Substitution of One Meat-Based Meal With Vegetarian and Vegan Alternatives Generates Lower Ammonia and Alters Metabolites in Cirrhosis: A Randomized Clinical Trial.

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.

The RIVET RCT: Rifamycin SV MMX improves muscle mass, physical function, and ammonia in cirrhosis and minimal encephalopathy.

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/ß-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-1ß 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.

Proton Pump Inhibitor Use and Complications of Cirrhosis Are Linked With Distinct Gut Microbial Bacteriophage and Eukaryotic Viral-Like Particle Signatures in Cirrhosis.

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.

Detection of SARS-CoV-2 RNA in wastewater from dormitory buildings in a university campus: comparison with individual testing results.

Wastewater-based epidemiology (WBE) for monitoring COVID-19 has been largely used to detect the spread of the disease at the community level. From February to December 2022, we collected 24-h composite sewage samples from dormitory buildings in George Mason University (Fairfax, Virginia, USA) housing approximately 5,200 resident students. SARS-CoV-2 RNA extraction was achieved using an automated system based on magnetic nanoparticles. Analysis of SARS-CoV-2 RNA was performed using reverse transcription quantitative PCR based on the Centers for Disease Control and Prevention (CDC) N1 and N2 assays. From the 362 samples collected, 86% showed positive detection of SARS-CoV-2 RNA. Wastewater monitoring was able to detect SARS-CoV-2 RNA in 96% of the samples from buildings housing students with COVID-19. Over the period of study, we observed significant correlations between the SARS-CoV-2 concentration (copy number mL-1) in wastewater and the number of positive cases on campus based on individual saliva testing. Although several reports have been published on the wastewater monitoring of COVID-19 in university campuses, our study is one of the very few that provides results that were obtained during the last phase of the pandemic (roughly the year 2022), when the large majority of students were vaccinated and back on campus.

Alterations in gut virome are associated with cognitive function and minimal hepatic encephalopathy cross-sectionally and longitudinally in cirrhosis.

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/ß 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.

Longitudinal transkingdom gut microbial approach towards decompensation in outpatients with 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/ß 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/ß 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/ß-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.

Genetic variation of Borreliella burgdorferi in Fairfax County, Virginia, targeting the OspC gene in white-footed mice.

Outer surface protein C (OspC) is a commonly used marker in population studies of Borreliella to differentiate types and establish evolution over time. Investigating the ospC genetic types of Borreliella burgdorferi across multiple organ tissues of white-footed mice has the potential to contribute to our understanding of Lyme disease and the wide spectrum of clinical presentation associated with infection. In this study, five unique tissue types were sampled from 90 mice and screened for B. burgdorferi infections. This initial screening revealed a 63% overall B. burgdorferi infection rate in the mice collected (57/90). A total of 163 tissues (30.4%) tested positive for B. burgdorferi infections and when mapped to Borreliella types, 143,894 of the initial 322,480 reads mapped to 10 of the reference sequences in the ospC strain library constructed for this study at a 97% MOI. Two tissue types, the ear and the tongue, each accounted for 90% of the observed Borreliella sequence diversity in the tissue samples surveyed. The largest amount of variation was observed in an individual ear tissue sample with six ospC sequence types, which is equivalent to 60% of the observed variation seen across all tested specimens, with statistically significant associations observed between tissue type and detected Borreliella. There is strong evidence for genetic variability in B. burgdorferi within local white-footed mouse populations and even within individual hosts by tissue type. These findings may shed light on drivers of infection sequalae in specific tissues in humans and highlights the need for expanded surveillance on the epigenetics of B. burgdorferi across reservoirs, ticks, and infected patients.

Reduced alcohol preference and intake after fecal transplant in patients with alcohol use disorder is transmissible to germ-free mice.

Alcohol use disorder is a major cause of morbidity, which requires newer treatment approaches. We previously showed in a randomized clinical trial that alcohol craving and consumption reduces after fecal transplantation. Here, to determine if this could be transmitted through microbial transfer, germ-free male C57BL/6 mice received stool or sterile supernatants collected from the trial participants pre-/post-fecal transplant. We found that mice colonized with post-fecal transplant stool but not supernatants reduced ethanol acceptance, intake and preference versus pre-fecal transplant colonized mice. Microbial taxa that were higher in post-fecal transplant humans were also associated with lower murine alcohol intake and preference. A majority of the differentially expressed genes (immune response, inflammation, oxidative stress response, and epithelial cell proliferation) occurred in the intestine rather than the liver and prefrontal cortex. These findings suggest a potential for therapeutically targeting gut microbiota and the microbial-intestinal interface to alter gut-liver-brain axis and reduce alcohol consumption in humans.

Association of serum metabolites and gut microbiota at hospital admission with nosocomial infection development in patients with cirrhosis.

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.

Pathogenic microbe detection in placental tissues supports placental pathobiome association with preterm birth risk in Pakistani women: A brief snapshot.

Preterm birth (PTB) poses great risk to neonatal health in Pakistan with few tertiary health care facilities. Role of intrauterine microbiome in maintaining healthy pregnancy has been highlighted. However, there is ongoing debate whether a true placental microbiome exist. We analyzed placental and vaginal microbiome through V3-V4 16srRNA sequencing and observed increased abundance of proteobacteria, with concomitant decline in the firmicutes population in preterm vagina. Simplistic placental microflora included many environmental microbes with PTB placenta carrying pathogenic microbes like ureaplasma and mycoplasma species. We observed contribution of environmental, vaginal and skin contamination in term versus pathobiome signatures in preterm placenta.

Area Deprivation Index and Gut-Brain Axis in Cirrhosis.

INTRODUCTION: Neighborhood deprivation has been associated with chronic diseases and with gut microbial alterations. Although cirrhosis is associated with gut microbiome changes and hepatic encephalopathy (HE), their association is unclear. METHODS: Demographics and cirrhosis details (model for end-stage liver disease [MELD], prior HE, and medications) were recorded from outpatients with cirrhosis. Area deprivation index (ADI), which ranks neighborhoods by socioeconomic disadvantage, was recorded as state decile and national percentile (high = worse for both) and dichotomized on the median. Patients underwent cognitive testing to diagnose minimal HE (MHE). Stool microbiota was analyzed using 16S ribosomal RNA for α/ß-diversity. Multivariable analysis was used to evaluate the factors independently associated with MHE. RESULTS: A total of 321 people with cirrhosis (60 years, 78% men, 75% non-Hispanic White, 24% non-Hispanic African American, 4% Hispanic) were included. 45% had prior HE and 56% MHE. For ADI, the national percentile was 49.1 ± 21.8 while the state decile was 6.1 ± 2.3. ADI was not associated with race, ethnicity, MELD, or HE-related variables on regression. Regarding microbiota, α-diversity was lower in MHE and prior HE patients but similar across ADI rankings. Low vs high ADIs were associated with different ß-diversity in univariable but not multivariable analyses. Multivariable analyses showed positive associations with MELD, prior HE, and lactate producers ( Lactobacillus and Lacticaseibacillus ) and negative associations with short-chain fatty acid producers ( Blautia , Lachnoclostridium , and Anaerobutyricum ) with MHE. DISCUSSION: Cirrhosis-related variables may be more influential in determining gut microbiome composition and cognitive impairment than ADI. Therefore, the focus should be on improving cirrhosis care, regardless of ADI, but studies evaluating other measures of social determinants are needed in cirrhosis.

Impact of bacterial infections and spontaneous bacterial peritonitis prophylaxis on phage-bacterial dynamics in cirrhosis.

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.

Results of a phase 1, randomized, placebo-controlled first-in-human trial of griffithsin formulated in a carrageenan vaginal gel.

HIV pre-exposure prophylaxis (PrEP) is dominated by clinical therapeutic antiretroviral (ARV) drugs. Griffithsin (GRFT) is a non-ARV lectin with potent anti-HIV activity. GRFT's preclinical safety, lack of systemic absorption after vaginal administration in animal studies, and lack of cross-resistance with existing ARV drugs prompted its development for topical HIV PrEP. We investigated safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of PC-6500 (0.1% GRFT in a carrageenan (CG) gel) in healthy women after vaginal administration. This randomized, placebo-controlled, parallel group, double-blind first-in-human phase 1 study enrolled healthy, HIV-negative, non-pregnant women aged 24-45 years. In the open label period, all participants (n = 7) received single dose of PC-6500. In the randomized period, participants (n = 13) were instructed to self-administer 14 doses of PC-6500 or its matching CG placebo (PC-535) once daily for 14 days. The primary outcomes were safety and PK after single dose, and then after 14 days of dosing. Exploratory outcomes were GRFT concentrations in cervicovaginal fluids, PD, inflammatory mediators and gene expression in ectocervical biopsies. This trial is registered with ClinicalTrials.gov, number NCT02875119. No significant adverse events were recorded in clinical or laboratory results or histopathological evaluations in cervicovaginal mucosa, and no anti-drug (GRFT) antibodies were detected in serum. No cervicovaginal proinflammatory responses and no changes in the ectocervical transcriptome were evident. Decreased levels of proinflammatory chemokines (CXCL8, CCL5 and CCL20) were observed. GRFT was not detected in plasma. GRFT and GRFT/CG in cervicovaginal lavage samples inhibited HIV and HPV, respectively, in vitro in a dose-dependent fashion. These data suggest GRFT formulated in a CG gel is a safe and promising on-demand multipurpose prevention technology product that warrants further investigation.

The Presence of Denitrifiers In Bacterial Communities of Urban Stormwater Best Management Practices (BMPs).

Stormwater best management practices (BMPs) are engineered structures that attempt to mitigate the impacts of stormwater, which can include nitrogen inputs from the surrounding drainage area. The goal of this study was to assess bacterial community composition in different types of stormwater BMP soils to establish whether a particular BMP type harbors more denitrification potential. Soil sampling took place over the summer of 2015 following precipitation events. Soils were sampled from four bioretention facilities, four dry ponds, four surface sand filters, and one dry swale. 16S rRNA gene analysis of extracted DNA and RNA amplicons indicated high bacterial diversity in the soils of all BMP types sampled. An abundance of denitrifiers was also indicated in the extracted DNA using presence/absence of nirS, nirK, and nosZ denitrification genes. BMP soil bacterial communities were impacted by the surrounding soil physiochemistry. Based on the identification of a metabolically-active community of denitrifiers, this study has indicated that denitrification could potentially occur under appropriate conditions in all types of BMP sampled, including surface sand filters that are often viewed as providing low potential for denitrification. The carbon content of incoming stormwater could be providing bacterial communities with denitrification conditions. The findings of this study are especially relevant for land managers in watersheds with legacy nitrogen from former agricultural land use.

Interaction of Microbiome, Diet, and Hospitalizations Between Brazilian and American Patients With Cirrhosis.

BACKGROUND & AIMS: Gut microbiota are affected by diet, country, and affect outcomes in cirrhosis. Western diets are associated with dysbiosis. Comparisons with other diets is needed. We aimed to compare cirrhosis patients from the United States with cirrhosis patients from Brazil with respect to diet, microbiota, and impact on hospitalizations. METHODS: Healthy controls and compensated/decompensated outpatients with cirrhosis from the United States and Brazil underwent dietary recall and stool for 16S ribosomal RNA sequencing. Demographics and medications/cirrhosis details were compared within and between countries. Patients with cirrhosis were followed up for 90-day hospitalizations. Regression for Shannon diversity was performed within cirrhosis. Regression for hospitalizations adjusting for clinical and microbial variables was performed. RESULTS: Model for end-stage liver disease (MELD), diabetes, ascites, and albumin were similar, but more Americans were men, had higher hepatic encephalopathy and alcohol/hepatitis C etiology, with lower nonalcoholic fatty liver disease than Brazilians. Brazilians had higher cereal, rice, and yogurt intake vs the United States. As disease progressed, cereals, rice/beans, coffee, and chocolate consumption was reduced. Microbial diversity was higher in Brazilians. Within cirrhosis, high diversity was related to Brazilian origin (P < .0001), age, and cereal intake (P = .05), while high MELD scores (P = .009) and ascites (P = .05) did the reverse. Regardless of stage, beneficial taxa and taxa associated with grant and yogurt intake were higher (Ruminococcaceae, Christensenellacae, and Prevotellaceae), while pathobionts (Porphyromonadaceae, Sutterellaceae, and Enterobacteriaceae) were lower in Brazilians. More Americans were hospitalized vs Brazilians (P = .002). On regression, MELD (P = .001) and ascites (P = .001) were associated with higher hospitalizations, while chocolate (P = .03) and Brazilian origin (P = .001) were associated with lower hospitalizations with/without microbiota inclusion. CONCLUSIONS: Brazilian cirrhotic patients follow a diet richer in cereals and yogurt, which is associated with higher microbial diversity and beneficial microbiota and could contribute toward lower hospitalizations compared with a Western-diet-consuming American cohort.

Stool microbiota are superior to saliva in distinguishing cirrhosis and hepatic encephalopathy using machine learning.

BACKGROUND & AIMS: Saliva and stool microbiota are altered in cirrhosis. Since stool is logistically difficult to collect compared to saliva, it is important to determine their relative diagnostic and prognostic capabilities. We aimed to determine the ability of stool vs. saliva microbiota to differentiate between groups based on disease severity using machine learning (ML). METHODS: Controls and outpatients with cirrhosis underwent saliva and stool microbiome analysis. Controls vs. cirrhosis and within cirrhosis (based on hepatic encephalopathy [HE], proton pump inhibitor [PPI] and rifaximin use) were classified using 4 ML techniques (random forest [RF], support vector machine, logistic regression, and gradient boosting) with AUC comparisons for stool, saliva or both sample types. Individual microbial contributions were computed using feature importance of RF and Shapley additive explanations. Finally, thresholds for including microbiota were varied between 2.5% and 10%, and core microbiome (DESeq2) analysis was performed. RESULTS: Two hundred and sixty-nine participants, including 87 controls and 182 patients with cirrhosis, of whom 57 had HE, 78 were on PPIs and 29 on rifaximin were included. Regardless of the ML model, stool microbiota had a significantly higher AUC in differentiating groups vs. saliva. Regarding individual microbiota: autochthonous taxa drove the difference between controls vs. patients with cirrhosis, oral-origin microbiota the difference between PPI users/non-users, and pathobionts and autochthonous taxa the difference between rifaximin users/non-users and patients with/without HE. These were consistent with the core microbiome analysis results. CONCLUSIONS: On ML analysis, stool microbiota composition is significantly more informative in differentiating between controls and patients with cirrhosis, and those with varying cirrhosis severity, compared to saliva. Despite logistic challenges, stool should be preferred over saliva for microbiome analysis. LAY SUMMARY: Since it is harder to collect stool than saliva, we wanted to test whether microbes from saliva were better than stool in differentiating between healthy people and those with cirrhosis and, among those with cirrhosis, those with more severe disease. Using machine learning, we found that microbes in stool were more accurate than saliva alone or in combination, therefore, stool should be preferred for analysis and collection wherever possible.

Multiple bacterial virulence factors focused on adherence and biofilm formation associate with outcomes in cirrhosis.

BACKGROUND & AIMS: Altered gut microbiota is associated with poor outcomes in cirrhosis, including infections and hepatic encephalopathy (HE). However, the role of bacterial virulence factors (VFs) is unclear. Aim: Define association of VFs with cirrhosis severity and infections, their linkage with outcomes, and impact of fecal microbiota transplant (FMT). METHODS: VF abundances were determined using metagenomic analysis in stools from controls and cirrhosis patients (compensated, HE-only, ascites-only, both and infected). Patients were followed for 90-day hospitalizations and 1-year death. Stool samples collected before/after a placebo-controlled FMT trial were also analyzed. Bacterial species and VFs for all species and selected pathogens (Escherichia, Klebsiella, Pseudomonas, Staphylococcus, Streptococcus, and Enterococcus spp) were compared between groups. Multi-variable analyses were performed for clinical biomarkers and VFs for outcome prediction. Changes in VFs pre/post-FMT and post-FMT/placebo were analyzed. Results: We included 233 subjects (40 controls, 43 compensated, 30 HE-only, 20 ascites-only, 70 both, and 30 infected). Decompensated patients, especially those with infections, had higher VFs coding for siderophores, biofilms, and adhesion factors versus the rest. Biofilm and adhesion VFs from Enterobacteriaceae and Enterococcus spp associated with death and hospitalizations independent of clinical factors regardless of when all VFs or selected pathogens were analyzed. FMT was associated with reduced VF post-FMT versus pre-FMT and post-placebo groups. CONCLUSIONS: Virulence factors from multiple species focused on adhesion and biofilms increased with decompensation and infections, associated with death and hospitalizations independent of clinical factors, and were attenuated with FMT. Strategies focused on targeting multiple virulence factors could potentially impact outcomes in cirrhosis. PRESENTATIONS: Portions of this manuscript were an oral presentation in the virtual International Liver Congress 2021. ABBREVIATIONS: VF: virulence factors, HE: hepatic encephalopathy, FMT: Fecal microbiota transplant, PPI: proton pump inhibitors, LPS: lipopolysaccharides, VFDB: Virulence factor database, OTU: operational taxonomic units, SBP: spontaneous bacterial peritonitis, UTI: urinary tract infections, MRSA: methicillin resistant Staphylococcus aureus, VRE: vancomycin-resistant Enterococcus, MAAsLin2: Microbiome Multivariable Associations with Linear Models, LPS: lipopolysaccharides, AKI: acute kidney injury.

The Effects of Consuming White Button Mushroom Agaricus bisporus on the Brain and Liver Metabolome Using a Targeted Metabolomic Analysis.

A targeted metabolomic analysis was performed on tissues derived from pigs fed diets supplemented with white button mushrooms (WBM) to determine the effect on the liver and brain metabolome. Thirty-one pigs were fed a grower diet alone or supplemented with either three or six servings of freeze-dried WBM for six weeks. Tissue metabolomes were analyzed using targeted liquid chromatography-mass spectrometry (LC-MS) combined with chemical similarity enrichment analysis (ChemRICH) and correlated to WBM-induced changes in fecal microbiome composition. Results indicated that WBM can differentially modulate metabolites in liver, brain cortex and hippocampus of healthy pigs. Within the glycero-phospholipids, there was an increase in alkyl-acyl-phosphatidyl-cholines (PC-O 40:3) in the hippocampus of pigs fed six servings of WBM. A broader change in glycerophospholipids and sphingolipids was detected in the liver with a reduction in several lipid species in pigs fed both WBM diets but with an increase in amino acids known as precursors of neurotransmitters in the cortex of pigs fed six servings of WBM. Metabolomic changes were positively correlated with increased abundance of Cryomorphaceae, Lachnospiraceae, Flammeovirgaceae and Ruminococcaceae in the microbiome suggesting that WBM can also positively impact tissue metabolite composition.

Distinct gut microbial compositional and functional changes associated with impaired inhibitory control in patients with cirrhosis.

Most cirrhosis etiologies, such as alcohol, hepatitis C, and obesity, involve behavior that require the loss of inhibitory control. Once cirrhosis develops, patients can also develop cognitive impairment due to minimal hepatic encephalopathy (MHE). Both processes could have distinct imprints on the gut-liver-brain axis. Determine the impact of inhibitory control versus traditional cirrhosis-related cognitive performance on gut microbial composition and function. Outpatients with cirrhosis underwent two tests for MHE: inhibitory control test (MHEICT, computerized associated with response inhibition) and psychometric hepatic encephalopathy score (MHEPHES, paper-pencil HE-specific associated with subcortical impairment) along with stool collection for metagenomics. MHEICT/not, MHEPHES/not, and discordant (positive on one test but negative on the other) were analyzed for demographics, bacterial species, and gut-brain modules (GBM) using multi-variable analyses. Ninety-seven patients [47 (49%) MHEPHES, 76 (78%) MHEICT, 41 discordant] were enrolled. MHEPHES/not: Cirrhosis severity was worse in MHEPHES without differences in alpha/beta diversity on bacterial species or GBMs. Pathobionts (Enterobacteriaceae) and γ-amino-butryic acid (GABA) synthesis GBM were higher in MHEPHES. MHEICT/not: We found similar cirrhosis severity and metagenomic alpha/beta diversity in MHEICT versus not. However, alpha/beta diversity of GBMs were different in MHEICT versus No-MHE patients. Alistipes ihumii, Prevotella copri, and Eubacterium spp. were higher, while Enterococcus spp. were uniquely lower in MHEICT versus no-MHE and discordant comparisons. GBMs belonging to tryptophan, menaquinone, GABA, glutamate, and short-chain fatty acid synthesis were also unique to MHEICT. Gut microbial signature of impaired inhibitory control, which is associated with addictive disorders that can lead to cirrhosis, is distinct from cirrhosis-related cognitive impairment.

Hepatic Branch Vagotomy Modulates the Gut-Liver-Brain Axis in Murine Cirrhosis.

BACKGROUND: Cirrhosis and hepatic encephalopathy (HE) are linked with an altered gut-liver-brain axis, however, the relative contribution of hepatic vagal innervation is unclear. We aimed to determine the impact of hepatic vagotomy on the gut microbiome, brain, and liver in murine cirrhosis. METHODS: 10-15-week-old male C57BL/6 mice with and without hepatic vagotomy underwent carbon tetrachloride (CCl4) gavage for 8 weeks. Frontal cortex [inflammation, glial/microglial activation, BDNF (brain-derived neurotrophic factor)], liver [histology including inflammation and steatosis, fatty acid synthesis (sterol-responsive binding protein-1) SREBP-1, insulin-induced gene-2 (Insig2) and BDNF], and colonic mucosal microbiota (16srRNA microbial sequencing) were evaluated on sacrifice. Conventional mice with and without cirrhosis were compared to vagotomized counterparts. RESULTS: Conventional control vs. cirrhosis: Cirrhosis resulted in dysbiosis, hepatic/neuro-inflammation with glial/microglial activation, and low brain BDNF vs. controls. Conventional control vs. vagotomy controls: Vagotomized control mice had a lower colonic dysbiosis than conventional mice but the rest of the hepatic/brain parameters were similar. Conventional cirrhosis vs. vagotomized cirrhosis: After vagotomy + cirrhosis, we found lower dysbiosis but continuing neuroinflammation in the absence of glial/microglial activation vs. conventional cirrhosis. Vagotomy + Cirrhosis groups showed higher hepatic steatosis due to higher SREBP1 and low Insig2 protein and altered activation of key genes involved in hepatic lipid metabolism and inflammation. BDNF levels in the brain were higher but low in the liver in vagotomy + cirrhosis, likely a protective mechanism. CONCLUSIONS: Hepatic vagal innervation affects the gut microbial composition, hepatic inflammation and steatosis, and cortical inflammation and BDNF expression and could be a critical modulator of the gut-liver-brain axis with consequences for HE development.