Recommendations to Improve Quality of Probiotic Systematic Reviews With Meta-Analyses.

Importance: Systematic reviews and meta-analyses often report conflicting results when assessing evidence for probiotic efficacy, partially because of the lack of understanding of the unique features of probiotic trials. As a consequence, clinical decisions on the use of probiotics have been confusing. Objective: To provide recommendations to improve the quality and consistency of systematic reviews with meta-analyses on probiotics, so evidence-based clinical decisions can be made with more clarity. Evidence Review: For this consensus statement, an updated literature review was conducted (January 1, 2020, to June 30, 2022) to supplement a previously published 2018 literature search to identify areas where probiotic systematic reviews with meta-analyses might be improved. An expert panel of 21 scientists and physicians with experience on writing and reviewing probiotic reviews and meta-analyses was convened and used a modified Delphi method to develop recommendations for future probiotic reviews. Findings: A total of 206 systematic reviews with meta-analysis components on probiotics were screened and representative examples discussed to determine areas for improvement. The expert panel initially identified 36 items that were inconsistently reported or were considered important to consider in probiotic meta-analyses. Of these, a consensus was reached for 9 recommendations to improve the quality of future probiotic meta-analyses. Conclusions and Relevance: In this study, the expert panel reached a consensus on 9 recommendations that should promote improved reporting of probiotic systematic reviews with meta-analyses and, thereby, assist in clinical decisions regarding the use of probiotics.

Clinical Manifestations and Stool Load of Atypical Enteropathogenic Escherichia coli Infections in United States Children and Adults.

BACKGROUND & AIMS: There is debate whether atypical enteropathogenic Escherichia coli (aEPEC) causes disease in adults. aEPEC is commonly detected in symptomatic and asymptomatic individuals. aEPEC, in contrast to typical EPEC, lacks bundle-forming pili, altering its pathogenicity. Here, we define for the first time the clinical manifestations of sporadic aEPEC infection in United States children and adults and determine whether EPEC load correlates with disease. METHODS: This is a retrospective case-control study of 380 inpatients/outpatients of all ages. EPEC load in stools was determined by quantitative polymerase chain reaction. RESULTS: Diarrhea, vomiting, abdominal pain, and fever were more prevalent in EPEC-positive cases than in EPEC-negative controls. aEPEC infection caused mostly acute, mild diarrhea lasting for 6 to 13 days. However, some had severe diarrhea with 10 to 40 bowel movements per day or had persistent/chronic diarrhea. Fever, vomiting, and abnormal serum sodium levels were more common in children. Adults more often reported abdominal pain and longer duration of diarrhea. Symptomatic aEPEC infection was associated with leukocytosis in 24% of patients. EPEC load >0.1% was associated with symptomatic infection; however, loads varied greatly. Co-infecting pathogens did not alter diarrhea severity or EPEC load. Longitudinal data reveal that some are colonized for months to years or are repeatedly infected. CONCLUSIONS: aEPEC is associated with a wide array of symptoms in adults, ranging from asymptomatic carriage to severe diarrhea. Higher EPEC loads are associated with presence of symptoms, but bacterial load does not predict disease or severity. Future studies are needed to understand bacterial and host factors that contribute to aEPEC pathogenicity to improve diagnostic tools and clinical care.

Spef1/CLAMP binds microtubules and actin-based structures and regulates cell migration and epithelia cell polarity.

During migration, cells invade, repair, and create barriers leading to the formation of new cellular contacts in target tissues. Cell migration requires many proteins that collectively form the cytoskeleton. The main cytoskeletal elements are actin filaments, microtubules (MTs), and intermediate filaments. These structures work in concert with a large number of accessory proteins that contribute in a variety of ways to regulate filament assembly and turnover, to alter the configuration or arrangement of filaments by bundling or crosslinking, to link the cytoskeleton to other structures in the cell, such as membranes and junctions, and to transport cargo along the filaments. Sperm flagella protein-1 (Spef1), also designated calponin homology and microtubules-associated protein (CLAMP), is a multifunctional protein that interacts with cytoskeletal structures, including MTs, actin filaments, and focal adhesions in epithelia. In this review, we outline Spef1/CLAMP structure and expression in several cellular models. The function of Spef1/CLAMP in flagellar and ciliary motility, MT-binding and stability, regulation of planar cell polarity, and potential contribution to the maintenance of actin-based structures, such as lamellipodia and filopodia during cell migration, are also discussed.

Durable reduction of Clostridioides difficile infection recurrence and microbiome restoration after treatment with RBX2660: results from an open-label phase 2 clinical trial.

BACKGROUND: Effective treatment options for recurrent Clostridioides difficile infection (rCDI) are limited, with high recurrence rates associated with the current standard of care. Herein we report results from an open-label Phase 2 trial to evaluate the safety, efficacy, and durability of RBX2660-a standardized microbiota-based investigational live biotherapeutic-and a closely-matched historical control cohort. METHODS: This prospective, multicenter, open-label Phase 2 study enrolled patients who had experienced either ≥ 2 recurrences of CDI, treated by standard-of-care antibiotic therapy, after a primary CDI episode, or ≥ 2 episodes of severe CDI requiring hospitalization. Participants received up to 2 doses of RBX2660 rectally administered with doses 7 days apart. Treatment success was defined as the absence of CDI diarrhea without the need for retreatment for 8 weeks after completing study treatment. A historical control group with matched inclusion and exclusion criteria was identified from a retrospective chart review of participants treated with standard-of-care antibiotics for recurrent CDI who matched key criteria for the study. The primary objective was to compare treatment success of RBX2660 to the historical control group. A key secondary outcome was the safety profile of RBX2660, including adverse events and CDI occurrence through 24 months after treatment. In addition, fecal samples from RBX2660-treated participants were sequenced to evaluate microbiome composition and functional changes from before to after treatment. RESULTS: In this Phase 2 open-label clinical trial, RBX2660 demonstrated a 78.9% (112/142) treatment success rate compared to a 30.7% (23/75) for the historical control group (p < 0.0001; Chi-square test). Post-hoc analysis indicated that 91% (88/97) of evaluable RBX2660 responders remained CDI occurrence-free to 24 months after treatment demonstrating durability. RBX2660 was well-tolerated with mostly mild to moderate adverse events. The composition and diversity of RBX2660 responders' fecal microbiome significantly changed from before to after treatment to become more similar to RBX2660, and these changes were durable to 24 months after treatment. CONCLUSIONS: In this Phase 2 trial, RBX2660 was safe and effective for reducing rCDI recurrence as compared to a historical control group. Microbiome changes are consistent with restorative changes implicated in resisting C. difficile recurrence. Clinical Trials Registration NCT02589847 (10/28/2015).

Gut microbiota and microbiota-based therapies for Clostridioides difficile infection.

Clostridioides difficile infection poses significant clinical challenges due to its recurrent nature. Current antibiotic management does not address the underlying issue, that of a disturbed gastrointestinal microbiome, called dysbiosis. This provides a supportive environment for the germination of C. difficile spores which lead to infection and toxin production as well as an array of other health conditions. The use of microbiome restoration therapies such as live biotherapeutics can reverse dysbiosis and lead to good clinical outcomes. Several such therapies are under clinical investigation.

Quantitative analysis and virulence phenotypes of atypical enteropathogenic Escherichia coli (EPEC) acquired from diarrheal stool samples from a Midwest US hospital.

Infectious diarrhea causes approximately 179 million illnesses annually in the US. Multiplex PCR assays for enteric pathogens detect enteropathogenic Escherichia coli (EPEC) in 12-29% of diarrheal stool samples from all age groups in developed nations. The aim of this study was to isolate and characterize EPEC from diarrhea samples identified as EPEC positive by BioFire Gastrointestinal Panel (GIP). EPEC is the second most common GIP-detected pathogen, equally present in sole and mixed infections peaking during summer months. EPEC bacterial load is higher in samples with additional pathogens. EPEC-GIP-positive stool samples were cultured on MacConkey II agar and analyzed by colony PCR for eaeA and bfpA to identify and classify EPEC isolates as typical (tEPEC) or atypical (aEPEC). EPEC were not recovered from the majority of stool samples with only 61 isolates obtained from 277 samples; most were aEPEC from adults. bfpA-mRNA was severely diminished in 3 of 4 bfpA-positive isolates. HeLa and SKCO-15 epithelial cells were infected with EPEC isolates and virulence-associated phenotypes, including adherence pattern, attachment level, pedestal formation, and tight junction disruption, were assessed. All aEPEC adherence patterns were represented with diffuse adherence predominating. Attachment rates of isolates adhering with defined adherence patterns were higher than tEPEC lacking bfpA (ΔbfpA). The majority of isolates formpedestals. All but one isolate initially increases but ultimately decreases transepithelial electrical resistance of SKCO-15 monolayers, similar to ΔbfpA. Most isolates severely disrupt occludin; ZO-1 disruption is variable. Most aEPEC isolates induce more robust virulence-phenotypes in vitro than ΔbfpA, but less than tEPEC-E2348/69.

The Efficacy and Safety of Fecal Microbiota Transplant for Recurrent Clostridium difficile Infection: Current Understanding and Gap Analysis.

The leading risk factor for Clostridioides (Clostridium) difficile infection (CDI) is broad-spectrum antibiotics, which lead to low microbial diversity, or dysbiosis. Current therapeutic strategies for CDI are insufficient, as they do not address the key role of the microbiome in preventing C. difficile spore germination into toxin-producing vegetative bacteria, which leads to symptomatic disease. Fecal microbiota transplant (FMT) appears to reduce the risk of recurrent CDI through microbiome restoration. However, a wide range of efficacy rates have been reported, and few placebo-controlled trials have been conducted, limiting our understanding of FMT efficacy and safety. We discuss the current knowledge gaps driven by questions around the quality and consistency of clinical trial results, patient selection, diagnostic methodologies, use of suppressive antibiotic therapy, and methods for adverse event reporting. We provide specific recommendations for future trial designs of FMT to provide improved quality of the clinical evidence to better inform treatment guidelines.

Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell-Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells.

Enteropathogenic Escherichia coli (EPEC) uses a type three secretion system to inject effector proteins into host intestinal epithelial cells, causing diarrhea. EPEC induces the formation of pedestals underlying attached bacteria, disrupts tight junction (TJ) structure and function, and alters apico-basal polarity by redistributing the polarity proteins Crb3 and Pals1, although the mechanisms are unknown. Here we investigate the temporal relationship of PAR polarity complex and TJ disruption following EPEC infection. EPEC recruits active aPKCζ, a PAR polarity protein, to actin within pedestals and at the plasma membrane prior to disrupting TJ. The EPEC effector EspF binds the endocytic protein sorting nexin 9 (SNX9). This interaction impacts actin pedestal organization, recruitment of active aPKCζ to actin at cell-cell borders, endocytosis of JAM-A S285 and occludin, and TJ barrier function. Collectively, data presented herein support the hypothesis that EPEC-induced perturbation of TJ is a downstream effect of disruption of the PAR complex and that EspF binding to SNX9 contributes to this phenotype. aPKCζ phosphorylates polarity and TJ proteins and participates in actin dynamics. Therefore, the early recruitment of aPKCζ to EPEC pedestals and increased interaction with actin at the membrane may destabilize polarity complexes ultimately resulting in perturbation of TJ.

Sperm Flagellar 1 Binds Actin in Intestinal Epithelial Cells and Contributes to Formation of Filopodia and Lamellipodia.

BACKGROUND & AIMS: Sperm flagellar 1 (also called CLAMP) is a microtubule-associated protein that regulates microtubule dynamics and planar cell polarity in multi-ciliated cells. We investigated the localization and function of sperm flagellar 1, or CLAMP, in human intestinal epithelia cells (IECs). METHODS: We performed studies with SKCO-15 and human intestinal enteroids established from biopsies from different intestinal segments (duodenal, jejunum, ileal, and colon) of a single donor. Enteroids were induced to differentiation after incubation with growth factors. The distribution of endogenous CLAMP in IECs was analyzed by immunofluorescence microscopy using total internal reflection fluorescence-ground state depletion and confocal microscopy. CLAMP localization was followed during the course of intestinal epithelial cell polarization as cells progressed from flat to compact, confluent monolayers. Protein interactions with endogenous CLAMP were determined in SKCO-15 cells using proximity ligation assays and co-immunoprecipitation. CLAMP was knocked down in SKCO-15 monolayers using small hairpin RNAs and cells were analyzed by immunoblot and immunofluorescence microscopy. The impact of CLAMP knock-down in migrating SKCO-15 cells was assessed using scratch-wound assays. RESULTS: CLAMP bound to actin and apical junctional complex proteins but not microtubules in IECs. In silico analysis predicted the calponin-homology domain of CLAMP to contain conserved amino acids required for actin binding. During IEC polarization, CLAMP distribution changed from primarily basal stress fibers and cytoplasm in undifferentiated cells to apical membranes and microvilli in differentiated monolayers. CLAMP accumulated in lamellipodia and filopodia at the leading edge of migrating cells in association with actin. CLAMP knock-down reduced the number of filopodia, perturbed filopodia polarity, and altered the organization of actin filaments within lamellipodia. CONCLUSIONS: CLAMP is an actin-binding protein, rather than a microtubule-binding protein, in IECs. CLAMP distribution changes during intestinal epithelial cell polarization, regulates the formation of filopodia, and appears to assist in the organization of actin bundles within lamellipodia of migrating IECs. Studies are needed to define the CLAMP domains that interact with actin and whether its loss from IECs affects intestinal function.

Results From a Randomized, Placebo-Controlled Clinical Trial of a RBX2660-A Microbiota-Based Drug for the Prevention of Recurrent Clostridium difficile Infection.

Background: Despite advancements, recurrent Clostridium difficile infections (CDI) remain an urgent public health threat with insufficient response rates to currently approved antibiotic therapies. Microbiota-based treatments appear effective, but rigorous clinical trials are required to optimize dosing strategies and substantiate long-term safety. Methods: This randomized, double-blind, placebo-controlled phase 2B trial enrolled adults with 2 or more CDI recurrences to receive: 2 doses of RBX2660, a standardized microbiota-based drug (group A); 2doses of placebo (group B); or 1 dose of RBX2660 followed by 1 dose of placebo (group C). Efficacy was defined as prevention of recurrent CDI for 8 weeks following treatment. Participants who had a recurrence within 8 weeks were eligible to receive up to 2 open-label RBX2660 doses. The primary endpoint was efficacy for group A compared to group B. Secondary endpoints included the efficacy of group C compared to group B, combined efficacy in the blinded and open-label phases, and safety for 24 months. Results: The efficacy for groups A, B, and C were 61%, 45%, and 67%, respectively. The primary endpoint was not met (P = .152). One RBX2660 dose (group C) was superior to placebo (group B; P = .048), and the overall efficacy (including open-label response) for RBX2660-treated participants was 88.8%. Adverse events did not differ significantly among treatment groups. Conclusions: One, but not 2, doses of RBX2660 was superior to placebo in this randomized, placebo-controlled trial. These data provide important insights for a larger phase 3 trial and continued clinical development of RBX2660. Clinical Trials Registration: NCT02299570.

EPEC NleH1 is significantly more effective in reversing colitis and reducing mortality than NleH2 via differential effects on host signaling pathways.

Enteropathogenic Escherichia coli (EPEC) is a foodborne pathogen that uses a type III secretion system to translocate effector molecules into host intestinal epithelial cells (IECs) subverting several host cell processes and signaling cascades. Interestingly, EPEC infection induces only modest intestinal inflammation in the host. The homologous EPEC effector proteins, NleH1 and NleH2, suppress the nuclear factor-κB (NF-κB) pathway and apoptosis in vitro. Increased apoptosis and activation of NF-κB and MAP kinases (MAPK) contribute to the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to determine if NleH1 and NleH2 also block MAPK pathways in vitro and in vivo, and to compare the effects of these bacterial proteins on a murine model of colitis. Cultured IECs were infected with various strains of EPEC expressing NleH1 and NleH2, or not, and the activation of ERK1/2 and p38 was determined. In addition, the impact of infection with various strains of EPEC on murine DSS colitis was assessed by change in body weight, colon length, histology, and survival. Activation of apoptosis and MAPK signaling were also evaluated. Our data show that NleH1, but not NleH2, suppresses ERK1/2 and p38 activation in vitro. Interestingly, NleH1 affords significantly greater protection against and hastens recovery from dextran sodium sulfate (DSS)-induced colitis compared to NleH2. Strikingly, colitis-associated mortality was abolished by infection with EPEC strains expressing NleH1. Interestingly, in vivo NleH1 suppresses activation of ERK1/2 and p38 and blocks apoptosis independent of the kinase domain that inhibits NF-κB. In contrast, NleH2 suppresses only caspase-3 and p38, but not ERK1/2. We conclude that NleH1 affords greater protection against and improves recovery from DSS colitis compared to NleH2 due to its ability to suppress ERK1/2 in addition to NF-κB, p38, and apoptosis. These findings warrant further investigation of anti-inflammatory bacterial proteins as novel treatments for IBD.

Modulation of epithelial cell polarity by bacterial pathogens.

Epithelial cells constitute a physical barrier that aids in protecting the host from microbial pathogens. Polarized epithelial cells contain distinct apical and basolateral membrane domains separated by intercellular junctions, including tight junctions (TJs), which contribute to the maintenance of apical-basal polarity. Polarity complexes also contribute to the establishment of TJ formation. Several pathogens perturb epithelial TJ barrier function and structure in addition to causing a loss of apical-basal polarity. Here, we review the impact of pathogenic bacteria on the disruption of cell-cell junctions and epithelial polarity.

EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity.

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins ß1-integrin and Na+ /K+ ATPase to the apical membrane of host cells. The Crumbs (Crb) polarity complex (Crb3/Pals1/Patj) is essential for epithelial cell polarisation and tight junction (TJ) assembly. Here, we demonstrate that EPEC displaces Crb3 and Pals1 from the apical membrane to the cytoplasm of cultured intestinal epithelial cells and colonocytes of infected mice. In vitro studies show that EspF, but not Map, alters Crb3, whereas both effectors modulate Pals1. EspF perturbs polarity formation in cyst morphogenesis assays and induces endocytosis and apical redistribution of Na+ /K+ ATPase. EspF binds to sorting nexin 9 (SNX9) causing membrane remodelling in host cells. Infection with ΔespF/pespFD3, a mutant strain that ablates EspF binding to SNX9, or inhibition of dynamin, attenuates Crb3 endocytosis caused by EPEC. In addition, infection with ΔespF/pespFD3 has no impact on Na+ /K+ ATPase endocytosis. These data support the hypothesis that EPEC perturbs apical-basal polarity in an EspF-dependent manner, which would contribute to EPEC-associated diarrhoea by disruption of TJ and altering the crucial positioning of membrane transporters involved in the absorption of ions and solutes.

Role of SHP2 protein tyrosine phosphatase in SERT inhibition by enteropathogenic E. coli (EPEC).

Enteropathogenic Escherichia coli (EPEC), one of the diarrheagenic E. coli pathotypes, is among the most important food-borne pathogens infecting children worldwide. Inhibition of serotonin transporter (SERT), which regulates extracellular availability of serotonin (5-HT), has been implicated previously in EPEC-associated diarrhea. EPEC was shown to inhibit SERT via activation of protein tyrosine phosphatase (PTPase), albeit the specific PTPase involved is not known. Current studies aimed to identify EPEC-activated PTPase and its role in SERT inhibition. Infection of Caco-2 monolayers with EPEC strain E2348/69 for 30 min increased the activity of Src-homology-2 domain containing PTPase (SHP2) but not SHP1 or PTPase 1B. Similarly, Western blot studies showed increased tyrosine phosphorylation of (p-tyrosine) SHP2, indicative of its activation. Concomitantly, EPEC infection decreased SERT p-tyrosine levels. This was associated with increased interaction of SHP2 with SERT, as evidenced by coimmunoprecipitation studies. To examine whether SHP2 directly influences SERT phosphorylation status or function, SHP2 cDNA plasmid constructs (wild type, constitutively active, or dominant negative) were overexpressed in Caco-2 cells by Amaxa electroporation. In the cells overexpressing constitutively active SHP2, SERT polypeptide showed complete loss of p-tyrosine. In addition, there was a decrease in SERT function, as measured by Na+Cl--sensitive [3H]5-HT uptake, and an increase in association of SERT with SHP2 in Caco-2 cells expressing constitutively active SHP2 compared with dominant-negative SHP2. Our data demonstrate that intestinal SERT is a target of SHP2 and reveal a novel mechanism by which a common food-borne pathogen uses cellular SHP2 to inhibit SERT.NEW & NOTEWORTHY The data presented in the current study reveal that intestinal serotonin transporter (SERT) is a target of the tyrosine phosphatase SHP2 and show a novel mechanism by which a common diarrheagenic pathogen, EPEC, activates cellular SHP2 to inhibit SERT function. These studies highlight host-pathogen interactions, which may be of therapeutic relevance in the management of diarrhea associated with enteric infections.

Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli.

Enteropathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl(-)/HCO3(-) exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl(-)/HCO3(-) exchange activity was measured as DIDS-sensitive (125)I(-) uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 µM) increased apical Cl(-)/HCO3(-) exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis.