*2.1. Gut Microbiota in Chronic Liver Diseases*

The human gastrointestinal (GI) tract is estimated to contain more than 10<sup>14</sup> microorganisms which collectively form the gut microbiota [4]. High motility and acidity within the esophagus and stomach limit colonization, however microbial numbers steadily increase throughout the small bowel, reaching the highest density in the colon where 10<sup>12</sup> bacteria can be found in every gram of dry feces [5].

The intestinal microbiota is composed primarily of bacteria (60% of dry fecal mass), but is also abundant in archaea, eukarya, and viral species [6]. Sequencing of 16S ribosomal DNA from mucosal and stool samples has shown that Firmicutes and Bacteroidetes are the two most abundant phyla in human feces, followed by Proteobacteria, Actinobacteria, Fusobacteria, and Verrucomicrobia species [7]. Mucosal and fecal microbiota harbor distinct microbial profiles, characterized by an abundance of Bacteroidetes in stool, while human colonic crypts are colonized mainly with Firmicutes and Proteobacteria [8].

The non-bacterial intestinal microbiota contributes significantly to health and disease, but has been largely unappreciated to date. Eukaryotes account for less than 0.03% of fecal microbes and are primarily composed of 200–300 fungal species [9]. The study of intestinal viromes has been limited due to challenges in isolation and culture; however, recent metagenomic analyses have revealed that bacteriophages comprise ~90% of the gut virome and contribute significantly to bacterial dynamics [10].

The gut microbiota is essential for proper digestion and, furthermore, plays an important role in facilitating gut immune responses against potential pathogens. Indeed, commensal *Bacteroides* and *Lactobacillus* spp. can stimulate the release and activation of antimicrobial peptides such as C-type lectins and pro-defensins from intestinal Paneth cells [11,12], activate intestinal B cells to express secretory IgA [13,14], and stimulate the production of protective mucus from colonic goblet cells [15]—all mechanisms that prevent bacterial translocation across the mucosa [16]. Bacteriophage adherence to mucus layers has also been hypothesized to protect against bacterial colonization and infiltration [17].

Disturbances within the gut microbiota, termed "dysbiosis", are linked to numerous diseases, many of which are hepatic in nature [18]. This is likely due to the bidirectional nature of the gut–liver axis: nutrient rich portal vein blood entering the liver originates from the gut, while hepatic bile

from gallbladder travels into the intestines to facilitate digestion [19]. Consequently, the insults that drive CLD, including caloric excess (NAFLD/NASH), alcoholism (ALD), and biliary damage (primary sclerosing cholangitis, primary biliary cirrhosis), can have significant effects on the gut microbiota, leading to intestinal permeability and exacerbation of inflammation and fibrosis.

Many studies have suggested an association between gut microbiome alteration and chronic liver diseases. Both Mouzaki et al. and Silva et al. demonstrated a reduction in *Bacteroidetes* and *Firmicutes* spp. in both NAFLD and NASH patients compared to adult healthy controls [20,21]. In pediatric studies, Zhu et al. measured a decrease in Firmicutes and increased Bacteroidetes in children with obesity or NASH [22]. A more recent, larger cohort study challenged these findings, finding a decrease of total Bacteroidetes in both NASH and NALFD pediatric patients, in agreement with adult studies [23].

In ALD, a reduction of *Lactobacillus* spp. has been recorded in both alcohol-consuming patients and mouse models [24,25]. Lactobacilli are beneficial bacteria commonly used in probiotics that have been shown to inhibit pathogen colonization [26]. Patients with ALD have also been found to have lower abundance of Bifidobacterium and Enterobacterium, and increased Proteobacteria, Fusobacteria, and Actinobacteria [27,28].

Changes in patient gut microbiota have also been measured in the context of worsening disease state. Indeed, significant differences in gut microbiota have been observed in NALFD subjects who had progressed to steatohepatitis or moderate fibrosis (F ≥ 2) when compared to patients with earlier stages of the disease. Boursier et al. found that NASH patients possessed a significantly larger abundance of *Bacteroides* and a reduction in *Prevotella* compared to NAFLD patients [29]. Recently, Bastian et al. also confirmed a significantly higher proportion of *Bacteroides* in fibrotic (F2–F4) patients compared to patients with minimal fibrosis (F0–F1). Two large studies by Loomba et al. and Caussy et al. also found a reduction in *Firmicutes* spp. and an enrichment of *Proteobacteria* spp. in patients with cirrhosis compared to those with minimal fibrosis [30]. In addition, Bajaj et al. recently demonstrated that periodontal therapy improves gut dysbiosis and systemic inflammation in cirrhotic patients [31]. Cirrhotic patients treated with scaling and root planning followed by oral hygiene showed a reduction in Enterobacteriaceae and Streptococcaceae, and a decrease of inflammatory markers interleukin (IL)-1β and IL-6 [31]. Together, these findings suggest that certain bacteria, likely Bacteriodes and *Proteobacteria* spp., and other factors such as oral health may play important roles in liver fibrosis progression.
