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Chronic liver disease is rising in western countries and liver cirrhosis is the 12th leading cause of death worldwide. Simultaneously, use of gastric acid suppressive medications is increasing. Here, we show that proton pump inhibitors promote progression of alcoholic liver disease, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis in mice by increasing numbers of intestinal Enterococcus spp. Translocating enterococci lead to hepatic inflammation and hepatocyte death. Expansion of intestinal Enterococcus faecalis is sufficient to exacerbate ethanol-induced liver disease in mice. Proton pump inhibitor use increases the risk of developing alcoholic liver disease among alcohol-dependent patients. Reduction of gastric acid secretion therefore appears to promote overgrowth of intestinal Enterococcus, which promotes liver disease, based on data from mouse models and humans. Recent increases in the use of gastric acid-suppressive medications might contribute to the increasing incidence of chronic liver disease.

Background: Whether human immunodeficiency virus (HIV) infection is associated with a change in the diversity of genital microbiota in women was investigated.Methods: Amplicon length heterogeneity polymerase chain reaction (LH-PCR) analysis and pyrosequencing of the 16S ribosomal RNA gene were used to analyze the diversity of the microbiota in HIV-positive (HIV(+)) and HIV-negative (HIV(-)) women with or without bacterial vaginosis (BV).Results: LH-PCR analysis revealed significantly more microbiota diversity in BV-positive (BV(+)) women than in BV-negative (BV(-)) women, but no significant difference was noted between HIV(+) women and HIV(-) women. Pyrosequencing revealed that Lactobacillus organisms constituted a median of 96% of the bacteria in BV(-) women. BV(+) women had a significantly higher number of taxa found at > or =1% of the total genital microbiota (median, 11 taxa). Common taxa in BV(+) women were Prevotella, Megasphaera, Gardnerella, Coriobacterineae, Lachnospira, and Sneathia. There was a trend (P = .07) toward the presence of a higher number of taxa in HIV(+)BV(+) women than in HIV(-)BV(+) women. Propionibacterineae, Citrobacter, and Anaerococcus were the taxa found only in HIV(+) women (P < .05).Conclusions: The present study demonstrated that both LH-PCR analysis and pyrosequencing differentiated microbiota in BV(+) women from that in BV(-) women and that pyrosequencing indicated a trend toward increased diversity in BV(+)HIV(+) women, suggesting that HIV infection is associated with changes in the diversity of genital microbiota. [ABSTRACT FROM AUTHOR]

Background: Tumor heterogeneity is a main contributor of resistance to anti-cancer targeted agents though it has proven difficult to study. Unfortunately, model systems to functionally characterize and mechanistically study dynamic responses to treatment across coexisting subpopulations of cancer cells remain a missing need in oncology., Methods: Using single cell cloning and expansion techniques, we established monoclonal cell subpopulations (MCPs) from a commercially available epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer cell line. We then used this model sensitivity to the EGFR inhibitor osimertinib across coexisting cell populations within the same tumor. Pathway-centered signaling dynamics associated with response to treatment and morphological characteristics of the MCPs were assessed using Reverse Phase Protein Microarray. Signaling nodes differentially activated in MCPs less sensitive to treatment were then pharmacologically inhibited to identify target signaling proteins putatively implicated in promoting drug resistance., Results: MCPs demonstrated highly heterogeneous sensitivities to osimertinib. Cell viability after treatment increased > 20% compared to the parental line in selected MCPs, whereas viability decreased by 75% in other MCPs. Reduced treatment response was detected in MCPs with higher proliferation rates, EGFR L858R expression, activation of EGFR binding partners and downstream signaling molecules, and expression of epithelial-to-mesenchymal transition markers. Levels of activation of EGFR binding partners and MCPs' proliferation rates were also associated with response to c-MET and IGFR inhibitors., Conclusions: MCPs represent a suitable model system to characterize heterogeneous biomolecular behaviors in preclinical studies and identify and functionally test biological mechanisms associated with resistance to targeted therapeutics., (© 2024. The Author(s).)

Introduction: Diet can affect ammoniagenesis in cirrhosis and hepatic encephalopathy (HE), but the impact of dietary preferences on metabolomics in cirrhosis is unclear. As most Western populations follow meat-based diets, we aimed to determine the impact of substituting a single meat-based meal with an equal protein-containing vegan/vegetarian alternative on ammonia and metabolomics in outpatients with cirrhosis on a meat-based diet., Methods: Outpatients with cirrhosis with and without prior HE on a stable Western meat-based diet were randomized 1:1:1 into 3 groups. Patients were given a burger with 20 g protein of meat, vegan, or vegetarian. Blood for metabolomics via liquid chromatography-mass spectrometry and ammonia was drawn at baseline and hourly for 3 hours after meal while patients under observation. Stool microbiome characteristics, changes in ammonia, and metabolomics were compared between/within groups., Results: Stool microbiome composition was similar at baseline. Serum ammonia increased from baseline in the meat group but not the vegetarian or vegan group. Metabolites of branched chain and acylcarnitines decreased in the meat group compared with the non-meat groups. Alterations in lipid profile (higher sphingomyelins and lower lysophospholipids) were noted in the meat group when compared with the vegan and vegetarian groups., Discussion: Substitution of a single meat-based meal with a non-meat alternatives results in lower ammoniagenesis and altered serum metabolomics centered on branched-chain amino acids, acylcarnitines, lysophospholipids, and sphingomyelins in patients with cirrhosis regardless of HE or stool microbiome. Intermittent meat substitution with vegan or vegetarian alternatives could be helpful in reducing ammonia generation in cirrhosis., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Background: Minimal hepatic encephalopathy (MHE) negatively affects the prognosis of cirrhosis, but treatment is not standard. Rifamycin SV MMX (RiVM) is a nonabsorbable rifampin derivative with colonic action., Methods: In a phase 2 placebo-controlled, double-blind randomized clinical trial patients with MHE were randomized to RiVM or placebo for 30 days with a 7-day follow-up. The primary endpoint was a change in stool cirrhosis dysbiosis ratio. Gut-brain (cognition, stool/salivary microbiome, ammonia, brain magnetic resonance spectroscopy), inflammation (stool calprotectin/serum cytokines), patient-reported outcomes (sickness impact profile: total/physical/psychosocial, high = worse), and sarcopenia (handgrip, bioelectric impedance) were secondary. Between/within groups and delta (post-pre) comparisons were performed., Results: Thirty patients (15/group) were randomized and completed the study without safety concerns. While cirrhosis dysbiosis ratio was statistically similar on repeated measures ANOVA (95% CI: -0.70 to 3.5), ammonia significantly reduced (95% CI: 4.4-29.6) in RiVM with changes in stool microbial α/β-diversity. MHE status was unchanged but only serial dotting (which tests motor strength) improved in RiVM-assigned patients. Delta physical sickness impact profile (95% CI: 0.33 = 8.5), lean mass (95% CI: -3.3 to -0.9), and handgrip strength (95% CI: -8.1 to -1.0) improved in RiVM versus placebo. Stool short-chain fatty acids (propionate, acetate, and butyrate) increased post-RiVM. Serum, urine, and stool bile acid profile changed to nontoxic bile acids (higher hyocholate/ursodeoxycholate and lower deoxycholate/lithocholate) post-RiVM. Serum IL-1β and stool calprotectin decreased while brain magnetic resonance spectroscopy showed higher glutathione concentrations in RiVM., Conclusions: RiVM is well tolerated in patients with MHE with changes in stool microbial composition and function, ammonia, inflammation, brain oxidative stress, and sarcopenia-related parameters without improvement in cognition. RiVM modulates the gut-brain axis and gut-muscle axis in cirrhosis., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Introduction: Proton pump inhibitors (PPIs) modulate the progression of cirrhosis to hepatic encephalopathy (HE) and can affect the bacterial microbiome. However, the impact of PPI on the virome in cirrhosis using viral-like particle (VLP) analysis is unclear., Methods: We determined the VLP in the stool microbiome in patients with cirrhosis cross-sectionally (ascites, HE, and PPI use analyzed) who were followed up for 6-month hospitalizations and through 2 clinical trials of PPI withdrawal and initiation., Results: In a cross-sectional study, PPI users had greater ascites prevalence and 6-month hospitalizations, but VLP α diversity was similar. Among phages, PPI users had lower Autographviridae and higher Streptococcus phages and Herelleviridae than nonusers, whereas opposite trends were seen in ascites and HE. Trends of eukaryotic viruses (higher Adenoviridae and lower Virgaviridae/Smacoviridae) were similar for PPI, HE, and ascites. Twenty-one percent were hospitalized, mostly due to HE. α Diversity was similar in the hospitalized/nonhospitalized/not groups. Higher Gokushovirinae and lower crAssphages were related to hospitalizations such as HE-related cross-sectional VLP changes. As part of the clinical trial, PPIs were added and withdrawn in 2 different decompensated groups over 14 days. No changes in α diversity were observed. Withdrawal reduced crAssphages, and initiation reduced Gokushovirinae and Bacteroides phages., Discussion: In cirrhosis, PPI use has a gut microbial VLP phage signature that is different from that in HE and ascites, and VLP changes are linked with hospitalizations over 6 months, independent of clinical biomarkers. Eukaryotic viral patterns were consistent across PPI use, HE, and ascites, indicating a relationship with the progression of cirrhosis. PPIs alone showed modest VLP changes with withdrawal or initiation. Distinct phage and eukaryotic viral patterns are associated with the use of PPIs in cirrhosis., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Patients with cirrhosis who have cognitive complaints are presumed to have hepatic encephalopathy (HE), which leads to unwarranted medications while ignoring the underlying disease process causing these complaints. Since neuropsychological testing, the current gold standard for HE diagnosis, is not readily available, an orderable test is needed. We aimed to develop and validate a rapid gut microbiota test to exclude HE and determine stakeholder input on this approach. Stool was collected from two cohorts: a two-center training cohort ( n  = 305, on/not on HE-related therapy) and a multicenter validation cohort ( n  = 30, on HE treatment). Stool microbiota was analyzed rapidly using nanopore analysis. Stakeholder (patients and clinicians) needs assessment was evaluated using semi-quantitative questionnaires. In the training cohort, machine learning using neural network identified a 20-species signature that differentiated HE vs no-HE with 84% specificity compared to the gold standard neuropsychological testing. This high specificity persisted regardless of whether patients were on HE-related therapy or not. In the validation cohort, application of this profile led to reevaluation of the HE diagnosis and treatment in > 40% of the patients. This approach was acceptable to patients (Veterans in the validation cohort) and clinician ( n  = 40 nationwide) stakeholders. We conclude that a machine learning stool signature based on 20 microbial species developed in a training set and validated in a separate multicenter prospective cohort differentiated those with vs. without HE, identified patients misdiagnosed with HE, and was acceptable to patients and clinician stakeholders.

Cognitive dysfunction due to minimal hepatic encephalopathy (MHE) adversely impacts patients with cirrhosis and more precise therapies are needed. Gut-brain axis changes are therapeutic targets, but prior studies have largely focused on bacterial changes. Our aim was to determine linkages between individual cognitive testing results and bacteria with the virome using a cross-sectional and longitudinal approach. We included cross-sectional ( n  = 138) and longitudinal analyses ( n  = 36) of patients with cirrhosis tested using three cognitive modalities, which were psychometric hepatic encephalopathy score (PHES), inhibitory control test (ICT), Stroop, and all three. Stool metagenomics with virome and bacteriome were analyzed studied cross-sectionally and in a subset followed for development/reversal of MHE repeated at 6 months (longitudinally only using PHES). Cross-sectional: We found no significant changes in α/β diversity in viruses or bacteria regardless of cognitive testing. Cognitively impaired patients were more likely to have higher relative abundance of bacteriophages linked with Streptococcus , Faecalibacterium , and Lactobacillus , which were distinct based on modality. These were also linked with cognition on correlation networks. Longitudinally, 27 patients remained stable while 9 changed their MHE status. Similar changes in phages that are linked with Streptococcus , Faecalibacterium , and Lactobacillus were seen. These phages can influence ammonia, lactate, and short-chain fatty acid generation, which are neuro-active. In conclusion, we found linkages between bacteriophages and cognitive function likely due to impact on bacteria that produce neuroactive metabolites cross-sectionally and longitudinally. These findings could help explore bacteriophages as options to influence treatment for MHE in cirrhosis.

Objective: First decompensation development is a critical milestone that needs to be predicted. Transkingdom gut microbial interactions, including archaeal methanogens, may be important targets and predictors but a longitudinal approach is needed., Design: Cirrhosis outpatients who provided stool twice were included. Group 1: compensated, group 2: 1 decompensation (decomp), group 3: >1 decompensationwere followed and divided into those who remained stable or decompensated. Bacteria, viral and archaeal presence, α/β diversity and taxa changes over time adjusted for clinical variables were analysed. Correlation networks between kingdoms were analysed., Results: 157 outpatients (72 group 1, 33 group 2 and 52 group 3) were followed and 28%-47% developed outcomes. Baseline between those who remained stable/developed outcome: While no α/β diversity differences were seen, commensals were lower and pathobionts were higher in those who decompensated. After decompensation: those experiencing their first decompensation showed greater decrease in α/β-diversity, bacterial change (↑ Lactobacillus spp, Streptococcus parasanguinis and ↓ beneficial Lachnospiraceae and Eubacterium hallii ) and viral change (↑Siphoviridae, ↓ Myoviridae) versus those with further decompensation. Archaea: 19% had Methanobacter brevii, which was similar between/within groups. Correlation networks: Baseline archaeal-viral-bacterial networks were denser and more homogeneous in those who decompensated versus the rest. Archaea-bacterial correlations collapsed post first decompensation. Lactobacillus phage Lc Nu and C2-like viruses were negatively linked with beneficial bacteria., Conclusion: In this longitudinal study of cirrhosis outpatients, the greatest transkingdom gut microbial changes were seen in those reaching the first decompensation, compared with subsequent decompensating events. A transkingdom approach may refine prediction and provide therapeutic targets to prevent cirrhosis progression., Competing Interests: Competing interests: Dr Bajaj’s institution received an investigator-initiated grant from BauschZH, WP, ALR and TW are employees of DiversigenNo other competing interests., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Background and Aims: Gut microbiota, including bacteria and phages, are altered in cirrhosis, but their role during infections and spontaneous bacterial peritonitis (SBP) prophylaxis is unclear. Our aim was determine metagenomic changes in gut bacteria; phages and their linkages centered around Gram-negative and Gram-positive pathobionts in patients with cirrhosis with/without infections or SBP prophylaxis., Approach and Results: We included uninfected (n = 231) and infected (n = 30, SBP n = 19 and urinary tract infection n = 11 before antibiotics) patients who gave stool for bacterial and phage metagenomics. We matched uninfected to infected patients 1:1 on a model for end-stage liver disease (MELD). We also analyzed subgroups of patients with ascites matched on an MELD (n = 73) to patients on SBP prophylaxis (n = 7) and then to SBP infection. Phage and bacterial taxa differences (DESeq2) and correlation networks centered around Escherichia coli and Enterococcus faecium were analyzed. Infections were mostly due to Enterobacteriaceae and Enterococcus spp. On metagenomics, higher fold changes of Enterobacteriaceae members, Enterococcus and Streptococcus spp., and Escherichia phages were seen in infected patients. Correlation networks showed more complex bacteria-phage linkages in infected patients compared with uninfected ones overall and centered around E. coli and E. faecium. SBP prophylaxis induced higher Gram-positive bacteria. In SBP, Enterococcus and Escherichia were higher versus ascites. Correlation networks around E. coli were complex in ascites but sparse with SBP prophylaxis, whereas the reverse was seen with E. faecium. Lytic phages and those associated with antibiotic resistance were correlated with E. faecium., Conclusion: In cirrhosis, there are significant changes in phage-bacterial linkages in infected patients and those on SBP prophylaxis compared to the remaining patients. SBP prophylaxis enriches complexity of E. faecium-centered but induces a collapse in E. coli-centered phage-bacterial correlations., (© 2022 American Association for the Study of Liver Diseases.)

Cirrhosis is complicated by a high rate of nosocomial infections (NIs), which result in poor outcomes and are challenging to predict using clinical variables alone. Our aim was to determine predictors of NI using admission serum metabolomics and gut microbiota in inpatients with cirrhosis. In this multicenter inpatient cirrhosis study, serum was collected on admission for liquid chromatography-mass spectrometry metabolomics, and a subset provided stool for 16SrRNA analysis. Hospital course, including NI development and death, were analyzed. Metabolomic analysis using analysis of covariance (ANCOVA) (demographics, Model for End-Stage Liver Disease [MELD] admission score, white blood count [WBC], rifaximin, and infection status adjusted) and random forest analyses for NI development were performed. Additional values of serum metabolites over clinical variables toward NI were evaluated using logistic regression. Stool microbiota and metabolomic correlations were compared in patients with and without NI development. A total of 602 patients (231 infection admissions) were included; 101 (17%) developed NIs, which resulted in worse inpatient outcomes, including intensive care unit transfer, organ failure, and death. A total of 127 patients also gave stool samples, and 20 of these patients developed NIs. The most common NIs were spontaneous bacterial peritonitis followed by urinary tract infection, Clostridioides difficile, and pneumonia. A total of 247 metabolites were significantly altered on ANCOVA. Higher MELD scores (odds ratio, 1.05; p < 0.0001), admission infection (odds ratio, 3.54; p < 0.0001), and admission WBC (odds ratio, 1.05; p = 0.04) predicted NI (area under the curve, 0.74), which increased to 0.77 (p = 0.05) with lower 1-linolenoyl-glycerolphosphocholine (GPC) and 1-stearoyl-GPC and higher N-acetyltryptophan and N-acetyl isoputreanine. Commensal microbiota were lower and pathobionts were higher in those who developed NIs. Microbial-metabolite correlation networks were complex and dense in patients with NIs, especially sub-networks centered on Ruminococcaceae and Pseudomonadaceae. NIs are common and associated with poor outcomes in cirrhosis. Admission gut microbiota in patients with NIs showed higher pathobionts and lower commensal microbiota. Microbial-metabolomic correlations were more complex, dense, and homogeneous among those who developed NIs, indicating greater linkage strength. Serum metabolites and gut microbiota on admission are associated with NI development in cirrhosis., (© 2022 American Association for the Study of Liver Diseases.)