Intestinal virome and therapeutic potential of bacteriophages in liver disease

Received 3 June 2021; received in revised form 20 July 2021; accepted 2 August 2021; available online xxx Humans harbour a large quantity of microbes in the intestinal tract and have evolved symbiotic relationships with many of them. However, several specific bacterial pathobionts are associated with liver disease pathogenesis. Although bacteriophages (phages) and eukaryotic viruses (collectively known as “the virome”) outnumber bacteria and fungi in the intestine, little is known about the intestinal virome in patients with liver disease. As natural predators of bacteria, phages can precisely edit the bacterial microbiota. Hence, there is interest in using them to target bacterial pathobionts in several diseases, including those of the liver. Herein, we will summarise changes in the faecal virome associated with fatty liver diseases and cirrhosis, and describe the therapeutic potential of phages and potential challenges to their clinical application. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).


Introduction
Work over the last few decades has increasingly shed light on the myriad of ways in which the different microbial communities that colonise our guts influence human health and disease. 1 These microbial communities are composed of bacteria, fungi, viruses, and archaea that together encode >100-fold more genes than the human genome. 2 The composition of the gut microbiome is significantly influenced, even from birth, by our surrounding environment and these encoded genes in turn have the potential to be of both benefit and harm. 3 Most of our understanding of how the gut microbiota affects human disease has been focused on bacteria, but with new advances in metagenomic methods, viruses are beginning to receive more attention.
About 90% of the human intestinal virome is made up of bacteriophages (phages or prokaryotic viruses), while the other 10% are eukaryotic viruses (Fig. 1). 4 Eukaryotic DNA and RNA viruses include plant and mammalian viruses, some of which can use intestinal cells as their host. Some eukaryotic viruses can affect human health by causing disease, like the well-known enteric pathogens Norovirus, Rotavirus, and Enterovirus, while others are not pathogenic. Plant viruses are likely derived from the diet. 5 The phageome consists of approximately 10 15 bacteriophages and is largely composed of the order Caudovirales (family Siphoviridae, Myoviridae, and Podoviridae) and family Microviridae. 6 In healthy individuals, the intestinal viral microbiome exhibits a high level of interpersonal heterogeneity with relative intrapersonal stability. 7,8 However, changes in the virome community can be seen with changes in lifestyle such as diet and with different disease states. 8,9 Deep sequencing of the intestinal viral microbiome in healthy individuals suggests that there is a small core group of phages shared among the majority of people, with a wider range of rarer phages that are unique to individuals. 10 Understanding how the composition of the intestinal viral microbiome differs amongst individuals with different disease states will help us elucidate the mechanism by which the viral microbiome influences disease. Differences in the viral microbiome have already been implicated in the pathogenesis of obesity, type 2 diabetes, colon cancer, inflammatory bowel disease, and more.
Herein, we will review current literature focused on the human intestinal virome in liver disease. An estimated 1.5 billion people have chronic liver disease worldwide and an estimated 1.2 million patients with cirrhosis will die every year, making it one of the leading causes of death globally. 11 Our existing strategies for reversing or preventing progression of liver disease are limited and often liver transplantation is the only therapy available to patients once they progress to endstage liver disease. In recent years, we have improved our understanding of how the intestinal microbiome contributes to liver disease and with that, there is increased interest in targeting the intestinal microbiome to treat liver disease. Hence, we will also review the use of phage therapy in gastrointestinal and liver diseases and summarise the key bacteria that may serve as potential targets for phage therapy in the future.

Intestinal virome in patients with liver disease
Both eukaryotic viruses and bacteriophages have been implicated in liver disease pathogenesis. Eukaryotic viruses include the known pathogenic and hepatotropic viruses, hepatitis A (Picornaviridae family) and E (Hepeviridae family), which can be transmitted by the oral-faecal route and detected in the stool. Both HAV and HEV exist in two forms, a non-enveloped form comprised of a capsid surrounding the RNA genome and a quasienveloped form that is masked within a layer of the host cell membrane. 12 The non-enveloped form, which is found in the stool and saliva of infected individuals, can survive harsh conditions such as transit through the gastrointestinal tract and cross the intestinal barrier into the blood via mechanisms that are not well understood. Recent work suggests that once in the blood, HAV harnesses endosomal gangliosides to infect hepatocytes and Kupffer cells, 13 where it replicates and exits back into the bloodstream in its quasienveloped form, which camouflages its antigenic proteins from neutralising antibodies. 14 Other known eukaryotic viruses that can be found in the intestinal virome and cause liver injury include Epstein-Barr virus (EBV), cytomegalovirus, and severe acute respiratory syndrome coronavirus 2. [15][16][17] Recent work by Jiang et al. investigating the intestinal virome in patients with alcoholic hepatitis and alcohol use disorder demonstrated that variations in intestinal viral taxa are associated with disease severity and mortality. 18 Comparing the intestinal viromes of patients with alcoholic hepatitis, alcohol use disorder, and controls, faecal samples from patients with alcohol use disorder had significantly higher viral diversity and richness compared with controls, and this was generally correlated with lower bacterial diversity. In patients with alcoholic hepatitis, Escherichia, Enterobacteria, and Enterococcus phages were overrepresented compared to controls, while Parabacteroides phages were underrepresented. Further, an increased abundance of Staphylococcus phages and Citrobacter phages were associated with increased disease severity.
Aside from differences in phage composition, faecal samples from patients with alcoholic hepatitis also contained significantly more mammalian viruses, such as those from the Parvoviridae and Herpesviridae families, than controls. Herpesviridae was only present in faecal samples from patients with alcoholic hepatitis, with most of the assigned reads attributed to EBV. 18 It is unclear why EBV is only detected in the guts of patients with alcoholic hepatitis, though a possible hypothesis is suppression of immunosurveillance in these patients. Alternatively, EBV reactivation might induce the development of hepatitis in alcoholic patients. Notably, a study of the intestinal virome in patients with non-alcoholic fatty liver disease (NAFLD) did not observe increased proportions of mammalian viruses compared to controls. 19 Further studies are needed to confirm and characterise the intestinal mammalian virus population in patients with alcoholassociated liver disease as this may shed light on its pathogenesis.
Another difference noted between NAFLD and alcohol-associated liver disease is that patients with NAFLD and fibrosis had significantly lower intestinal viral diversity and proportionately fewer phages compared with controls. 19 Incorporating faecal viral diversity in addition to clinical data into a model to non-invasively predict histologic fibrosis severity significantly improved the model's diagnostic accuracy. Additionally, the abundance of several Lactococcus and Leuconostoc phages were inversely correlated with severity of liver fibrosis, whereas the abundance of Lactobacillus phages was positively correlated with the severity of liver fibrosis. Though the abundance of some phages was inversely correlated with their respective bacterial hosts, viral diversity did not correlate with bacterial diversity. It is difficult to draw conclusions regarding how liver disease affects the phage/bacteria relationship with data from a single timepoint.
One study evaluated phage/bacteria interactions across 2 timepoints in patients with compensated cirrhosis before and after 8 weeks of treatment with rifaximin. 20 This study reported a significant reduction in the genus-level richness of the bacterial but not viral population after rifaximin use. Decreased complexity of bacterial-phage interactions was also seen after rifaximin, with complete collapse of bacterial-phage interactions seen in phages directed against pathobionts such as Streptococcus, Pseudomonas, and Enterobacteriaceae spp. These changes are most likely secondary to the direct impact of rifaximin on the bacterial population, and it is unclear how much cirrhosis contributed to these dynamics. Cross-sectional analysis revealed that phage-bacterial correlation network complexity was highest in controls, lowest in patients with cirrhosis taking only lactulose, and improved in patients taking both lactulose and rifaximin. 20 A notable technical difference between these studies is that this study performed metagenomic sequencing of faecal DNA whereas the prior 2 studies used filtration techniques to isolate RNA-and DNA-containing viral particles from stool, followed by metagenomic sequencing.
Research on the intestinal virome is in its infancy and a causative link between changes in the

Key point
Patients with liver disease exhibit differences in both the diversity and composition of their intestinal viromes compared with healthy controls, though the impact of these differences on the bacterial microbiome requires further study. phageome and disease has not been established. It remains to be seen whether changes in the virome are disease drivers, or whether they are the result of disease. Future longitudinal studies are required to confirm virome changes in independent cohorts of patients, and to test the stability of the faecal virome and its correlation with liver disease severity over time. The analysis of the virome depends on metagenomic sequencing, methods for virome research have not been standardised, and only a small fraction of all sequences can be assigned to known viral taxa in public databanks. Improvements in bioinformatic analysis will lead to a better understanding of the dynamics of phage-bacteria interactions. This will allow us to answer the question of whether changes in phages drive bacterial dysbiosis or vice versa.

Phage biology and their therapeutic potential
Bacteriophages -natural predators of bacteria Phages are viruses that infect bacteria and are considered to be the most numerous group of viruses on the planet, with an estimated 10 31 total phage particles. 21 Shortly after the discovery of phages by Frederick Twort in 1915, 22 they were used to treat bacterial haemorrhagic dysentery. Phage therapy is the practice of using preparations of infectious phages to treat bacterial infections, which has the advantage over antibiotics of targeting specific bacterial species or strains while self-replicating and spreading to infect additional target bacterial cells. Phage therapy became very popular throughout the world to treat a wide range of diseases caused by both Gram-positive and Gram-negative pathogens, such as Staphylococcus, Streptococcus, Vibrio, Klebsiella, Enterobacter, Shigella, Escherichia, Pseudomonas and Providencia to name a few. 23 Commercial production of phage cocktails was initiated in France by what would later become L'Oreal, 24 followed by the Eliava Institute of Bacteriophage, Microbiology and Virology (EIBMV) (Tbilisi, Georgia) and the Hirszfeld Institute of Immunology and Experimental Therapy (HIIET) (Wroclaw, Poland). In the US, pharmaceutical giant Eli Lilly (Indianapolis, IN) produced 7 phage cocktails. 24 After the discovery and use of antibiotics, phage therapy fell out of favour in many Western countries, particularly the US. Much of the concern regarding the efficacy of phages as a therapeutic stemmed from reproducibility issues where the same successful cocktail of phages used on one patient did not work for all patients. This was presumably due to the narrow host range of the selected phages. Another problem was inflammatory responses to the phage cocktail. 25 This was more likely due to contamination of the lysate by bacterial endo and exotoxins used to grow the phages in production rather than an immune response to the phages. Likewise, there was concern that the rapid lysis of cells by phageencoded lytic enzymes can cause septic shock; however, this argument also applies to bactericidal antibiotics.
Phages come in all shapes and sizes, with genomes consisting of either double-stranded or single-stranded DNA or RNA. They were originally defined based on morphology and categorised into 21 morphotypes, before nucleic acid sequencing technologies started being used for taxonomic classification. 26,27 Phages can be tailed, polyhedral, filamentous, pleomorphic, enveloped or not, but it is the double-stranded DNA tailed phages in the order Caudovirales, that are both the most common and the most commonly used for therapeutic

Bacteriophages (>90%)
Phageome with 10 15 phages Lytic and lysogenic phages purposes. In addition to morphotype and nucleic acid type, there are 2 main lifestyle categories of phages, lytic (a.k.a., virulent, obligately lytic) and temperate. Lytic phages can replicate only via the lytic life cycle that ends with the destruction of the infected bacterial cell and release of progeny phages ( Fig. 2A) while temperate phages can choose between the lytic and lysogenic life cycles (Fig. 2B). The latter includes the integration of phage DNA into the bacterial chromosome and its passive replication. [28][29][30] Temperate phages show reduced lytic abilities, may incorporate and transfer (i.e. transduce) bacterial DNA, including drug resistance and pathogenicity genes, and can convert a bacterium into a "lysogen" (i.e., bacterial cell with a viral genome integrated into the bacterial chromosome) that becomes immune to superinfection by the same phage or related phages. Therefore, temperate phages have historically not been used as therapeutics. 31 However, temperate phages can be modified to become obligately lytic 32,33 and since temperate phages are commonly found in bacteria, their modification could expand the arsenal of therapeutic phages, at least for some pathogens with limited or no known lytic phages. 34 Although the host range of a phage tends to be quite narrow (e.g., strain/serotype-or species-specific), 35 there are lytic phages that can infect more distantly related bacteria. 36 Host specificity is largely determined by receptor-binding proteins, a.k.a. anti-receptors. [37][38][39][40][41][42][43] These phage-encoded proteins enable high affinity binding of phage virions to receptors located on the outer surface of bacterial cells, such as lipopolysaccharide, lipoteichoic acid, capsular polysaccharide, flagella and pili. 44 Swapping domains or altering the sequence of specific regions of receptor-binding proteins results in altered host range specificity, enabling the phage to attach to different strains or different bacterial genera. [45][46][47][48] Phages typically encode a single receptor-binding protein, but can encode more than one, resulting in polyvalency (i.e., the ability to bind to more than 1 receptor and potentially more than 1 host organism/strain). 49 Resurgence of phage therapy The widespread overprescribing of antibiotics by physicians coupled with the overuse of antibiotics in the livestock industry are key factors that are thought to have led to the global spread of antibiotic-resistant bacteria. 50,51 This poses a serious public health problem since there are few, or in some cases, no drugs available to treat lifethreatening bacterial infections. Antibiotics are not entirely safe either, as they can cause allergic reactions and severe side effects, including organ damage and a clearing of the normal commensal gut microbiota, leaving the gut vulnerable to secondary infections by opportunistic pathogens such as Clostridioides difficile. 52 Because phages are host specific rather than broad spectrum like many antibiotics, phage therapy has the potential to have fewer off-target effects on beneficial bacterial microbiome species. Phages are now being used to treat livestock infections, [53][54][55] to prevent food spoilage, [56][57][58][59][60][61] in human compassionate use cases, and in clinical trials.

Phage therapy in gastrointestinal diseases
Over the last 2 decades, several clinical trials have been performed with T4-like phages (Table 1). Oral administration of phage cocktails is considered safe in both healthy adults and children, with only occasional side effects independent of phage dosage. [62][63][64][65] To determine the safety and efficacy of phage therapy for gastrointestinal infections, a T4like coliphage cocktail was given orally for 4 days to children hospitalised with acute diarrhoea. Nonbacterial causes of diarrhoea were not ruled out. No adverse events were reported, suggesting the overall safety of the phage cocktail. 66 However, substantial intestinal replication of phages was not observed, and phage treatment did not lead to an improvement in quantitative diarrhoea parameters, such as stool output and frequency, compared to placebo. 66  One successful case was reported in 2016, in which a 68-year-old male patient was suffering from necrotising pancreatitis complicated by a pancreatic pseudocyst infected with multidrugresistant Acinetobacter baumannii. 67 Phages were applied by intracavitary and intravenous routes, and the patient completely recovered after 5 months. 67 Although this is only a case report, the obvious clinical improvements suggest that phage therapy might be useful for treating bacterial infections, and especially those caused by multidrugresistant bacteria.
Phage therapy may also be a promising way to precisely edit the gut microbiota. Two randomised, placebo-controlled trials have been performed to determine the safety and efficacy of phages in adults suffering from mild to moderate gastrointestinal distress (e.g., gas, bloating, diarrhoea, constipation, etc) (NCT03269617; NCT04511221). Over the 28-day study, oral administration of the coliphage cocktail was shown to be safe and well tolerated. Patients experienced a similar reduction in gastrointestinal symptom severity during both the treatment and placebo periods, suggesting that the phage therapy was ineffective, but there was also no evidence that patients' initial symptoms were secondary to overgrowth of the bacteria targeted by the administered phage. Future studies can evaluate the efficacy of phage therapy by documenting the interactions of phages in these cocktails with the specifically targeted bacterial strains obtained from treated patients.
In addition to these clinical trials, there are also some encouraging preclinical data (Table 1). Adherent-invasive E. coli (AIEC) have been shown to be involved in the pathogenesis of inflammatory bowel diseases. 68,69 Administration of a phage cocktail against these E. coli strains reduced intestinal AIEC colonisation in transgenic mice expressing the human AIEC receptor. 70 Furthermore, wild-type mice colonised with AIEC were protected from dextran sodium sulfate-induced colitis upon phage treatment, with less E. coli in faeces, as well as in ileal and colonic sections. 70 To evaluate the ability of the phage cocktail to target AIEC strains in patients, ileal biopsies from patients with Crohn's disease were spiked with an AIEC strain. Active phage replication was detected 5 hours and 24 hours after phage administration, confirming the killing potential of phages in such an environment. 70 A phase I/IIa placebo-controlled clinical trial was therefore initiated, to assess the safety and efficacy of the phage cocktails in patients with inactive Crohn's disease (NCT03808103).

Potential for phage therapy in liver disease
Known bacterial pathobionts driving liver disease as potential targets for phage therapy Our existing knowledge of the taxonomic differences in the bacterial microbiota of patients with liver disease can help guide further investigation into potential targets for phage therapy. In the following subsections, we summarise faecal bacterial changes in selected human liver diseases ( Table 2).
Non-alcoholic fatty liver disease NAFLD encompasses a spectrum of diseases ranging from excessive fat deposition in the liver in the absence of significant alcohol use (simple steatosis or NAFL) that can progress to liver inflammation (non-alcoholic steatohepatitis [NASH]), and eventually fibrosis. 71 Several studies have found a decreased faecal abundance of Faecalibacterium and specifically Faecalibacterium prausnitzii in both obese and non-obese patients with NASH. [72][73][74] Ruminococcus was enriched in obese patients with NASH in 1 study, 75 but reduced in other studies of obese 73 and non-obese patients. 74 Ruminococcus obeum was specifically found to be reduced in patients with NAFLD in 1 study. 76 Advanced fibrosis secondary to NASH is associated with an overall decrease in intestinal bacterial diversity and an increase in the relative abundance of Gram-negative bacteria such as Bacteroides and Escherichia. [76][77][78][79][80] Although no causative role of these bacterial strains for steatohepatitis has been demonstrated in preclinical models, Yuan et al. demonstrated that an ethanolproducing Klebsiella pneumoniae (K. pneumoniae) strain was present in 60% of a Chinese cohort of patients with NAFLD and that introducing this strain into mice induced steatohepatitis. 81 Alcohol-associated liver disease Heavy alcohol use leads to a spectrum of liver diseases beginning with steatosis, which can be reversible or can progress to steatohepatitis and fibrosis in susceptible patients. 82  correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis, and oral administration of cytolysin-positive E. faecalis promotes ethanol-induced liver injury in mice. 87 Autoimmune hepatitis Autoimmune hepatitis is a chronic inflammatory liver disease whose pathogenesis is poorly understood, though genetic susceptibility and loss of tolerance against liver antigens are proposed mechanisms. 88 Patients with autoimmune hepatitis have an overrepresentation of potential pathobionts, including Veillonella species such as Veillonella dispar, in their faecal microbiomes. 89 Translocation of Enterococcus gallinarum (E. gallinarum) to the liver triggered an autoimmune response in mice genetically predisposed to autoimmunity. Subsequent antibiotic treatment prevented the formation of pathogenic autoantibodies and T cells, thus improving mortality. 90 E. gallinarum DNA was detected in the livers of most patients with autoimmune hepatitis but in none of the healthy control livers. 90 Primary sclerosing cholangitis Primary sclerosing cholangitis (PSC) is a cholestatic liver disease characterised by inflammation of the bile ducts leading to stricturing and sclerosis and eventually progressive biliary fibrosis and cirrhosis. Several recent studies compared the faecal bacterial microbiota of patients with PSC and healthy controls. 91 Patients with PSC are consistently shown to have lower bacterial microbiome diversity than healthy controls. Additionally, Veillonella has been shown to be enriched in the stool of patients with PSC compared to healthy controls in multiple studies. [92][93][94][95][96][97] Enterococcus, Streptococcus and Lactobacillus are also frequently enriched in patients with PSC, whereas there is a relative depletion of short-chain fatty acid-producing Firmicutes, such as Faecalibacterium and Coprococcus. Germ-free mice inoculated with faecal matter from patients with PSC were more susceptible to hepatobiliary injury by diethyldithiocarbamate and harboured K. pneumoniae, Proteus mirabilis, and E. gallinarum in their mesenteric lymph nodes. 98 Further, specific K. pneumoniae strains could induce pore formation on human intestinal epithelial organoids, suggesting that increased bacterial translocation could be a potential mechanism of increased susceptibility to hepatobiliary injury. 98 Cirrhosis Patients with cirrhosis have decreased proportions of beneficial, autochthonous taxa, such as Lachnospiraceae and Ruminococcaceae, and overrepresentation of potentially pathogenic bacteria such as Enterobacteriaceae, Staphylococcaceae, and Enterococcaceae, whose abundance correlates with disease progression and endotoxemia. 85,99,100 Another study observed a higher relative abundance of bacteria normally associated with oral flora in the intestinal microbiome of patients with cirrhosis, as well as increased Veillonella and Streptococcus species compared to controls. 2 Changes in the composition of the intestinal bacterial microbiome have also been correlated with the severity of liver disease. The ratio of autochthonous taxa, such as Ruminococcaceae, Lachnospiraceae, and Clostridiales, to non-autochthonous taxa, such as Enterobacteriaceae and Bacteroidaceae, was much higher in healthy individuals than patients with cirrhosis and inversely correlated with model for end-stage liver disease score and degree of hepatic decompensation. 101 Moreover, an increased relative abundance of pathogenic bacteria was associated with the development of complications such as hepatic encephalopathy.
Patients with cirrhosis not only exhibit an increased relative abundance of pathogenic bacteria in their intestinal bacterial microbiomes, they are also at increased risk of bacterial translocation, a process whereby bacteria migrate from the intestinal lumen to extraintestinal sites. 102 Aerobic Gram-negative bacteria, such as E. coli, K. pneumoniae, Pseudomonas aeruginosa, and other Enterobacteriaceae, translocate much more readily than anaerobic bacteria. 103 Notably, these species have also been implicated in decompensation of cirrhosis. 104 Increasingly, studies are comparing patients with cirrhosis based on their disease aetiology. While many intestinal bacterial communities are shared across the spectrum of liver disease aetiologies, differences in the patterns of dysbiosis across the different aetiologies of cirrhosis may provide a better picture of the mechanisms underlying these associations.

Preclinical phage utilisation in liver disease
Although no clinical trial using phage therapy for patients with liver disease has been published, 2 preclinical studies used phage therapy to treat liver disease. Duan et al. demonstrated that intestinal levels of E. faecalis are significantly increased in patients with alcoholic hepatitis. 87 Furthermore, the presence of a specific strain of E. faecalis that produces the bacterial exotoxin cytolysin correlates with increased severity of disease and mortality in patients with alcoholic hepatitis. Transplantation of faeces from cytolysin-positive patients with alcoholic hepatitis worsened ethanol-induced liver disease in gnotobiotic mice, whereas treatment of these mice with specific phages targeting cytolytic E. faecalis by oral gavage, reversed the exacerbation of liver disease. No improvement in liver disease was seen in the gnotobiotic mice treated with phages targeting non-cytolytic E. faecalis. This preclinical study demonstrates the utility of targeting specific species of the intestinal bacterial microbiome to modify disease progression. Another study showed that selective elimination of the ethanol-producing K. pneumoniae strain using phages prior to faecal transplantation into mice prevented development of diet-induced steatohepatitis. 81 These studies are good examples of how elimination of pathobionts by phages can improve liver disease in mouse models.

Conclusion and future directions
Recent advances in the field of microbiota research have identified a few bacterial strains that correlate with liver disease in patients, are causatively linked to disease pathogenesis, and could thus act as therapeutic targets. Despite the renewed interest in phage therapy, there are many roadblocks preventing it from becoming standard of care. One major roadblock is the narrow host range of phages, which limits wide therapeutic utility and the use of the same phages in different patients. One possibility is to use a cocktail of multiple phages. Limited host range can also be addressed through natural or engineered alterations in phage-encoded receptor-binding proteins, capable of targeting different hosts. 42,43,[45][46][47]49,105 To avoid using phages with undesirable off-target effects (i.e., reducing commensal bacteria), experiments should analyse the effects of any potential therapeutic phage on the composition of the microbiome. Phage host range will never be as broad as standard of care broad-spectrum antibiotics. In addition, phages can be made to bind multiple receptors (i.e. polyvalent), thereby extending the host range of a single virion. 46 By targeting more than 1 receptor, we avoid many of the obstacles that bacteria have evolved to prevent phage adsorption (e.g. mutations to or physically blocking receptors with extracellular polysaccharides). Blocking receptors using biofilms can be avoided by expressing an extracellular polysaccharidedegrading enzyme in the phage. 106 Other obstacles to the widespread use of phage therapeutics relate to their pharmacokinetics and pharmacodynamics within the human body. For example, some phages administered systemically can be cleared rapidly from the circulation. 107 It is therefore critical to determine the dose of phages being administered and their clearance from the site where they are applied, to ensure that phages are present at the specific site long enough to lyse bacteria.
Screening patients for the presence and sufficient abundance of target bacteria in the intestine, as well testing the susceptibility of target bacteria to phages, will be crucial for the successful clinical application of phage therapy. Combining a personalised treatment approach with phages' ability to precisely edit the microbiota could make phagebased therapies powerful new tools for the treatment of many diseases, including those of the liver. Authors' contributions C.H. was responsible for drafting the chapters "Introduction", "Intestinal virome in patients with liver disease", "Known bacteria driving liver disease as potential targets for phage therapy", "Preclinical phage utilisation in liver disease", coordinating the writing and compiling the final version. Y.D. was responsible for drafting the chapter "Phage therapy in gastrointestinal diseases". D.E.F. was responsible for drafting the chapters "Bacteriophages -natural predators of bacteria", "Resurgence of phage therapy and parts of "Conclusion and future directions". B.S. was responsible for drafting the chapters "Brief Summary" and parts of "Conclusion and future directions". All authors revised and approved the manuscript.