Characterization of gut microbiome composition in Iranian patients with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.

Gut microbiota dysbiosis is intimately associated with development of non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Nevertheless, the gut microbial community during the course of NAFLD and NASH is yet to be comprehensively profiled. This study evaluated alterations in fecal microbiota composition in Iranian patients with NAFLD and NASH compared with healthy individuals. This cross-sectional study enrolled 15 NAFLD, 15 NASH patients, and 20 healthy controls, and their clinical parameters were examined. The taxonomic composition of the fecal microbiota was determined by sequencing the V3-V4 region of 16S rRNA genes of stool samples. Compared to the healthy controls, NAFLD and NASH patients presented reduced bacterial diversity and richness. We noticed a reduction in the relative abundance of Bacteroidota and a promotion in the relative abundance of Proteobacteria in NAFLD and NASH patients. L-histidine degradation I pathway, pyridoxal 5'-phosphate biosynthesis I pathway, and superpathway of pyridoxal 5'-phosphate biosynthesis and salvage were more abundant in NAFLD patients than in healthy individuals. This study examined fecal microbiota dysbiosis in NAFLD and NASH patients and presented consistent results to European countries. These condition- and ethnicity-specific data could provide different diagnostic signatures and therapeutic targets.

Extracellular vesicles and pasteurized cells derived from Akkermansia muciniphila protect against high-fat induced obesity in mice.

BACKGROUND: Several studies have shown that probiotics have beneficial effects on weight control and metabolic health. In addition to probiotics, recent studies have investigated the effects of paraprobiotics and postbiotics. Therefore, we evaluated the preventive effects of live and pasteurized Akkermansia muciniphila MucT (A. muciniphila) and its extracellular vesicles (EVs) on HFD-induced obesity. RESULTS: The results showed that body weight, metabolic tissues weight, food consumption, and plasma metabolic parameters were increased in the HFD group, whereas A. muciniphila preventive treatments inhibited these HFD. The effects of pasteurized A. muciniphila and its extracellular vesicles were more noticeable than its active form. The HFD led to an increase in the colonic, adipose tissue, and liver inflammations and increased the expression of genes involved in lipid metabolism and homeostasis. Nevertheless, these effects were inhibited in mice that were administered A. muciniphila and its EVs. The assessment of the gut microbiota revealed significant differences in the microbiota composition after feeding with HFD. However, all treatments restored the alterations in some bacterial genera and closely resemble the control group. Also, the correlation analysis indicated that some gut microbiota might be associated with obesity-related indices. CONCLUSIONS: Pasteurized A. muciniphila and its EVs, as paraprobiotic and postbiotic agents, were found to play a key role in the regulation of metabolic functions to prevent obesity, probably by affecting the gut-adipose-liver axis.

The Protective Effects of Live and Pasteurized Akkermansia muciniphila and Its Extracellular Vesicles against HFD/CCl4-Induced Liver Injury.

Akkermansia muciniphila, as a member of the gut microbiota, has been proposed as a next-generation probiotic. Liver fibrosis is the main determinant of liver dysfunction and mortality in patients with chronic liver disease. In this study, we aimed to determine the beneficial effects of live and pasteurized A. muciniphila and its extracellular vesicles (EVs) on the prevention of liver fibrosis. The response of hepatic stellate cells (HSCs) to live and pasteurized A. muciniphila and its EVs was examined in quiescent, lipopolysaccharide (LPS)-activated LX-2 cells. Liver fibrosis was induced in 8-week-old C57BL/6 mice, using a high-fat diet (HFD) and carbon tetrachloride (CCl4) administration for 4 weeks. The mice were concomitantly treated via oral gavage with three forms of bacteria. The relative expression of different fibrosis and inflammatory markers was assessed in the tissues. Histological markers, serum biochemical parameters, and cytokine production were also analyzed, and their correlations with the relative abundance of targeted fecal bacteria were examined. All A. muciniphila preparations exhibited protective effects against HSC activation; however, EVs showed the greatest activity in HSC regression. Oral gavage with A. muciniphila ameliorated the serum biochemical and inflammatory cytokines and improved liver and colon histopathological damages. The relative expression of fibrosis and inflammatory biomarkers was substantially attenuated in the tissues of all treated mice. The composition of targeted stool bacteria in the live A. muciniphila group was clearly different from that in the fibrosis group. This study indicated that A. muciniphila and its derivatives could successfully protect against HFD/CCl4-induced liver injury. However, further studies are needed to prove the beneficial effects of A. muciniphila on the liver. IMPORTANCE Akkermansia muciniphila, as a member of the gut microbiota, has been proposed as a next-generation probiotic. Liver fibrosis is the main determinant of liver dysfunction and mortality in patients with chronic liver disease. In this study, we aimed to determine the beneficial effects of live and pasteurized A. muciniphila and its extracellular vesicles (EVs) on the prevention of liver fibrosis. The results of the present study indicated that oral administration of live and pasteurized A. muciniphila and its EVs could normalize the fecal targeted bacteria composition, improve the intestinal permeability, modulate inflammatory responses, and subsequently prevent liver injury in HFD/CCl4-administered mice. Following the improvement of intestinal and liver histopathology, HFD/CCl4-induced kidney damage and adipose tissue inflammation were also ameliorated by different A. muciniphila treatments.

Comparative effects of alive and pasteurized Akkermansia muciniphila on normal diet-fed mice.

Recently, Akkermansia muciniphila an anaerobic member of the gut microbiota, has been proposed as a next-generation probiotic. The aim of this study was evaluation of the effect of alive and pasteurized A. muciniphila on health status, intestinal integrity, immune response, lipid metabolism, and gut microbial composition in normal-diet fed mice as well as direct effects of the bacterium on Caco-2 cell line. A total of 30 mice were distributed into three different groups, control, alive, and pasteurized A. muciniphila-treated group. After acclimation, control and treatment groups were administrated with PBS and 109 CFU/200µL of bacterial suspension for 5 weeks, respectively. Besides, Caco-2 separately exposed to alive, pasteurized A. muciniphila and PBS for 24 h. The results showed that administration of A. muciniphila leads to reduction in body, liver, and white adipose weight. Histology data revealed both treatments had no adverse effects in colon, liver, and adipose tissues as well as induced better gut structure. Moreover, biochemical parameters and inflammatory biomarkers in plasma demonstrated that pasteurized A. muciniphila had more pronounce effect. Furthermore, alive A. muciniphia had better effects on the modulation of gene expression related to fatty acid synthesis, energy homeostasis, and immune response in the liver; meanwhile, these effects in the adipose was more in the pasteurized A. muciniphila administration. More importantly, the improvement of gut health by enhancing strengthen intestinal integrity and maintaining immune homeostasis was seen in both treatments; notably, pasteurized A. muciniphila had more effective. Similarly, treatment with the pasteurized form more effectively upregulated tight junction and regulated immune response-related genes in Caco-2 cell line. Both treatments triggered the improvement of microbiota communities, particularly the alive form. Therefore, both forms of A. muciniphila could modulate lipid and immune homeostasis, improved some gut microbiota, and promoted the overall health, while all these effects were dominantly observed in pasteurized form. In conclusion, pasteurized A. muciniphila can be considered as new medical supplement to maintain health state and prevent diseases in normal mice through different mechanisms.

Probiotic-Induced Tolerogenic Dendritic Cells: A Novel Therapy for Inflammatory Bowel Disease?

Inflammatory bowel diseases (IBDs) are immune-mediated, chronic relapsing diseases with a rising prevalence worldwide in both adult and pediatric populations. Treatment options for immune-mediated diseases, including IBDs, are traditional steroids, immunomodulators, and biologics, none of which are capable of inducing long-lasting remission in all patients. Dendritic cells (DCs) play a fundamental role in inducing tolerance and regulating T cells and their tolerogenic functions. Hence, modulation of intestinal mucosal immunity by DCs could provide a novel, additional tool for the treatment of IBD. Recent evidence indicates that probiotic bacteria might impact immunomodulation both in vitro and in vivo by regulating DCs' maturation and producing tolerogenic DCs (tolDCs) which, in turn, might dampen inflammation. In this review, we will discuss this evidence and the mechanisms of action of probiotics and their metabolites in inducing tolDCs in IBDs and some conditions associated with them.

Assessment of fecal Akkermansia muciniphila in patients with osteoporosis and osteopenia: a pilot study.

OBJECTIVES: Osteoporosis is characterized by slow deterioration in bone mass and disruption of its structure, leading to an increased risk of bone fractures. Gut microbiota plays an important role in the transport and absorption of nutrients needed for bone health. Akkermansia muciniphila is one of the gut microbiota members that its beneficial role in prevention of metabolic disorder was suggested. The aim of the current pilot study was the assessment of fecal A. muciniphila in patients with osteoporosis and osteopenia. METHODS: A total of 36 subjects including eight with osteoporosis (three men and five women), eight with osteopenia (two men and six women), and 20 normal controls (six men and 14 women) were selected. Microbial genome was extracted from fresh stool samples. The bacterial load was determined by quantitative real-time PCR using 16S rRNA specific primers. RESULTS: The participants' mean age in the osteoporosis, osteopenia and control groups were 61.71, 45 and 45.05 years, respectively. The majority of osteoporosis patients were post-menopause women, while in osteopenia group was pre-menopause. There were significant differences in terms of age, T-score, Z-score, and menopause among groups (P value < 0.05). The presence of A. muciniphila was higher in the healthy group compared to osteopenia group; however, these differences were not statistically significant. CONCLUSIONS: In conclusion, however, there was no statistically significant difference between the study groups; it seems that the load of A. muciniphila may be related to bone health. Further in vivo and in vitro studies are needed to investigate the immunological and biochemical pathways.

The Anti-fibrotic Effects of Heat-Killed Akkermansia muciniphila MucT on Liver Fibrosis Markers and Activation of Hepatic Stellate Cells.

Hepatic stellate cell (HSC) activation is a key phenomenon in development of liver fibrosis. Recently, Akkermansia muciniphila has been introduced as a next-generation microbe residing in the mucosal layer of the human gut. Due to the probable risks associated with the use of live probiotics, the tendency to use heat-killed bacteria has been raised. Herein, we investigated the potential anti-fibrotic effects of heat-killed A. muciniphila MucT on activation of HSCs. The human LX-2 cells were stimulated by various concentrations of LPS to evaluate the optimal concentration for HSC activation. Cell viability of LX-2 cells treated with LPS and heat-killed A. muciniphila MucT was measured by MTT assay. Scanning electron microscopy was used to analyze the morphology of heat-killed bacteria. Quiescent and LPS-stimulated LX-2 cells were coinfected with heat-killed A. muciniphila MucT. The gene expression of α-SMA, TIMP, Col1, TGF-ß, TLR4, and PPARγ was analyzed using quantitative real-time PCR. Our results showed that LPS treatment led to a significant increase in fibrosis markers in a concentration-independent manner (P < 0.0001), and significantly downregulated the expression of PPARγ (P < 0.0001). The heat-killed A. muciniphila MucT could significantly modulate the expression of fibrosis markers particularly in MOI 10 (P < 0.0001), and reversed the HSC activation in LPS-stimulated LX-2 cells. In conclusion, we demonstrated that heat-killed A. muciniphila MucT was safe and capable to ameliorate LPS-induced HSC activation through modulation of fibrosis markers. Further in vivo studies are required to validate the anti-fibrotic properties of heat-killed A. muciniphila MucT.

Global scientific output trend for Akkermansia muciniphila research: a bibliometric and scientometric analysis.

BACKGROUND: Akkermansia muciniphila is an anaerobic bacterium residing in the healthy intestinal tract of host and its quantity has a negative correlation with various host diseases. This study for the first time provides a holistic bibliometric aspect of Akkermansi muciniphila research in the literature and shows the hot topic terms of these articles in any period of time. METHODS: Scopus database was selected to retrieve documents relevant to Akkermansia muciniphila in any language up to 2019. The bibliometric profile of Akkermansia muciniphila articles including subject area, year distribution, citations, institutions, journals, authors, and countries was systematically characterized and the collaboration networks of authors and countries as well as the burst detection algorithm of the words in the titles, abstracts and keywords were visualized. RESULTS: There is a progressive growing trend in research on Akkermansia from 2004 to 2019 with a total of 566 articles during this period. Out of 353 original articles, there are 194 animal studies (155 studies on mice) and 112 human studies. Also, 65 various diseases were investigated in these studies. The most focused conditions are obesity (71 articles) and type2 diabetes (39 articles). The United States is the leading country on Akkermansia publications (n = 132), followed by China (n = 95). Frontiers in Microbiology is the most dominant journal with 23 Akkermansia publications. In addition, "cancer" is the hot topic of recent Akkermansia research. CONCLUSION: Akkermansia research is of progressive interest during the last decade and the studies on this subject move towards its relationship with cancer and its promising effect on health.

Akkermansia muciniphila-Derived Extracellular Vesicles as a Mucosal Delivery Vector for Amelioration of Obesity in Mice.

Recent evidence suggests that probiotics can restore the mucosal barrier integrity, ameliorate inflammation, and promote homeostasis required for metabolism in obesity by affecting the gut microbiota composition. In this study, we investigated the effect of Akkermansia muciniphila and its extracellular vesicles (EVs) on obesity-related genes in microarray datasets and evaluated the cell line and C57BL/6 mice by conducting RT-PCR and ELISA assays. A. muciniphila-derived EVs caused a more significant loss in body and fat weight of high-fat diet (HFD)-fed mice, compared with the bacterium itself. Moreover, treatment with A. muciniphila and EVs had significant effects on lipid metabolism and expression of inflammatory markers in adipose tissues. Both treatments improved the intestinal barrier integrity, inflammation, energy balance, and blood parameters (i.e., lipid profile and glucose level). Our findings showed that A. muciniphila-derived EVs contain various biomolecules, which can have a positive impact on obesity by affecting the involved genes. Also, our results showed that A. muciniphila and its EVs had a significant relationship with intestinal homeostasis, which highlights their positive role in obesity treatment. In conclusion, A. muciniphila-derived EVs can be used as new therapeutic strategies to ameliorate HFD-induced obesity by affecting various mechanisms.

The Helicobacter pylori resistance rate to clarithromycin in Iran.

BACKGROUND: Clarithromycin is the most commonly suggested antibiotic in Helicobacter pylori eradication regimens. Increasing antibiotic resistance rate to clarithromycin is the main reason for therapeutic failure. The resistance is associated with mutations in the 23S rRNA gene. AIM: The purpose of this study was to determine the clarithromycin resistance rate of isolated H. pylori strains from referral patients in Kashan, Iran. METHODS: In total 95 H. pylori strains isolated from gastric mucosa of 246 patients with different clinical signs underwent gastroscopy in Kashan, Iran in 2013. The Epsilometer test was used to determine the minimum inhibitory concentrations (MICs) of clarithromycin (CLR) in isolated H. pylori strains. Occurrence of mutation in 23S rRNA gene of clarithromycin-resistant strains was investigated with polymerase chain reaction-sequencing method. RESULTS: Antibiotic susceptibility pattern showed that 32 strains (33.7%) were resistant to clarithromycin (MICs ≥1 µg/ml), 33.7% were intermediate resistant, while 31 of H. pylori strains (32.6%) were susceptible. The 23S rRNA gene mutations at positions A2143G and A2142G were detected in H. pylori clarithromycin-resistant strains. CONCLUSION: The resistance rate to clarithromycin in Iran is high. H. pylori clarithromycin-resistant strains were not associated with sex, age, and disease. The A2143G mutation in clarithromycin-resistant H. pylori strains was the predominant finding.