent these 3 components. We will summarize what is known and discuss preliminary observations suggesting how such enteropathogen directly and/ or indirectly impairs commensal microbiota biofilm architecture, disrupts mucus layer and damages host epithelium physiology and survival.

Background and Aim: Irritable bowel syndrome is characterized by abdominal pain and altered bowel habits and may occur following stressful events or infectious gastroenteritis such as giardiasis. Recent findings revealed a link between cholecystokinin (CCK), neurotrophin synthesis, and intestinal hyperalgesia. The aim was to investigate the role of CCK in visceral hypersensitivity using mouse models challenged with a bout of infection with Giardia lamblia or psychological stress, either alone or in combination., Methods: Abdominal pain was evaluated by visceromoter response to colorectal distension. Nerve fibers in intestinal tissues were stained using immunohistochemistry (PGP9.5). Human neuroblastoma SH-SY5Y cells incubated with bacterial-free mouse gut supernatant or recombinant CCK-8S were assessed for neurite outgrowth and nerve growth factor (NGF) production., Results: Intestinal hypersensitivity was induced by either stress or Giardia infection, and a trend of increased pain was seen following dual stimuli. Increased CCK levels and PGP9.5 immunoreactivity were found in colonic mucosa of mice following stress and/or infection. Inhibitors to the CCK-A receptor (L-364718) or CCK-B receptor (L-365260) blocked visceral hypersensitivity caused by stress, but not when induced by giardiasis. Nerve fiber elongation and NGF synthesis were observed in SH-SY5Y cells after incubation with colonic supernatants from mice given the dual stimuli, or after treatment with CCK-8S. Increased nerve fiber length by colonic supernatant and CCK-8S was attenuated by L-365260 or neutralizing anti-NGF., Conclusions: This new model successfully recapitulates intestinal hypernociception induced by stress or Giardia. Colonic CCK contributes to visceral hypersensitivity caused by stress, but not by Giardia, partly via NGF-dependent neurite outgrowth., (© 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background: Distinct CD8+ T-cell subsets such as interleukin-17-expressing Tc17 and Foxp3-expressing Tcreg are functionally similar to CD4+ T cells. Though CD4+ T cells are dysregulated in patients with inflammatory bowel disease (IBD), CD8+ T cells are not well investigated. Vitamin D is an environmental factor which influences T-cell subsets. We assessed the prevalence of CD8+ T-cell subsets among peripheral blood mononuclear cells (PBMC) and lamina propria mononuclear cells (LPMC) of patients with Crohn's disease, patients with ulcerative colitis, and healthy controls. We then tested the effect of 1α,25-dihydroxyvitamin D3 on CD8+ T-cell subsets., Methods: A total of 73 patients with Crohn's disease, 49 patients with ulcerative colitis, and 47 healthy controls were studied. LPMC or PBMC were isolated and flow cytometry was performed. CD3+ T cells, isolated from PBMC, were cultured with or without 1α,25-dihydroxyvitamin D3, before flow cytometry., Results: In LPMC, the prevalence of Tcreg was higher in patients with IBD (P < 0.05), whereas Tc17 were higher in patients with ulcerative colitis compared with patients with Crohn's disease and healthy controls (P < 0.05). In PBMC, both Tcreg and Tc17 were higher in patients with IBD (P < 0.01). Double-expressing interferon-γ+ interleukin-17+ and Foxp3+ interleukin-17+ CD8+ T cells were also identified indicating possible CD8+ plasticity. 1α,25-dihydroxyvitamin D3 decreased interferon-γ-expressing Tc1 (P < 0.05), but had no effect on Tc17 or Tcreg., Conclusions: The prevalence of novel CD8+ T-cell subsets is altered in patients with IBD. Double-expressing cells indicate plasticity and were identified in patients with IBD. Vitamin D may have a limited effect on CD8+ T cells by decreasing interferon-γ expression.

The zebrafish model system is helping researchers improve the health and welfare of people and animals and has become indispensable for advancing biomedical research. As genetic engineering is both resource intensive and time-consuming, sharing successfully developed genetically modified zebrafish lines throughout the international community is critical to research efficiency and to maximizing the millions of dollars in research funding. New restrictions on importation of zebrafish into Canada based on putative susceptibility to infection by the spring viremia of carp virus (SVCV) have been imposed on the scientific community. In this commentary, we review the disease profile of SVCV in fish, discuss the findings of the Canadian government's scientific assessment, how the interpretations of their assessment differ from that of the Canadian research community, and describe the negative impact of these regulations on the Canadian research community and public as it pertains to protecting the health of Canadians.

Irritable bowel syndrome (IBS) is the most frequent functional gastrointestinal disorder. It is characterized by abdominal hypersensitivity, leading to discomfort and pain, as well as altered bowel habits. While it is common for IBS to develop following the resolution of infectious gastroenteritis [then termed postinfectious IBS (PI-IBS)], the mechanisms remain incompletely understood. Giardia duodenalis is a cosmopolitan water-borne enteropathogen that causes intestinal malabsorption, diarrhea, and postinfectious complications. Cause-and-effect studies using a human enteropathogen to help investigate the mechanisms of PI-IBS are sorely lacking. In an attempt to establish causality between giardiasis and postinfectious visceral hypersensitivity, this study describes a new model of PI-IBS in neonatal rats infected with G. duodenalis At 50 days postinfection with G. duodenalis (assemblage A or B), long after the parasite was cleared, rats developed visceral hypersensitivity to luminal balloon distension in the jejunum and rectum, activation of the nociceptive signaling pathway (increased c-fos expression), histological modifications (villus atrophy and crypt hyperplasia), and proliferation of mucosal intraepithelial lymphocytes and mast cells in the jejunum, but not in the rectum. G. duodenalis infection also disrupted the intestinal barrier, in vivo and in vitro, which in turn promoted the translocation of commensal bacteria. Giardia-induced bacterial paracellular translocation in vitro correlated with degradation of the tight junction proteins occludin and claudin-4. The extensive observations associated with gut hypersensitivity described here demonstrate that, indeed, in this new model of postgiardiasis IBS, alterations to the gut mucosa and c-fos are consistent with those associated with PI-IBS and, hence, offer avenues for new mechanistic research in the field., (Copyright © 2016 the American Physiological Society.)