Salivary microbiota‐immune profiling in cirrhosis: Could this be the noninvasive strategy that will revolutionize prognostication in hepatology?
Authors: Vishal C. Patel, Debbie L. Shawcross
Journal: Hepatology (2015)

Potential conflict of interest: Dr. Shawcross consults for, advises for, is on the speakers' bureau for, and received grants from Norgine. Dr. Patel received grants from Norgine. See Article on Page 1260. Humans are colonized by a diverse range of microbes, the most abundantly inhabited being the gastrointestinal tract, which anatomically commences at the oral mucosa, with the colonic lumen being the most densely populated. The gut microbiota consist mainly of bacteria, but it is worth remembering that fungi, viruses, archaea, and protozoa are also resident and active. As a composite, these microorganisms outnumber human cells by a factor of 10. We can carry up to 2 kg of microbes within our gut, and these tens of trillions of microorganisms have been referred to as the "last human organ."1 Whereas these microbes consist of over 3 million genes, or an impressive 150 times the number contained within our own genome, two thirds of the entire gut microbiome is unique to each individual.2 Furthermore, although the gut microbiome consists of greater than 1,000 species of bacteria, approximately 170 species predominate in any given individual, with Bacteroidetes and Firmicutes being the dominant phyla.3 The diverse and complex role played by the gut microbiome includes the development and modulation of the innate and adaptive immune systems, both locally within the intestinal mucosa with defense against pathogens and tumorigenesis and systemically. Dietary and homeostatic functions pertain to the biosynthesis of vitamins, metabolism of glucose, bile salts, and xyloglucans, as well as the liberation of short‐chain fatty acids (SCFAs) from otherwise indigestible dietary starches, mainly by bacteria from the Bacteroidetes phylum. SCFAs have immune‐signaling and anti‐inflammatory properties and are a vital source of energy for intestinal mucosal cells, helping to maintain intestinal integrity and barrier function. Therefore, a change to the gut microbiome impacts on a multitude of vital homeostatic functions, many of which the human host cannot perform independently. Technological advances in genomic sequencing and bioanalytics are beginning to revolutionize our understanding of the gut microbiome in relation to hepatological diseases. Further insights are now being gained with the unraveling of some of the complex mechanisms that contribute to the commensal and synergistic interactions between the host and our microbiome, as well as the downstream mechanistic effects in both normal physiology and when these processes are disturbed inducing pathology. It is well established that patients with cirrhosis have enteric dysbiosis with small bowel bacterial overgrowth and translocation of bacteria and their products (such as lipopolysaccharide [LPS], flagellin, peptidoglycan, and bacterial DNA) across a more permeable gut epithelial barrier, which is exacerbated by underlying portal hypertension (PH) and endothelial dysfunction.4 This culminates in systemic inflammation and endotoxemia, which induces innate immune dysfunction by Toll‐like receptor signaling, predisposing to infection, and development of decompensating complications, such as hemorrhage, sepsis, and hepatic encephalopathy (HE; Fig. 1).5 Indeed, it is widely recognized that intestinal decontamination with nonabsorbable antibiotics, such as rifaximin (RIF), is an effective treatment for HE.6 This implicates gut dysbiosis and the ensuing metabolic sequelae involving intestinal absorption of nitrogenous compounds in the progression of neurocognitive dysfunction.Figure 1: Oral‐gut‐liver–brain axis in the pathogenesis of HE. Patients with cirrhosis have salivary and enteric dysbiosis with small bowel bacterial overgrowth and translocation of bacteria and their products (such as LPS and bacterial DNA) across a leaky gut, which is exacerbated by underlying PH and endothelial dysfunction. This culminates in hyperammonemia, systemic inflammation, and endotoxemia, which induces innate immune dysfunction, predisposing to infection and the development of decompensating complications, such as hemorrhage, sepsis, and HE.Enteric dysbiosis is also causally linked to the pathogenesis of cirrhosis and the progression to advanced liver disease. Utilizing quantitative metagenomics, it has been recently shown that 75,245 microbial genes differ in abundance between patients with cirrhosis and healthy individuals and, of relevance, that over 50% of these taxonomically assigned bacterial species are of buccal origin, suggesting an invasion of the gut from the mouth in cirrhosis.7 Bajaj et al., in this issue of Hepatology, reveal that salivary dysbiosis, represented by a reduction in autochthonous bacteria, is indeed present and, to an extent, reflects the changes observed in the fecal microbiota in cirrhosis, with an associated systemic and salivary‐specific inflammatory milieu.8 Of note, salivary dysbiosis was more pronounced in the high number of patients (37%; 38 of 102) that went on to be hospitalized during the 90‐day follow‐up. This implies a causative role, particularly when considering that cirrhotic saliva is enriched with genes related to endotoxin synthesis proteins, as well as nucleic acid and vitamin metabolism, the latter of which are purported to modulate intestinal barrier integrity and oxidative stress. This work confirms that there are clear differences in systemic inflammatory response, as represented by detection in serum of various proinflammatory cytokines in those patients with cirrhosis with previous HE, compared to no HE. However, the group of patients with cirrhosis with previous HE did have more advanced liver disease with ascites, which may propagate bacterial translocation with more‐severe PH, and very significant differences in Model for End‐Stage Liver Disease (MELD) and Child‐Pugh scores. What is somewhat unexpected, though, is the finding that there was no significant additive effect in dysbiosis ratios of saliva or stool in previous HE patients in relation to the presence of ascites, administration of RIF or proton pump inhibitors, or the presence of diabetes mellitus, given what has previously been reported in regard to the generation of endotoxemia, susceptibility to infection, and impact on gut microbiota.9 This warrants further investigation in appropriately powered studies. However, the contribution to the systemic inflammatory process from salivary dysbiosis, compared to the gut, is likely to be small, and this is attributed mainly to the quantum difference in the number of bacteria between the two sites translating into a relative effect, with the gut being the main driver of inflammation more distantly. However, it is interesting that, above and beyond confirming the presence of dysbiosis in the saliva of individuals with cirrhosis, Bajaj et al. now present novel evidence of a buccal proinflammatory milieu with higher salivary interleukin (IL)−1β and IL‐6 concentrations and a resultant increase in secretory immunoglobulin A (IgA). This is shown to be accompanied by a reduction in histatins 1 and 5 and lysozyme, in keeping with impairment of local innate defenses of the oral cavity. These impaired salivary defenses are associated with an oral proinflammatory environment, and this represents a global mucosal‐immune interface change in cirrhosis given what is already known about similar dysbiotic changes affecting the gut microbiota. These findings also have potential implications for susceptibility to infections of the respiratory tract given its anatomical proximity to the oral mucosa. Salivary dysbiosis may perhaps contribute, as an example, to the increased predilection for ventilator‐associated pneumonia in the event that these patients with cirrhosis require such invasive organ support. Prospective studies have clearly demonstrated that critically ill patients with cirrhosis affected by ventilator‐associated pneumonia have a more severe clinical presentation, worse clinical outcomes, and significantly increased 28‐ and 90‐day mortality,10 and it is possible that the oral dysbiotic field changes described in this issue of Hepatology have some responsibility that warrant further study. So, how does this study contribute to our understanding of the role of the enteric microbiome in the pathogenesis of inflammation and immune dysfunction in cirrhosis, and how might this allow therapies to be developed that ultimately lead to improvement in clinically relevant outcomes? It is clear that further evaluation of the salivary microbiome—and perhaps that existing within other sites in the host—is an exciting prospect given this biofluid's representativeness of the intestinal flora. From a practical perspective, saliva is also more readily accessible and "socially acceptable" to our patients when compared with fecal sampling. Well‐designed studies are warranted to better understand the role of the enteric microbiome in the human host in the pathogenesis and progression of liver disease and must be undertaken by profiling the genes expressed and the downstream metabolites and effector proteins to make the leap from correlation to causality. This will better inform us in the development of microbial‐modulating therapies, including pre‐, pro‐, and synbiotics, immunomodulatory diets, and even fecal microbial transplantation, with the ultimate goal being the restoration of enteric homeostasis. Application of advanced molecular techniques in the near future will not only become a powerful tool that potentially offers superior prognostication, compared to the traditional scoring systems such as MELD, but will also bring into reality the concept of personalized medicine by utilizing metagenomic fingerprinting, enabling individualized immune profiling.