Giardia Antagonizes Beneficial Functions of Indigenous and Therapeutic Intestinal Bacteria during Malnutrition.

Undernutrition in children commonly disrupts the structure and function of the small intestinal microbial community, leading to enteropathies, compromised metabolic health, and impaired growth and development. The mechanisms by which diet and microbes mediate the balance between commensal and pathogenic intestinal flora remain elusive. In a murine model of undernutrition, we investigated the direct interactions Giardia lamblia, a prevalent small intestinal pathogen, on indigenous microbiota and specifically on Lactobacillus strains known for their mucosal and growth homeostatic properties. Our research reveals that Giardia colonization shifts the balance of lactic acid bacteria, causing a relative decrease in Lactobacillus spp . and an increase in Bifidobacterium spp . This alteration corresponds with a decrease in multiple indicators of mucosal and nutritional homeostasis. Additionally, protein-deficient conditions coupled with Giardia infection exacerbate the rise of primary bile acids and susceptibility to bile acid-induced intestinal barrier damage. In epithelial cell monolayers, Lactobacillus spp . mitigated bile acid-induced permeability, showing strain-dependent protective effects. In vivo, L. plantarum, either alone or within a Lactobacillus spp consortium, facilitated growth in protein-deficient mice, an effect attenuated by Giardia , despite not inhibiting Lactobacillus colonization. These results highlight Giardia's potential role as a disruptor of probiotic functional activity, underscoring the imperative for further research into the complex interactions between parasites and bacteria under conditions of nutritional deficiency.

Gut microbiome responds to alteration in female sex hormone status and exacerbates metabolic dysfunction.

Women are at significantly greater risk of metabolic dysfunction after menopause, which subsequently leads to numerous chronic illnesses. The gut microbiome is associated with obesity and metabolic dysfunction, but its interaction with female sex hormone status and the resulting impact on host metabolism remains unclear. Herein, we characterized inflammatory and metabolic phenotypes as well as the gut microbiome associated with ovariectomy and high-fat diet feeding, compared to gonadal intact and low-fat diet controls. We then performed fecal microbiota transplantation (FMT) using gnotobiotic mice to identify the impact of ovariectomy-associated gut microbiome on inflammatory and metabolic outcomes. We demonstrated that ovariectomy led to greater gastrointestinal permeability and inflammation of the gut and metabolic organs, and that a high-fat diet exacerbated these phenotypes. Ovariectomy also led to alteration of the gut microbiome, including greater fecal ß-glucuronidase activity. However, differential changes in the gut microbiome only occurred when fed a low-fat diet, not the high-fat diet. Gnotobiotic mice that received the gut microbiome from ovariectomized mice fed the low-fat diet had greater weight gain and hepatic gene expression related to metabolic dysfunction and inflammation than those that received intact sham control-associated microbiome. These results indicate that the gut microbiome responds to alterations in female sex hormone status and contributes to metabolic dysfunction. Identifying and developing gut microbiome-targeted modulators to regulate sex hormones may be useful therapeutically in remediating menopause-related diseases.

Unveiling and harnessing the human gut microbiome in the rising burden of non-communicable diseases during urbanization.

The world is witnessing a global increase in the urban population, particularly in developing Asian and African countries. Concomitantly, the global burden of non-communicable diseases (NCDs) is rising, markedly associated with the changing landscape of lifestyle and environment during urbanization. Accumulating studies have revealed the role of the gut microbiome in regulating the immune and metabolic homeostasis of the host, which potentially bridges external factors to the host (patho-)physiology. In this review, we discuss the rising incidences of NCDs during urbanization and their links to the compositional and functional dysbiosis of the gut microbiome. In particular, we elucidate the effects of urbanization-associated factors (hygiene/pollution, urbanized diet, lifestyles, the use of antibiotics, and early life exposure) on the gut microbiome underlying the pathogenesis of NCDs. We also discuss the potential and feasibility of microbiome-inspired and microbiome-targeted approaches as novel avenues to counteract NCDs, including fecal microbiota transplantation, diet modulation, probiotics, postbiotics, synbiotics, celobiotics, and precision antibiotics.

KSHV Viral Protein Kinase Interacts with USP9X to Modulate the Viral Lifecycle.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy and one of the most common childhood cancers. Immunosuppressed patients, including HIV-infected patients, are more prone to KSHV-associated disease. KSHV encodes a viral protein kinase (vPK) that is expressed from ORF36. KSHV vPK contributes to the optimal production of infectious viral progeny and upregulation of protein synthesis. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used a bottom-up proteomics approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Subsequently, we validated this interaction using a co-immunoprecipitation assay. We report that both the ubiquitin-like and the catalytic domains of USP9X are important for association with vPK. To uncover the biological relevance of the USP9X/vPK interaction, we investigated whether the knockdown of USP9X would modulate viral reactivation. Our data suggest that depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Understanding how USP9X influences the reactivation of KSHV will provide insights into how cellular deubiquitinases regulate viral kinase activity and how viruses co-opt cellular deubiquitinases to propagate infection. Hence, characterizing the roles of USP9X and vPK during KSHV infection constitutes a first step toward identifying a potentially critical interaction that could be targeted by future therapeutics. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy. KSHV encodes a viral protein kinase (vPK) that aids viral replication. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used an affinity purification approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Overall, our data suggest a proviral role for USP9X.

Characterizing the metabolic effects of the selective inhibition of gut microbial ß-glucuronidases in mice.

The hydrolysis of xenobiotic glucuronides by gut bacterial glucuronidases reactivates previously detoxified compounds resulting in severe gut toxicity for the host. Selective bacterial ß-glucuronidase inhibitors can mitigate this toxicity but their impact on wider host metabolic processes has not been studied. To investigate this the inhibitor 4-(8-(piperazin-1-yl)-1,2,3,4-tetrahydro-[1,2,3]triazino[4',5':4,5]thieno[2,3-c]isoquinolin-5-yl)morpholine (UNC10201652, Inh 9) was administered to mice to selectively inhibit a narrow range of bacterial ß-glucuronidases in the gut. The metabolomic profiles of the intestinal contents, biofluids, and several tissues involved in the enterohepatic circulation were measured and compared to control animals. No biochemical perturbations were observed in the plasma, liver or gall bladder. In contrast, the metabolite profiles of urine, colon contents, feces and gut wall were altered compared to the controls. Changes were largely restricted to compounds derived from gut microbial metabolism. This work establishes that inhibitors targeted towards bacterial ß-glucuronidases modulate the functionality of the intestinal microbiota without adversely impacting the host metabolic system.

Metagenomics combined with activity-based proteomics point to gut bacterial enzymes that reactivate mycophenolate.

Mycophenolate mofetil (MMF) is an important immunosuppressant prodrug prescribed to prevent organ transplant rejection and to treat autoimmune diseases. MMF usage, however, is limited by severe gastrointestinal toxicity that is observed in approximately 45% of MMF recipients. The active form of the drug, mycophenolic acid (MPA), undergoes extensive enterohepatic recirculation by bacterial ß-glucuronidase (GUS) enzymes, which reactivate MPA from mycophenolate glucuronide (MPAG) within the gastrointestinal tract. GUS enzymes demonstrate distinct substrate preferences based on their structural features, and gut microbial GUS enzymes that reactivate MPA have not been identified. Here, we compare the fecal microbiomes of transplant recipients receiving MMF to healthy individuals using shotgun metagenomic sequencing. We find that neither microbial composition nor the presence of specific structural classes of GUS genes are sufficient to explain the differences in MPA reactivation measured between fecal samples from the two cohorts. We next employed a GUS-specific activity-based chemical probe and targeted metaproteomics to identify and quantify the GUS proteins present in the human fecal samples. The identification of specific GUS enzymes was improved by using the metagenomics data collected from the fecal samples. We found that the presence of GUS enzymes that bind the flavin mononucleotide (FMN) is significantly correlated with efficient MPA reactivation. Furthermore, structural analysis identified motifs unique to these FMN-binding GUS enzymes that provide molecular support for their ability to process this drug glucuronide. These results indicate that FMN-binding GUS enzymes may be responsible for reactivation of MPA and could be a driving force behind MPA-induced GI toxicity.

Human Tumor Targeted Cytotoxic Mast Cells for Cancer Immunotherapy.

The diversity of autologous cells being used and investigated for cancer therapy continues to increase. Mast cells (MCs) are tissue cells that contain a unique set of anti-cancer mediators and are found in and around tumors. We sought to exploit the anti-tumor mediators in MC granules to selectively target them to tumor cells using tumor specific immunoglobin E (IgE) and controllably trigger release of anti-tumor mediators upon tumor cell engagement. We used a human HER2/neu-specific IgE to arm human MCs through the high affinity IgE receptor (FcεRI). The ability of MCs to bind to and induce apoptosis of HER2/neu-positive cancer cells in vitro and in vivo was assessed. The interactions between MCs and cancer cells were investigated in real time using confocal microscopy. The mechanism of action using cytotoxic MCs was examined using gene array profiling. Genetically manipulating autologous MC to assess the effects of MC-specific mediators have on apoptosis of tumor cells was developed using siRNA. We found that HER2/neu tumor-specific IgE-sensitized MCs bound, penetrated, and killed HER2/neu-positive tumor masses in vitro. Tunneling nanotubes formed between MCs and tumor cells are described that parallel tumor cell apoptosis. In solid tumor, human breast cancer (BC) xenograft mouse models, infusion of HER2/neu IgE-sensitized human MCs co-localized to BC cells, decreased tumor burden, and prolonged overall survival without indications of toxicity. Gene microarray of tumor cells suggests a dependence on TNF and TGFß signaling pathways leading to apoptosis. Knocking down MC-released tryptase did not affect apoptosis of cancer cells. These studies suggest MCs can be polarized from Type I hypersensitivity-mediating cells to cytotoxic cells that selectively target tumor cells and specifically triggered to release anti-tumor mediators. A strategy to investigate which MC mediators are responsible for the observed tumor killing is described so that rational decisions can be made in the future when selecting which mediators to target for deletion or those that could further polarize them to cytotoxic MC by adding other known anti-tumor agents. Using autologous human MC may provide further options for cancer therapeutics that offers a unique anti-cancer mechanism of action using tumor targeted IgE's.

The mother of all synbiotics: Just a spoonful of sugar makes the bugs stick around.

Success of live biotherapeutics depends upon sustained and durable engraftment of beneficial microbes with robust functional output. In this issue of Cell Host & Microbe, Button et al. (2022) report that a human milk oligosaccharide-Bifidobacterium synbiotic delivers by supporting functional engraftment in healthy adults without antibiotic administration.

A Proximal-to-Distal Survey of Healthy Adult Human Small Intestine and Colon Epithelium by Single-Cell Transcriptomics.

BACKGROUND & AIMS: Single-cell transcriptomics offer unprecedented resolution of tissue function at the cellular level, yet studies analyzing healthy adult human small intestine and colon are sparse. Here, we present single-cell transcriptomics covering the duodenum, jejunum, ileum, and ascending, transverse, and descending colon from 3 human beings. METHODS: A total of 12,590 single epithelial cells from 3 independently processed organ donors were evaluated for organ-specific lineage biomarkers, differentially regulated genes, receptors, and drug targets. Analyses focused on intrinsic cell properties and their capacity for response to extrinsic signals along the gut axis across different human beings. RESULTS: Cells were assigned to 25 epithelial lineage clusters. Multiple accepted intestinal stem cell markers do not specifically mark all human intestinal stem cells. Lysozyme expression is not unique to human Paneth cells, and Paneth cells lack expression of expected niche factors. Bestrophin 4 (BEST4)+ cells express Neuropeptide Y (NPY) and show maturational differences between the small intestine and colon. Tuft cells possess a broad ability to interact with the innate and adaptive immune systems through previously unreported receptors. Some classes of mucins, hormones, cell junctions, and nutrient absorption genes show unappreciated regional expression differences across lineages. The differential expression of receptors and drug targets across lineages show biological variation and the potential for variegated responses. CONCLUSIONS: Our study identifies novel lineage marker genes, covers regional differences, shows important differences between mouse and human gut epithelium, and reveals insight into how the epithelium responds to the environment and drugs. This comprehensive cell atlas of the healthy adult human intestinal epithelium resolves likely functional differences across anatomic regions along the gastrointestinal tract and advances our understanding of human intestinal physiology.

The Emulsifier Carboxymethylcellulose Induces More Aggressive Colitis in Humanized Mice with Inflammatory Bowel Disease Microbiota Than Polysorbate-80.

Commonly used synthetic dietary emulsifiers, including carboxymethylcellulose (CMC) and polysorbate-80 (P80), promote intestinal inflammation. We compared abilities of CMC vs. P80 to potentiate colitis and impact human microbiota in an inflammatory environment using a novel colitis model of ex-germ-free (GF) IL10-/- mice colonized by pooled fecal transplant from three patients with active inflammatory bowel diseases. After three days, mice received 1% CMC or P80 in drinking water or water alone for four weeks. Inflammation was quantified by serial fecal lipocalin 2 (Lcn-2) and after four weeks by blinded colonic histologic scores and colonic inflammatory cytokine gene expression. Microbiota profiles in cecal contents were determined by shotgun metagenomic sequencing. CMC treatment significantly increased fecal Lcn-2 levels compared to P80 and water treatment by one week and throughout the experiment. Likewise, CMC treatment increased histologic inflammatory scores and colonic inflammatory cytokine gene expression compared with P80 and water controls. The two emulsifiers differentially affected specific intestinal microbiota. CMC did not impact bacterial composition but significantly decreased Caudoviricetes (bacteriophages), while P80 exposure non-significantly increased the abundance of both Actinobacteria and Proteobacteria. Commonly used dietary emulsifiers have different abilities to induce colitis in humanized mice. CMC promotes more aggressive inflammation without changing bacterial composition.

Targeted inhibition of gut bacterial ß-glucuronidase activity enhances anticancer drug efficacy.

Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial ß-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.

Relationship Between the Gut Microbiome and Systemic Chemotherapy.

The intestinal microbiome encodes vast metabolic potential, and multidisciplinary approaches are enabling a mechanistic understanding of how bacterial enzymes impact the metabolism of diverse pharmaceutical compounds, including chemotherapeutics. Microbiota alter the activity of many drugs and chemotherapeutics via direct and indirect mechanisms; some of these alterations result in changes to the drug's bioactivity and bioavailability, causing toxic gastrointestinal side effects. Gastrointestinal toxicity is one of the leading complications of systemic chemotherapy, with symptoms including nausea, vomiting, diarrhea, and constipation. Patients undergo dose reductions or drug holidays to manage these adverse events, which can significantly harm prognosis, and can result in mortality. Selective and precise targeting of the gut microbiota may alleviate these toxicities. Understanding the composition and function of the microbiota may serve as a biomarker for prognosis, and predict treatment efficacy and potential adverse effects, thereby facilitating personalized medicine strategies for cancer patients.

Discovering the Microbial Enzymes Driving Drug Toxicity with Activity-Based Protein Profiling.

It is increasingly clear that interindividual variability in human gut microbial composition contributes to differential drug responses. For example, gastrointestinal (GI) toxicity is not observed in all patients treated with the anticancer drug irinotecan, and it has been suggested that this variability is a result of differences in the types and levels of gut bacterial ß-glucuronidases (GUSs). GUS enzymes promote drug toxicity by hydrolyzing the inactive drug-glucuronide conjugate back to the active drug, which damages the GI epithelium. Proteomics-based identification of the exact GUS enzymes responsible for drug reactivation from the complexity of the human microbiota has not been accomplished, however. Here, we discover the specific bacterial GUS enzymes that generate SN-38, the active and toxic metabolite of irinotecan, from human fecal samples using a unique activity-based protein profiling (ABPP) platform. We identify and quantify gut bacterial GUS enzymes from human feces with an ABPP-enabled proteomics pipeline and then integrate this information with ex vivo kinetics to pinpoint the specific GUS enzymes responsible for SN-38 reactivation. Furthermore, the same approach also reveals the molecular basis for differential gut bacterial GUS inhibition observed between human fecal samples. Taken together, this work provides an unprecedented technical and bioinformatics pipeline to discover the microbial enzymes responsible for specific reactions from the complexity of human feces. Identifying such microbial enzymes may lead to precision biomarkers and novel drug targets to advance the promise of personalized medicine.

Targeting Regorafenib-Induced Toxicity through Inhibition of Gut Microbial ß-Glucuronidases.

Regorafenib (Stivarga) is an oral small molecule kinase inhibitor used to treat metastatic colorectal cancer, hepatocellular carcinomas, and gastrointestinal stromal tumors. Diarrhea is one of the most frequently observed adverse reactions associated with regorafenib. This toxicity may arise from the reactivation of the inactive regorafenib-glucuronide to regorafenib by gut microbial ß-glucuronidase (GUS) enzymes in the gastrointestinal tract. We sought to unravel the molecular basis of regorafenib-glucuronide processing by human intestinal GUS enzymes and to examine the potential inhibition of these enzymes. Using a panel of 31 unique gut microbial GUS enzymes derived from the 279 mapped from the human gut microbiome, we found that only four were capable of regorafenib-glucuronide processing. Using crystal structures as a guide, we pinpointed the molecular features unique to these enzymes that confer regorafenib-glucuronide processing activity. Furthermore, a pilot screen identified the FDA-approved drug raloxifene as an inhibitor of regorafenib reactivation by the GUS proteins discovered. Novel synthetic raloxifene analogs exhibited improved potency in both in vitro and ex vivo studies. Taken together, these data establish that regorafenib reactivation is exclusively catalyzed by gut microbial enzymes and that these enzymes are amenable to targeted inhibition. Our results unravel key molecular details of regorafenib reactivation in the GI tract and provide a potential pathway to improve clinical outcomes with regorafenib.

Inhibition of Aurora A Kinase in Combination with Chemotherapy Induces Synthetic Lethality and Overcomes Chemoresistance in Myc-Overexpressing Lymphoma.

BACKGROUND: Aberrant Myc expression plays a critical role in various tumors, including non-Hodgkin lymphoma (NHL). Myc-positive lymphoma is clinically aggressive, more resistant to chemotherapy, and associated with high mortality. OBJECTIVE: The current study aimed to show inhibition of aurora A kinase (AURKA) may overcome resistance to chemotherapy and improve outcomes in Myc-overexpressing lymphoma. METHODS: Myc-overexpressing lymphoma cell lines were evaluated by trypan blue, annexin V/propidium iodide staining, and western blotting for cytotoxicity, cell cycle, apoptosis, and Myc-associated protein expression, respectively, in the presence of cyclophosphamide with or without MLN8237, an AURKA inhibitor. Immunofluorescence for apoptosis-inducing factor (AIF) and acridine orange staining were used to analyze levels of autophagy. EµMyc genetically modified mouse model and xenograft models bearing Myc-overexpressing lymphoma cells were used to determine the efficacy of cyclophosphamide, MLN8237, or the combination in chemosensitive and chemoresistant tumors. RESULTS: In our in vitro experiments using chemoresistant lymphoma cells, MLN8237 and cyclophosphamide showed synergistic effects. Mice bearing lymphoma xenograft had rapid disease progression with median survival of ~ 35 days when treated with cyclophosphamide alone. In contrast, the combination of cyclophosphamide and MLN8237 induced complete tumor regression in all mice, which led to improvement in survival compared with the single agent control (p = 0.022). Kinome analysis of tumors treated with MLN8237 showed global suppression of various kinases. CONCLUSION: Our data demonstrate that AURKA inhibition induces synthetic lethality and overcomes chemoresistance in Myc-overexpressing lymphoma. The combination of MLN8237 and conventional chemotherapy showed promising safety and anti-tumor activities in preclinical models of Myc-positive NHL.

Structure, function, and inhibition of drug reactivating human gut microbial ß-glucuronidases.

Bacterial ß-glucuronidase (GUS) enzymes cause drug toxicity by reversing Phase II glucuronidation in the gastrointestinal tract. While many human gut microbial GUS enzymes have been examined with model glucuronide substrates like p-nitrophenol-ß-D-glucuronide (pNPG), the GUS orthologs that are most efficient at processing drug-glucuronides remain unclear. Here we present the crystal structures of GUS enzymes from human gut commensals Lactobacillus rhamnosus, Ruminococcus gnavus, and Faecalibacterium prausnitzii that possess an active site loop (Loop 1; L1) analogous to that found in E. coli GUS, which processes drug substrates. We also resolve the structure of the No Loop GUS from Bacteroides dorei. We then compare the pNPG and diclofenac glucuronide processing abilities of a panel of twelve structurally diverse GUS proteins, and find that the new L1 GUS enzymes presented here process small glucuronide substrates inefficiently compared to previously characterized L1 GUS enzymes like E. coli GUS. We further demonstrate that our GUS inhibitors, which are effective against some L1 enzymes, are not potent towards all. Our findings pinpoint active site structural features necessary for the processing of drug-glucuronide substrates and the inhibition of such processing.

Human herpesvirus-encoded kinase induces B cell lymphomas in vivo.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that is the etiological agent of the endothelial cell cancer Kaposi's sarcoma (KS) and 2 B cell lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV ORF36, also known as viral protein kinase (vPK), is a viral serine/threonine kinase. We previously reported that KSHV vPK enhances cell proliferation and mimics cellular S6 kinase to phosphorylate ribosomal protein S6, a protein involved in protein synthesis. We created a mouse model to analyze the function of vPK in vivo. We believe this is the first mouse tumor model of a viral kinase encoded by a pathogenic human virus. We observed increased B cell activation in the vPK transgenic mice compared with normal mice. We also found that, over time, vPK transgenic mice developed a B cell hyperproliferative disorder and/or a high-grade B cell non-Hodgkin lymphoma at a greatly increased incidence compared with littermate controls. This mouse model shows that a viral protein kinase is capable of promoting B cell activation and proliferation as well as augmenting lymphomagenesis in vivo and may therefore contribute to the development of viral cancers.

Microbial Glucuronidase Inhibition Reduces Severity of Diclofenac-Induced Anastomotic Leak in Rats.

BACKGROUND: The non-steroidal anti-inflammatory drug diclofenac has been associated with intestinal anastomotic leakage, although the underlying pathophysiology is unclear. Previous data suggest that reactivation of biliary diclofenac metabolites by microbial ß-glucuronidases in the gut plays a role in harming the intestinal mucosa, and that microbiome-targeted glucuronidase inhibition prevents this damage. Here, the microbial glucuronidase inhibitor Inh1 was examined for its ability to reduce diclofenac-induced anastomotic leakage in rats. METHODS: Ninety male Wistar rats were allocated to five groups. In the two diclofenac groups, group DCF received diclofenac (3 mg/kg per day) and group DCF-Inh1 additionally received 800 mcg/kg per day of glucuronidase inhibitor Inh1 solution orally. In non-diclofenac groups, animals received either Inh1 (800 mcg/kg per day; group Inh1) solution, the vehicle (methylcellulose; group Veh), or no solution (group Ctrl). All solutions were provided from the day of surgery until sacrifice on day three. Plasma concentrations of diclofenac were determined. Outcomes were anastomotic leakage, leak severity, and anastomotic strength. RESULTS: Anastomotic leak rates were 89% in group DCF and 44% in group DCF-Inh1 (p = 0.006). Leak severity was reduced in group DCFic-Inh1 (p = 0.029). In non-diclofenac cohorts, mostly minor leakage signs were observed in 25% in group Ctrl, 39% in group Inh1 (0.477), and 24% in group Veh (p = 1.000). Bursting pressure and breaking strength were not significantly different. Plasma concentrations of diclofenac were not changed by Inh1. CONCLUSION: Microbial glucuronidase inhibitor reduces diclofenac-induced anastomotic leakage severity, which suggests a harmful effect of diclofenac metabolite reactivation in the gut. This finding improves the understanding of the pathogenesis of anastomotic leakage.