Microbiota modulation in human health and disease: focus on the gut:liver:brain axis

Intestinal microbiota dysbiosis and modification of intestinal permeability leading to bacterial translocation, have been implicated in the development of numerous liver diseases or worsening of hepatic disorders, such as cirrhosis, portal hypertension, hepatic encephalopathy (HE) and acute-on-chronic-liver failure. There is strong evidence that the pathogenesis of cirrhosis and HE is linked to a dysbiotic gut microbiota and accumulation of microbial by-products, such as ammonia, indoles, oxindoles and endotoxins, which the liver fails to detoxify. Indeed, current main line clinical treatments target microbiota dysbiosis by decreasing numbers of pathogenic bacteria and reducing blood endotoxemia and ammonia levels. Despite the large amount of existing data, there is still a need to study in more detail the composition and the metabolic output of the gut microbiota and its crosstalk with host physiological function in liver failure associated HE. Aim of this thesis was to investigate the microbiota effects of the main current therapies used in clinical practice to treat HE. Impact of a prebiotic (lactulose), a probiotic (VLS#3) and an antibiotic (rifaximin) to modulate the gut microbiotia of cirrhotic patients both in terms of composition and metabolic output was investigated using pH controlled anaerobic batch cultures. Combining high-throughput Illumina sequencing of V3-V4 16S rRNA region, Fluorescent In Situ Hybridization coupled with flow cytometry and GC-MS, changes in faecal microbiota composition and metabolic output were measured. Significant metabolic rather than microbial changes were observed. Short chain fatty acids (acetate, propionate and acetate) production was promoted over time by lactulose and lactulose plus VSL#3 treatment and this increase was accompanied by a concomitant reduction of ammonia level and an increase in bifidobacteria. Rifaximin and its combination with lactulose was able to strongly reduce Streptococcaceae abundance, a known hallmark of cirrhotic dysbiosis, and concomitantly increase of Bifidobacteriales. Moreover I investigated how the use of VSL#3 impacted on the microbiota of paediatric patients and young adults affected by portal vein hypertension and minimal HE. VSL#3 supplementation resulted in a trend toward improved cognitive function and patients wellbeing. A trend towards an increased relative abundance in Actinobacteria and a 2 concomitant decrease in Bacteroidetes, known to be overabundant in HE dysbiosis, was observed . The results suggested also a slight increase in Ruminococcus and Faecalibacterium abundance. Indeed the data suggest an amelioration of dysbiotic condition by VSL#3 that could evolve in a decreased severity of cirrhosis progression. However, as the current pilot study was limited by sample size, these observation await confirmation in an adequately powered clinical trial. In an effort to design more efficacious microbiota modulatory tools, I also characterized a Lactobacillus brevis strain isolated from an alpine traditional cheese for its potential as a next-generation probiotics thanks to its ability to produce and secrete high amounts of the neurotransmitter γ-aminobutyric acid (GABA). Lb. brevis FEM 1874 was able to efficiently convert glutamate to GABA by the increased expression of the GAD operon genes resulting in high GABA accumulation in the culture medium. Moreover, FEM 1874 proved resistant to acidic pH, pancreatic fluids and bile acids, good indicators for probiotic survival in the gastro-intestinal tract. FEM 1874 was also able to ferment prebiotic fibres indicating the potential of using a synbiotic formulation targeting the gut:brain axis. Overall, the research herein showed the potential of microbiota modulatory formulations to target the dysbiosis related to gut:liver:brain axis disruption in liver disease and inducing metabolic changes capable of ameliorating related clinical symptoms.