Early treatment with rifaximin during epileptogenesis reverses gut alterations and reduces seizure duration in a mouse model of acquired epilepsy.

The gut microbiota is altered in epilepsy and is emerging as a potential target for new therapies. We studied the effects of rifaximin, a gastrointestinal tract-specific antibiotic, on seizures and neuropathology and on alterations in the gut and its microbiota in a mouse model of temporal lobe epilepsy (TLE). Epilepsy was induced by intra-amygdala kainate injection causing status epilepticus (SE) in C57Bl6 adult male mice. Sham mice were injected with vehicle. Two cohorts of SE mice were fed a rifaximin-supplemented diet for 21 days, starting either at 24 h post-SE (early disease stage) or at day 51 post-SE (chronic disease stage). Corresponding groups of SE mice (one each disease stage) were fed a standard (control) diet. Cortical ECoG recording was done at each disease stage (24/7) for 21 days in all SE mice to measure the number and duration of spontaneous seizures during either rifaximin treatment or control diet. Then, epileptic mice ± rifaximin and respective sham mice were sacrificed and brain, gut and feces collected. Biospecimens were used for: (i) quantitative histological analysis of the gut structural and cellular components; (ii) markers of gut inflammation and intestinal barrier integrity by RTqPCR; (iii) 16S rRNA metagenomics analysis in feces. Hippocampal neuronal cell loss was assessed in epileptic mice killed in the early disease phase. Rifaximin administered for 21 days post-SE (early disease stage) reduced seizure duration (p < 0.01) and prevented hilar mossy cells loss in the hippocampus compared to epileptic mice fed a control diet. Epileptic mice fed a control diet showed a reduction of both villus height and villus height/crypt depth ratio (p < 0.01) and a decreased number of goblet cells (p < 0.01) in the duodenum, as well as increased macrophage (Iba1)-immunostaining in the jejunum (p < 0.05), compared to respective sham mice. Rifaximin's effect on seizures was associated with a reversal of gut structural and cellular changes, except for goblet cells which remained reduced. Seizure duration in epileptic mice was negatively correlated with the number of mossy cells (p < 0.01) and with villus height/crypt depth ratio (p < 0.05). Rifaximin-treated epileptic mice also showed increased tight junctions (occludin and ZO-1, p < 0.01) and decreased TNF mRNA expression (p < 0.01) in the duodenum compared to epileptic mice fed a control diet. Rifaximin administered for 21 days in chronic epileptic mice (chronic disease stage) did not change the number or duration of seizures compared to epileptic mice fed a control diet. Chronic epileptic mice fed a control diet showed an increased crypt depth (p < 0.05) and reduced villus height/crypt depth ratio (p < 0.01) compared to respective sham mice. Rifaximin treatment did not affect these intestinal changes. At both disease stages, rifaximin modified α- and ß-diversity in epileptic and sham mice compared to respective mice fed a control diet. The microbiota composition in epileptic mice, as well as the effects of rifaximin at the phylum, family and genus levels, depended on the stage of the disease. During the early disease phase, the abundance of specific taxa was positively correlated with seizure duration in epileptic mice. In conclusion, gut-related alterations reflecting a dysfunctional state, occur during epilepsy development in a TLE mouse model. A short-term treatment with rifaximin during the early phase of the disease, reduced seizure duration and neuropathology, and reversed some intestinal changes, strengthening the therapeutic effects of gut-based therapies in epilepsy.

Rifaximin and alternative agents in the management of irritable bowel syndrome: A comprehensive review.

Rifaximin, a broad-spectrum antibiotic, boasts a unique chemical composition and pharmacokinetic profile, rendering it highly effective in treating irritable bowel syndrome (IBS). Its minimal systemic absorption confines its impact to the gastrointestinal (GI) tract, where it yields significant therapeutic benefits. This review examines rifaximin's physico-chemical attributes and its role in managing IBS symptoms. Its molecular structure facilitates intestinal lumen retention postoral administration, minimizing systemic exposure and adverse effects. This targeted action is crucial in addressing the gut microbiota's role in IBS pathophysiology. By modifying microbial populations and their metabolite production, rifaximin mitigates symptoms like bloating, irregular bowel habits, and abdominal pain associated with IBS. It achieves this by reducing pathogenic bacteria and altering bacterial metabolism, enhancing mucosal and immune function. Clinical trials affirm rifaximin's superiority over placebo and conventional therapies in alleviating overall IBS symptoms and addressing small intestine bacterial overgrowth (SIBO). Despite its promising efficacy and sustained symptom relief, further research is essential to optimize long-term effectiveness and dosing regimens. Rifaximin stands as a vital treatment option for IBS due to its distinctive properties and clinical utility; yet, ongoing investigation is imperative for maximizing its therapeutic benefits.

Modulation of the microbiota across different intestinal segments by Rifaximin in PI-IBS mice.

BACKGROUND: Rifaximin has been increasingly applied in irritable bowel syndrome (IBS) treatment. Whether there were differences in the effects of rifaximin on microbiota from different intestinal segments, especially the small intestine where rifaximin predominantly acted, has not been confirmed. METHODS: In this study, we used Trichinella spiralis infection to induce post infectious irritable bowel syndrome (PI-IBS) and measured visceral sensitivity of mice by means of abdominal withdrawal reflex (AWR) tests to colorectal distention (CRD). We compared the effects of rifaximin on the composition of ileal, colonic mucosal and fecal microbiota in PI-IBS mice. RESULTS: Rifaximin significantly reduced AWR scores and increased pain threshold in PI-IBS mice, and this effect was associated with the change in the relative abundance of ileal mucosal microbiota. Rifaximin could obviously decrease ileum mucosal microbiota alpha diversity assessed by Shannon microbial diversity index. Meanwhile, the analysis of beta diversity and relative abundance of microbiota at phylum, family and genus levels showed that rifaximin could improve the microbiota structure of ileal mucosa. However, for colonic mucosal and fecal microbiota, this effect of rifaximin was not obvious. Rifaximin could reshape the correlation of genera between different intestinal segments. CONCLUSION: Rifaximin improved visceral hypersensitivity in PI-IBS mice. Rifaximin mainly affected ileal mucosal microbiota, and its improvement effect on IBS might be closely related to the improvement of ileal microbiota structure.

The effect of antibiotic versus no treatment at dry-off on udder health and milk yield in subsequent lactation: A retrospective analysis of Austrian health recording data from dairy herds.

Bovine mastitis is the most commonly diagnosed disease of dairy cows worldwide and causes extensive economic losses to milk producers. Intramammary infection status before dry-off plays a decisive role with respect to udder health and milk yield in the subsequent lactation. The aim of this study was to compare the effect of antibiotic dry cow therapy (DCT) versus no treatment at dry-off on milk yield, somatic cell count (SCC), inflammation of the mammary gland (IMG), and the incidence of clinical mastitis in the subsequent lactation. Dairy herd data from 251 Austrian dairy farms were recorded over an observation period of 12 mo and subsequently analyzed. The data set included 5,018 dairy cows: 2,078 were treated with antibiotics (abDCT group) and 2,940 were not treated (noDCT group) at dry-off. The abDCT group was subdivided, based on the antimicrobial active substances used for drying off, into 4 different groups (penicillins, cloxacillin, cephalosporins, and rifaximin). Based on bacteriological culture results, infections were grouped into those caused by major, minor, and other pathogens. Additionally, the IMG was defined via SCC from milk recording data using a cutoff of 200,000 cells/mL before drying off and after calving. The incidence of clinical mastitis cases within 30 and 90 d in milk was calculated using veterinary diagnosis data. To investigate the effect of different dry cow therapies on the following parameters: milk yield, SCC, and diagnosed clinical mastitis cases, different linear mixed models were constructed. Overall, the abDCT group was determined to have a significantly higher milk yield over 305 d in milk in the subsequent lactation (increase of 6.18%), compared with the noDCT group (increase of 4.29%). Both groups (abDCT and noDCT) demonstrated a decrease in the first SCC after calving compared with the SCC before dry-off, although the treated cows had a significantly higher reduction. Regarding the different antibiotic groups, with exception of the rifaximin treated cows, all antibiotic groups showed a significant difference from not treated cows with respect to SCC. Additionally, we were able to demonstrate that cows with IMG before dry-off had a 2.073 times higher chance of an increased SCC (>200,000 cells/mL) after calving. With respect to the veterinary diagnosis data, neither the IMG before drying off nor the type of DCT had a significant influence on the probability of developing clinical mastitis within 30 or 90 d in milk. Only a small number of treatments was accompanied with a bacteriological examination before drying off. However, the existing data in this study indicates that the intramammary infection status before dry-off in combination with different dry cow treatments influences udder health and milk yield after calving. Nevertheless, further studies with larger data sets of bacteriological examinations are necessary to enable a more in-depth investigation into the effects of different antibiotic substances used for DCT.

Effect of subinhibitory doses of rifaximin on in vitro Pseudomonas aeruginosa adherence and biofilm formation to biotic and abiotic surface models.

BACKGROUND: The adhesion of Pseudomonas aeruginosa to biotic and abiotic surfaces is responsible for the persistence and development of bacterial infection. OBJECTIVES: To fill the gap in the knowledge regarding the relationship between rifaximin susceptibility and biofilm formation, and to investigate the effect of subinhibitory doses of rifaximin on the adhesion and biofilm formation. MATERIAL AND METHODS: A total of 10 isolates of P. aeruginosa were obtained from 110 urine samples of urinary tract infection (UTI) patients. Biofilm formation on polystyrene microtiter plates, minimum inhibitory concentrations (MICs) of rifaximin against the 10 isolates of P. aeruginosa (Pa1-Pa10), the effect of sub-MICs of rifaximin (0.5 × MIC, 0.25 × MIC, 0.125 × MIC, and 0.06 × MIC) on biofilm formation by the Pa4 isolate to polystyrene microtiter plates, and the adhesion to human epithelial cells (HECs) in vitro were evaluated. RESULTS: The MICs of rifaximin against 10 isolates ranged from 62.5 µg/mL to 1000 µg/mL. The Pa4 isolate produced the highest level of biofilm formation, while the MIC of Pa4 was 125 µg/mL. There was no correlation between bacterial susceptibility to rifaximin and biofilm formation (r: -0.016; p > 0.05). Sub-MIC doses of rifaximin significantly reduced the biofilm formation on abiotic surfaces, while only 0.5 × MIC, 0.25 × MIC and 0.12 × MIC of rifaximin reduced the adhesion to HECs significantly (p < 0.05) in a dose-dependent manner. CONCLUSIONS: This pioneering study demonstrated the negative effect of sub-MIC doses of rifaximin on biofilm formation and adhesion to abiotic and biotic surfaces in vitro.

Rifaximin protects against circadian rhythm disruption-induced cognitive impairment through preventing gut barrier damage and neuroinflammation.

Circadian rhythm disruption (CRD) is a potential risk factor for developing Alzheimer's disease (AD). However, the mechanistic link between CRD and AD is still not fully understood. CRD may lead to intestinal barrier impairment. Several studies in animals and humans suggest a connection between gut microbiota disturbance, intestinal barrier damage and neurodegenerative diseases. In this study, we investigated the effect of CRD on cognition in mice and explored the role of intestinal barrier and inflammatory responses in this process. CRD modulates the composition of gut microbiota, impairs intestinal barrier integrity, and induces both peripheral and central inflammation and cognitive impairment in mice. Rifaximin, a non-absorbable antibiotic which modulates the gut microbial composition and increases intestinal barrier integrity, effectively suppresses inflammatory responses, and rescues cognitive impairment induced by CRD. Furthermore, the impairment in hippocampal neurogenesis, tau hyperphosphorylation, and loss in synaptic proteins in CRD mice is also reversed by Rifaximin. These data identify that the impaired intestinal barrier integrity related to gut microbiota disturbance plays a key role in CRD-induced inflammatory responses and cognitive impairments in mice, and Rifaximin is effective in preventing CRD-induced cognitive deficit through protecting the gut barrier and ameliorating neuroinflammation.

Rifaximin-α reduces gut-derived inflammation and mucin degradation in cirrhosis and encephalopathy: RIFSYS randomised controlled trial.

BACKGROUND & AIMS: Rifaximin-α is efficacious for the prevention of recurrent hepatic encephalopathy (HE), but its mechanism of action remains unclear. We postulated that rifaximin-α reduces gut microbiota-derived endotoxemia and systemic inflammation, a known driver of HE. METHODS: In a placebo-controlled, double-blind, mechanistic study, 38 patients with cirrhosis and HE were randomised 1:1 to receive either rifaximin-α (550 mg BID) or placebo for 90 days. PRIMARY OUTCOME: 50% reduction in neutrophil oxidative burst (OB) at 30 days. SECONDARY OUTCOMES: changes in psychometric hepatic encephalopathy score (PHES) and neurocognitive functioning, shotgun metagenomic sequencing of saliva and faeces, plasma and faecal metabolic profiling, whole blood bacterial DNA quantification, neutrophil toll-like receptor (TLR)-2/4/9 expression and plasma/faecal cytokine analysis. RESULTS: Patients were well-matched: median MELD (11 rifaximin-α vs. 10 placebo). Rifaximin-α did not lead to a 50% reduction in spontaneous neutrophil OB at 30 days compared to baseline (p = 0.48). However, HE grade normalised (p = 0.014) and PHES improved (p = 0.009) after 30 days on rifaximin-α. Rifaximin-α reduced circulating neutrophil TLR-4 expression on day 30 (p = 0.021) and plasma tumour necrosis factor-α (TNF-α) (p <0.001). Rifaximin-α suppressed oralisation of the gut, reducing levels of mucin-degrading sialidase-rich species, Streptococcus spp, Veillonella atypica and parvula, Akkermansia and Hungatella. Rifaximin-α promoted a TNF-α- and interleukin-17E-enriched intestinal microenvironment, augmenting antibacterial responses to invading pathobionts and promoting gut barrier repair. Those on rifaximin-α were less likely to develop infection (odds ratio 0.21; 95% CI 0.05-0.96). CONCLUSION: Rifaximin-α led to resolution of overt and covert HE, reduced the likelihood of infection, reduced oralisation of the gut and attenuated systemic inflammation. Rifaximin-α plays a role in gut barrier repair, which could be the mechanism by which it ameliorates bacterial translocation and systemic endotoxemia in cirrhosis. CLINICAL TRIAL NUMBER: ClinicalTrials.gov NCT02019784. LAY SUMMARY: In this clinical trial, we examined the underlying mechanism of action of an antibiotic called rifaximin-α which has been shown to be an effective treatment for a complication of chronic liver disease which effects the brain (termed encephalopathy). We show that rifaximin-α suppresses gut bacteria that translocate from the mouth to the intestine and cause the intestinal wall to become leaky by breaking down the protective mucus barrier. This suppression resolves encephalopathy and reduces inflammation in the blood, preventing the development of infection.

Rifaximin modulates TRH and TRH-like peptide expression throughout the brain and peripheral tissues of male rats.

BACKGROUND: The TRH/TRH-R1 receptor signaling pathway within the neurons of the dorsal vagal complex is an important mediator of the brain-gut axis. Mental health and protection from a variety of neuropathologies, such as autism, Attention Deficit Hyperactivity Disorder, Alzheimer's and Parkinson's disease, major depression, migraine and epilepsy are influenced by the gut microbiome and is mediated by the vagus nerve. The antibiotic rifaximin (RF) does not cross the gut-blood barrier. It changes the composition of the gut microbiome resulting in therapeutic benefits for traveler's diarrhea, hepatic encephalopathy, and prostatitis. TRH and TRH-like peptides, with the structure pGlu-X-Pro-NH2, where "X" can be any amino acid residue, have reproduction-enhancing, caloric-restriction-like, anti-aging, pancreatic-ß cell-, cardiovascular-, and neuroprotective effects. TRH and TRH-like peptides occur not only throughout the CNS but also in peripheral tissues. To elucidate the involvement of TRH-like peptides in brain-gut-reproductive system interactions 16 male Sprague-Dawley rats, 203 ± 6 g, were divided into 4 groups (n = 4/group): the control (CON) group remained on ad libitum Purina rodent chow and water for 10 days until decapitation, acute (AC) group receiving 150 mg RF/kg powdered rodent chow for 24 h providing 150 mg RF/kg body weight for 200 g rats, chronic (CHR) animals receiving RF for 10 days; withdrawal (WD) rats receiving RF for 8 days and then normal chow for 2 days. RESULTS: Significant changes in the levels of TRH and TRH-like peptides occurred throughout the brain and peripheral tissues in response to RF. The number of significant changes in TRH and TRH-like peptide levels in brain resulting from RF treatment, in descending order were: medulla (16), piriform cortex (8), nucleus accumbens (7), frontal cortex (5), striatum (3), amygdala (3), entorhinal cortex (3), anterior (2), and posterior cingulate (2), hippocampus (1), hypothalamus (0) and cerebellum (0). The corresponding ranking for peripheral tissues were: prostate (6), adrenals (4), pancreas (3), liver (2), testis (1), heart (0). CONCLUSIONS: The sensitivity of TRH and TRH-like peptide expression to RF treatment, particularly in the medulla oblongata and prostate, is consistent with the participation of these peptides in the therapeutic effects of RF.

Rifaximin protects SH-SY5Y neuronal cells from iron overload-induced cytotoxicity via inhibiting STAT3/NF-κB signaling.

Acute or chronic liver disease-caused liver failure is the cause of hepatic encephalopathy (HE), characterized by neuropsychiatric manifestations. Liver diseases potentially lead to peripheral iron metabolism dysfunction and surges of iron concentration in the brain, contributing to the pathophysiological process of degenerative disorders of the central nervous system. In this study, the mechanism of rifaximin treating HE was investigated. Ferric ammonium citrate (FAC)-induced iron overload significantly reduced the proliferation and boosted the apoptosis in SH-SY5Y cells through increasing reactive oxygen species (ROS) levels and inducing iron metabolism disorder. Rifaximin treatment could rectify the FAC-induced iron overload and lipopolysaccharide (LPS)-induced iron deposition, therefore, effectively protecting SH-SY5Y cells from ROS-induced cell injury and apoptosis. Signal transducer and activator of transcription 3 (STAT3)/nuclear factor-kappa B (NF-κB) signaling is involved in the protective function of rifaximin against LPS-induced iron deposition. The therapeutic effect of rifaximin on HE associated with acute hepatic failure in mouse model was ascertained. In conclusion, Rifaximin could effectively protect SH-SY5Y cells against injury caused by iron overload through the rectification of the iron metabolism disorder via the STAT3/NF-κB signaling pathway.

Rifaximin Protects against Malathion-Induced Rat Testicular Toxicity: A Possible Clue on Modulating Gut Microbiome and Inhibition of Oxidative Stress by Mitophagy.

Testicular dysfunction is caused by chronic exposure to environmental pollution, such as malathion, which causes oxidative stress, promoting cell damage. Autophagy is a key cellular process for eliminating malfunctioning organelles, such as the mitochondria (mitophagy), an eminent source of reactive oxygen species (ROS). Autophagy is crucial for protection against testicular damage. Rifaximin (RFX) is a non-absorbable antibiotic that can reshape the gut microbiome, making it effective in different gastrointestinal disorders. Interestingly, the gut microbiome produces short chain fatty acids (SCFAs) in the circulation, which act as signal molecules to regulate the autophagy. In this study, we investigated the regulatory effects of RFX on gut microbiota and its circulating metabolites SCFA and linked them with the autophagy in testicular tissues in response to malathion administration. Moreover, we divided the groups of rats that used malathion and RFX into a two-week group to investigate the mitophagy process and a four-week group to study mitochondriogenesis. The current study revealed that after two weeks of cotreatment with RFX, apoptosis was inhibited, oxidative stress was improved, and autophagy was induced. More specifically, PINK1 was overexpressed, identifying mitophagy activation. After four weeks of cotreatment with RFX, there was an increase in acetate and propionate-producing microflora, as well as the circulating levels of SCFAs. In accordance with this, the expression of PGC-1α, a downstream to SCFAs action on their receptors, was activated. PGC-1α is an upstream activator of mitophagy and mitochondriogenesis. In this sense, the protein expression of TFAM, which regulates the mitochondrial genome, was upregulated along with a significant decrease in apoptosis and oxidative stress. Conclusion: we found that RFX has a positive regulatory effect on mitophagy and mitochondria biogenesis, which could explain the novel role played by RFX in preventing the adverse effects of malathion on testicular tissue.

Long-Term Effects of Rifaximin on Patients with Hepatic Encephalopathy: Its Possible Effects on the Improvement in the Blood Ammonia Concentration Levels, Hepatic Spare Ability and Refractory Ascites.

Background and Objectives: To investigate the long-term efficacy of rifaximin (RFX) for hyperammonemia and efficacy for refractory ascites in patients with cirrhosis. Materials and Methods: We enrolled 112 patients with liver cirrhosis who were orally administered RFX in this study. Changes in the clinical data of patients were evaluated up to 36 months after RFX administration. The primary endpoint was a change in blood ammonia levels. Secondary endpoints included changes in clinical symptoms, Child−Pugh (CP) score, number of hospitalizations, degree of refractory ascites, adverse events, and the relationship between RFX administration and the renin-angiotensin-aldosterone system. Results: An improved rate of overt hepatic encephalopathy (HE) of 82.7% was observed 3 months after RFX administration, which significantly induced a progressive decrease in blood ammonia concentration and an improved CP score up to 36 months. No serious RFX treatment-related adverse events were observed. 36.5% in patients after RFX administration improved refractory ascites. After RFX administration, patients with satisfactory control of hepatic ascites without addition of diuretic had lower renin concentration than those with poor control (p < 0.01). At less than 41 pg/mL renin concentration, the control of refractory ascites was significantly satisfactory (p < 0.0001). Conclusions: RFX reduced blood ammonia concentration and improved hepatic spare ability and the quality of life of patients with long-term HE to up to 36 months. Our study revealed the effects of RFX against refractory ascites, suggesting that renin concentration may be a predictive marker for assessing ascites control.

[Effect of Gegen Qinlian Decoction on gut microbiota of irritable bowel syndrome with diarrhea rats].

This study aims to explore the effect of Gegen Qinlian Decoction on gut microbiota of irritable bowel syndrome with diarrhea(IBS-D) rats. A total of 36 male SD rats were randomly classified into the control group, model group, rifaximin group(150 mg·kg~(-1)), and high-dose(8.125 g·kg~(-1)), medium-dose(4.062 5 g·kg~(-1)) and low-dose(2.031 3 g·kg~(-1)) Gegen Qinlian Decoction groups with the random number table method, 6 in each group. After modeling, rats were treated for 8 days. The general state, bristol stool chart(BSC) score, and the minimum volume threshold for abdominal withdrawal reflex were recorded. Pathological changes of colon tissues were observed based on hematoxylin and eosin(HE) staining, and gut microbiota was analyzed based on 16 S rRNA sequencing. Compared with the model group, rifaximin group and high-dose and medium-dose Gegen Qinlian Decoction groups showed low BSC score(P<0.01) and high minimum volume threshold for abdominal lifting(P<0.05). HE staining showed that Gegen Qinlian Decoction could relieve intestinal inflammation. 16 S rRNA sequencing suggested obvious variation of gut microbiota in IBS-D rats. Gegen Qinlian Decoction significantly raised the richness index and diversity index of gut microbiota, regulated the number of the flora, and improved alpha diversity and beta diversity. Species composition of gut microbiota and LEfSe analysis showed that Gegen Qinlian Decoction could significantly increase the ratio of Bacteroidota to Firmicutes, elevate the abundance of probiotics such as Clostridia and Lachnospirales, and reduce the abundance of conditional pathogens such as Bacteroidales, and Prevotellaceae. PICRUSt2 analysis indicated that Gegen Qinlian Decoction was mainly related to multiple metabolic pathways such as carbohydrate metabolism and amino acid metabolism. In summary, Gegen Qinlian Decoction can significantly reduce visceral hypersensitivity of IBS-D rats, alleviate intestinal inflammation, and relieve clinical symptoms such as diarrhea. The mechanism is the likelihood that it regulates the composition and structure of gut microbiota and improves its metabolic pathway as well.

Interaction of bacterial metagenome and virome in patients with cirrhosis and hepatic encephalopathy.

OBJECTIVE: Altered bacterial composition is associated with disease progression in cirrhosis but the role of virome, especially phages, is unclear. DESIGN: Cross-sectional and pre/post rifaximin cohorts were enrolled. Cross-sectional: controls and cirrhotic outpatients (compensated, on lactulose (Cirr-L), on rifaximin (Cirr-LR)) were included and followed for 90-day hospitalisations. Pre/post: compensated cirrhotics underwent stool collection pre/post 8 weeks of rifaximin. Stool metagenomics for bacteria and phages and their correlation networks were analysed in controls versus cirrhosis, within cirrhotics, hospitalised/not and pre/post rifaximin. RESULTS: Cross-sectional: 40 controls and 163 cirrhotics (63 compensated, 43 Cirr-L, 57 Cirr-LR) were enrolled. Cirr-L/LR groups were similar on model for end-stage liver disease (MELD) score but Cirr-L developed greater hospitalisations versus Cirr-LR (56% vs 30%, p=0.008). Bacterial alpha/beta diversity worsened from controls through Cirr-LR. While phage alpha diversity was similar, beta diversity was different between groups. Autochthonous bacteria linked negatively, pathobionts linked positively with MELD but only modest phage-MELD correlations were seen. Phage-bacterial correlation network complexity was highest in controls, lowest in Cirr-L and increased in Cirr-LR. Microviridae and Faecalibacterium phages were linked with autochthonous bacteria in Cirr-LR, but not Cirr-L hospitalised patients had greater pathobionts, lower commensal bacteria and phages focused on Streptococcus, Lactococcus and Myoviridae. Pre/post: No changes in alpha/beta diversity of phages or bacteria were seen postrifaximin. Phage-bacterial linkages centred around urease-producing Streptococcus species collapsed postrifaximin. CONCLUSION: Unlike bacteria, faecal phages are sparsely linked with cirrhosis characteristics and 90-day outcomes. Phage and bacterial linkages centred on urease-producing, ammonia-generating Streptococcus species were affected by disease progression and rifaximin therapy and were altered in patients who experienced 90-day hospitalisations.

Rifaximin-mediated gut microbiota regulation modulates the function of microglia and protects against CUMS-induced depression-like behaviors in adolescent rat.

BACKGROUND: Chronic unpredictable mild stress (CUMS) can not only lead to depression-like behavior but also change the composition of the gut microbiome. Regulating the gut microbiome can have an antidepressant effect, but the mechanism by which it improves depressive symptoms is not clear. Short-chain fatty acids (SCFAs) are small molecular compounds produced by the fermentation of non-digestible carbohydrates. SFCAs are ubiquitous in intestinal endocrine and immune cells, making them important mediators of gut microbiome-regulated body functions. The balance between the pro- and anti-inflammatory microglia plays an important role in the occurrence and treatment of depression caused by chronic stress. Non-absorbable antibiotic rifaximin can regulate the structure of the gut microbiome. We hypothesized that rifaximin protects against stress-induced inflammation and depression-like behaviors by regulating the abundance of fecal microbial metabolites and the microglial functions. METHODS: We administered 150 mg/kg rifaximin intragastrically to rats exposed to CUMS for 4 weeks and investigated the composition of the fecal microbiome, the content of short-chain fatty acids in the serum and brain, the functional profiles of microglia and hippocampal neurogenesis. RESULTS: Our results show that rifaximin ameliorated depressive-like behavior induced by CUMS, as reflected by sucrose preference, the open field test and the Morris water maze. Rifaximin increased the relative abundance of Ruminococcaceae and Lachnospiraceae, which were significantly positively correlated with the high level of butyrate in the brain. Rifaximin increased the content of anti-inflammatory factors released by microglia, and prevented the neurogenic abnormalities caused by CUMS. CONCLUSIONS: These results suggest that rifaximin can regulate the inflammatory function of microglia and play a protective role in pubertal neurodevelopment during CUMS by regulating the gut microbiome and short-chain fatty acids.

Effects of Rifaximin on Luminal and Wall-Adhered Gut Commensal Microbiota in Mice.

Rifaximin is a broad-spectrum antibiotic that ameliorates symptomatology in inflammatory/functional gastrointestinal disorders. We assessed changes in gut commensal microbiota (GCM) and Toll-like receptors (TLRs) associated to rifaximin treatment in mice. Adult C57BL/6NCrl mice were treated (7/14 days) with rifaximin (50/150 mg/mouse/day, PO). Luminal and wall-adhered ceco-colonic GCM were characterized by fluorescent in situ hybridization (FISH) and microbial profiles determined by terminal restriction fragment length polymorphism (T-RFLP). Colonic expression of TLR2/3/4/5/7 and immune-related markers was assessed (RT-qPCR). Regardless the period of treatment or the dose, rifaximin did not alter total bacterial counts or bacterial biodiversity. Only a modest increase in Bacteroides spp. (150 mg/1-week treatment) was detected. In control conditions, only Clostridium spp. and Bifidobacterium spp. were found attached to the colonic epithelium. Rifaximin showed a tendency to favour their adherence after a 1-week, but not 2-week, treatment period. Minor up-regulation in TLRs expression was observed. Only the 50 mg dose for 1-week led to a significant increase (by 3-fold) in TLR-4 expression. No changes in the expression of immune-related markers were observed. Rifaximin, although its antibacterial properties, induces minor changes in luminal and wall-adhered GCM in healthy mice. Moreover, no modulation of TLRs or local immune systems was observed. These findings, in normal conditions, do not rule out a modulatory role of rifaximin in inflammatory and or dysbiotic states of the gut.

Gastrointestinal (non-systemic) antibiotic rifaximin differentially affects chronic stress-induced changes in colon microbiome and gut permeability without effect on behavior.

Chronic stress is often accompanied by gastrointestinal symptoms, which might be due to stress-induced shift of gut microbiome to pathogenic bacteria. It has been hypothesized that stress alters gut permeability and results in mild endotoxemia which exaggerates HPA activity and contributes to anxiety and depression. To reveal the relationship between microbiome composition, stress-induced gastrointestinal functions and behavior, we treated chronically stressed mice with non-absorbable antibiotic, rifaximin. The "two hits" stress paradigm was used, where newborn mice were separated from their mothers for 3 h daily as early life adversity (maternal separation, MS) and exposed to 4 weeks chronic variable stress (CVS) as adults. 16S rRNA based analysis of gut microbiome revealed increases of Bacteroidetes and Proteobacteria and more specifically, Clostridium species in chronically stressed animals. In mice exposed to MS + CVS, we found extenuation of colonic mucosa, increased bacterial translocation to mesenteric lymph node, elevation of plasma LPS levels and infiltration of F4/80 positive macrophages into the colon lamina propria. Chronically stressed mice displayed behavioral signs of anxiety-like behavior and neophobia. Rifaximin treatment decreased Clostridium concentration, gut permeability and LPS plasma concentration and increased colonic expression of tight junction proteins (TJP1, TJP2) and occludin. However, these beneficial effects of rifaximin in chronically stressed mice was not accompanied by positive changes in behavior. Our results suggest that non-absorbable antibiotic treatment alleviates stress-induced local pathologies, however, does not affect stress-induced behavior.

Effects of Vitamin D3 on Intestinal Flora in a Mouse Model of Inflammatory Bowel Disease Treated with Rifaximin.

BACKGROUND Rifaximin is an antimicrobial agent used to treat inflammatory bowel disease (IBD). Vitamin D3 can control IBD due to its effects on inflammatory cytokines. The purpose of this study was to assess the effect of vitamin D3 on the intestinal flora of a dextran sulfate sodium (DSS)-induced mouse model treated with rifaximin. MATERIAL AND METHODS The mouse model of IBD was developed using DSS (4%) administered via the drinking water. Twenty-four male C57BL6 mice were divided into the control group with a normal diet (N=6), the DSS group with a normal diet (N=6), the DSS group with a normal diet treated with rifaximin (N=6), and the DSS group with a normal diet treated with rifaximin and vitamin D3 (N=6). After 14 days, the colonic tissue was studied histologically. Serum levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1ß (IL-1ß) and enzyme-linked immunosorbent assay (ELISA) were used to measure the level of IL-6 and P65, and phospho-p65 was measured by western blot. 16S rRNA gene sequencing was used to analyze fecal samples. RESULTS In the DSS mouse model of IBD, rifaximin reduced the inflammation severity of the colon and reduced the expression of phospho-p65, p65, TNF-alpha, and IL-6. In the DSS+rifaximin+vitamin D3 group, the therapeutic influences of rifaximin, in terms of weight loss and colonic disease activity, were significantly reduced, and the gut microbiota of the mice were completely changed in composition and diversity. CONCLUSIONS In a mouse model of IBD, treatment with vitamin D3 significantly increased the metabolism of rifaximin and reduced its therapeutic effects.

Effective Combination Therapy of Angiotensin-II Receptor Blocker and Rifaximin for Hepatic Fibrosis in Rat Model of Nonalcoholic Steatohepatitis.

The progression of nonalcoholic steatohepatitis (NASH) is complicated. The multiple parallel-hits theory is advocated, which includes adipocytokines, insulin resistance, endotoxins, and oxidative stress. Pathways involving the gut-liver axis also mediate the progression of NASH. Angiotensin-II receptor blockers (ARB) suppress hepatic fibrosis via the activation of hepatic stellate cells (HSCs). Rifaximin, a nonabsorbable antibacterial agent, is used for the treatment of hepatic encephalopathy and has been recently reported to improve intestinal permeability. We examined the inhibitory effects on and mechanism of hepatic fibrogenesis by combining ARB and rifaximin administration. Fischer 344 rats were fed a choline-deficient/l-amino acid-defined (CDAA) diet for 8 weeks to generate the NASH model. The therapeutic effect of combining an ARB and rifaximin was evaluated along with hepatic fibrogenesis, the lipopolysaccharide-Toll-like receptor 4 (TLR4) regulatory cascade, and intestinal barrier function. ARBs had a potent inhibitory effect on hepatic fibrogenesis by suppressing HSC activation and hepatic expression of transforming growth factor-ß and TLR4. Rifaximin reduced intestinal permeability by rescuing zonula occludens-1 (ZO-1) disruption induced by the CDAA diet and reduced portal endotoxin. Rifaximin directly affect to ZO-1 expression on intestinal epithelial cells. The combination of an ARB and rifaximin showed a stronger inhibitory effect compared to that conferred by a single agent. ARBs improve hepatic fibrosis by inhibiting HSCs, whereas rifaximin improves hepatic fibrosis by improving intestinal permeability through improving intestinal tight junction proteins (ZO-1). Therefore, the combination of ARBs and rifaximin may be a promising therapy for NASH fibrosis.

Efficacy of Aerosolized Rifaximin versus Tobramycin for Treatment of Pseudomonas aeruginosa Pneumonia in Mice.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that can cause chronic lung infections in patients with cystic fibrosis (CF). The current preferred treatment for CF lung infections includes inhaled tobramycin (TOB); however, studies suggest TOB cannot effectively inhibit biofilm formation. Using an NIH small compounds drug library approved for safe use in humans, we identified rifaximin (RFX), a semisynthetic, rifamycin family, nonsystemic antibiotic that inhibits alginate production and growth in P. aeruginosa Inhibition of alginate production was further analyzed using the uronic acid carbazole assay and a promoter reporter assay that measures the transcription of the alginate biosynthetic operon. Compared to TOB, RFX significantly reduced alginate production in laboratory and CF sputum isolates of P. aeruginosa In addition, RFX showed a narrow range of MICs when measured with multidrug-resistant bacterial species of clinical relevance, synergistic activities with TOB or amikacin against clinical isolates, as well as reduction toward in vitro preformed biofilms. In C57BL/6 mice, penetration of nebulized TOB into the lungs was shown at a higher level than that of RFX. Further, in vivo assessment using a DBA/2 mouse lung infection model found increased survival rates with a single-dose treatment of nebulized RFX and decreased P. aeruginosa PAO1 bioburden with a multiple-dose treatment of RFX plus TOB. In addition, mice treated with a single exposure to dimethyl sulfoxide (DMSO), a solvent that dissolves RFX, showed no apparent toxicity. In summary, RFX may be used to supplement TOB inhalation therapy to increase efficacy against P. aeruginosa biofilm infections.

Rifaximin prevents ethanol-induced liver injury in obese KK-Ay mice through modulation of small intestinal microbiota signature.

Exacerbation of alcoholic hepatitis (AH) with comorbid metabolic syndrome is an emerging clinical problem, where microbiota plays a profound role in the pathogenesis. Here, we investigated the effect of rifaximin (RFX) on liver injury following chronic-binge ethanol (EtOH) administration in KK-Ay mice, a rodent model of metabolic syndrome. Female, 8-wk-old KK-Ay mice were fed Lieber-DeCarli diet (5% EtOH) for 10 days, following a single EtOH gavage (4 g/kg body wt). Some mice were given RFX (0.1 g/L, in liquid diet) orally. Small intestinal contents were collected from mice without binge. Intestinal microbiota was quantified using aerobic and anaerobic culturing techniques and further analyzed by 16S rRNA sequencing in detail. EtOH feeding/binge caused hepatic steatosis, oxidative stress, and induction of inflammatory cytokines in KK-Ay mice, which were markedly prevented by RFX treatment. Hepatic mRNA levels for cluster of differentiation 14, Toll-like receptor (TLR) 4, TLR2, and NADPH oxidase 2 were increased following EtOH feeding/binge, and administration of RFX completely suppressed their increase. The net amount of small intestinal bacteria was increased over threefold after chronic EtOH feeding as expected; however, RFX did not prevent this net increase. Intriguingly, the profile of small intestinal microbiota was dramatically changed following EtOH feeding in the order level, where the Erysipelotrichales predominated in the relative abundance. In sharp contrast, RFX drastically blunted the EtOH-induced increases in the Erysipelotrichales almost completely, with increased proportion of the Bacteroidales. In conclusion, RFX prevents AH through modulation of small intestinal microbiota/innate immune responses in obese KK-Ay mice.NEW & NOTEWORTHY Here we demonstrated that rifaximin (RFX) prevents chronic-binge ethanol (EtOH)-induced steatohepatitis in KK-Ay mice. Chronic EtOH feeding caused small intestinal bacterial overgrowth, with drastic alteration in the microbiota profile predominating the order Erysipelotrichales. RFX minimized this EtOH induction in Erysipelotrichales with substitutive increases in Bacteroidales. RFX also prevented EtOH-induced increases in portal lipopolysaccharide, and hepatic cluster of differentiation 14, toll-like receptor (TLR) 2, and TLR4 mRNA levels, suggesting the potential involvement of microbiota-related innate immune responses.