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Intestinal virome and therapeutic potential of bacteriophages in liver disease

Open AccessPublished:August 23, 2021DOI:https://doi.org/10.1016/j.jhep.2021.08.003

      Summary

      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.

      Keywords

      • -
        Over 90% of the human intestinal virome is composed of bacteriophages, with eukaryotic plant and mammalian viruses making up the remaining fraction.
      • -
        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.
      • -
        Use of phage therapy for the treatment of multidrug resistant infections shows promise in case reports, and several clinical trials involving phage therapy are underway.
      • -
        Our knowledge of taxonomic differences in the bacterial microbiomes of patients with different aetiologies of liver disease could help identify potential targets for phage therapy.
      • -
        Preclinical data suggest that selective targeting of bacterial strains such as Enterococcus faecalis or Klebsiella pneumoniae by phage therapy can modify liver disease progression, such as in alcoholic hepatitis or steatohepatitis, respectively.

      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.
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      • Over 90% of the human intestinal virome is composed of bacteriophages, with eukaryotic plant and mammalian viruses making up the remaining fraction.
      Figure thumbnail gr1
      Fig. 1Intestinal virome.
      The intestine and liver are intimately connected and communicate via the portal vein and the common bile duct. The intestinal microbiota contains bacteria, fungi, archaea and viruses. Viruses in the intestinal virome are predominantly phages (also called phageome), but also contain some eukaryotic viruses. Lytic phages can lyse bacteria and contribute to changes in the bacterial microbiota.
      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.
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      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 end-stage 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 quasi-enveloped form that is masked within a layer of the host cell membrane.
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      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.
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      • 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.
      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.
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      Intestinal virome in patients with alcoholic hepatitis.
      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.
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      Intestinal virome in patients with alcoholic hepatitis.
      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.
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      Intestinal virome signature associated with severity of nonalcoholic fatty liver disease.
      Further studies are needed to confirm and characterise the intestinal mammalian virus population in patients with alcohol-associated 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.
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      Intestinal virome signature associated with severity of nonalcoholic fatty liver disease.
      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.
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      Interaction of bacterial metagenome and virome in patients with cirrhosis and hepatic encephalopathy.
      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.
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      Interaction of bacterial metagenome and virome in patients with cirrhosis and hepatic encephalopathy.
      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 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 1031 total phage particles.
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      Shortly after the discovery of phages by Frederick Twort in 1915,
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      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.
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      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.
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      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.
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      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 phage-encoded 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.
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      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 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.
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      Figure thumbnail gr2
      Fig. 2Bacteriophage life cycles.
      Depicted are the 2 main types of tailed phage life cycles, lytic (A) and lysogenic (B). Lytic growth consists of replicating the genome, expressing structural proteins, packaging the genome into particles, assembling the mature virions, and ending in lysis and cell death of the host bacterium. In contrast, lysogenic growth is a storage state whereby the phage integrates its genome into the bacterial host chromosome, surviving by vertical transmission through host cell division. Lysogenic phages can also undergo lytic growth under certain conditions. Proteins related to the repressors cI and cro (brown colour) are responsible for switching from lysogenic to lytic development.
      Although the host range of a phage tends to be quite narrow (e.g., strain/serotype- or species-specific),
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      there are lytic phages that can infect more distantly related bacteria.
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      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.
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      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.
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      Antimicrobial resistance: a global multifaceted phenomenon.
      This poses a serious public health problem since there are few, or in some cases, no drugs available to treat life-threatening 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.
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      Clinical practice. Antibiotic-associated diarrhea.
      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,
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      Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts.
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      to prevent food spoilage,
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      Application of a bacteriophage cocktail to reduce Salmonella Typhimurium U288 contamination on pig skin.
      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.
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      Oral T4-like phage cocktail application to healthy adult volunteers from Bangladesh.
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      Safety analysis of a Russian phage cocktail: from MetaGenomic analysis to oral application in healthy human subjects.
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      • et al.
      Oral application of Escherichia coli bacteriophage: safety tests in healthy and diarrheal children from Bangladesh.
      To determine the safety and efficacy of phage therapy for gastrointestinal infections, a T4-like coliphage cocktail was given orally for 4 days to children hospitalised with acute diarrhoea. Non-bacterial causes of diarrhoea were not ruled out. No adverse events were reported, suggesting the overall safety of the phage cocktail.
      • Sarker S.A.
      • Sultana S.
      • Reuteler G.
      • Moine D.
      • Descombes P.
      • Charton F.
      • et al.
      Oral phage therapy of acute bacterial diarrhea with two coliphage preparations: a randomized trial in children from Bangladesh.
      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.
      • Sarker S.A.
      • Sultana S.
      • Reuteler G.
      • Moine D.
      • Descombes P.
      • Charton F.
      • et al.
      Oral phage therapy of acute bacterial diarrhea with two coliphage preparations: a randomized trial in children from Bangladesh.
      This could be explained by the fact that only half of patients actually harboured Escherichia coli (E. coli) strains susceptible to the administered phages, and E. coli only represented 5% of total faecal bacteria. Overall, this trial confirmed the safety of phage treatments in children with diarrhoea. Though the trial failed to show efficacy, this could potentially be explained by low E. coli abundance in the stool samples or symptoms caused by a non-bacterial infection (e.g. viral gastroenteritis).
      Table 1Overview of recent studies of phage-related treatment in gastrointestinal diseases. (English literature only).
      Type of studyPhage (target)Dose and methodPatientsResult and conclusionRef.
      Clinical trialPhage T4 (E. coli)105 PFU/ml, dose A,

      103 PFU/ml, dose B,

      Oral administration
      15 healthy individualsSafe, but E. coli abundance not changed
      • Bruttin A.
      • Brüssow H.
      Human volunteers receiving Escherichia coli phage T4 orally: a safety test of phage therapy.
      Clinical trialT4-like Phages (E. coli)3x109 PFU/ml, dose A,

      3x107 PFU/ml, dose B,

      Oral administration
      15 healthy individualsSafe, gut microbiota profile not affected
      • Sarker S.A.
      • McCallin S.
      • Barretto C.
      • Berger B.
      • Pittet A.-C.
      • Sultana S.
      • et al.
      Oral T4-like phage cocktail application to healthy adult volunteers from Bangladesh.
      Clinical trialCommercial phage cocktail ColiProteus (E. coli)20 ml for adults, 10 ml for children, and 10-fold dilution

      Oral administration
      5 healthy adults, 10 healthy childrenOverall safe, with occasional reported side effects independent of dosage
      • McCallin S.
      • Alam Sarker S.
      • Barretto C.
      • Sultana S.
      • Berger B.
      • Huq S.
      • et al.
      Safety analysis of a Russian phage cocktail: from MetaGenomic analysis to oral application in healthy human subjects.
      Clinical trialT4-like Phages or ColiProteus (E. coli)108 or 106 PFU for older children (T4-like phages),

      107 or 105 PFU for younger children (T4-like phages),

      5x108 or 109 PFU for all (ColiProteus),

      Oral administration
      20 older children, 20 younger childrenBoth cocktails are safe
      • Sarker S.A.
      • Berger B.
      • Deng Y.
      • Kieser S.
      • Foata F.
      • Moine D.
      • et al.
      Oral application of Escherichia coli bacteriophage: safety tests in healthy and diarrheal children from Bangladesh.
      Clinical trialT4-like Phages or ColiProteus (E. coli)3.6x108 PFU (T4-like phages),

      1.4x109 PFU (ColiProteus),

      Oral administration
      120 children with diarrhoeaSafe, but lack of efficacy
      • Sarker S.A.
      • Sultana S.
      • Reuteler G.
      • Moine D.
      • Descombes P.
      • Charton F.
      • et al.
      Oral phage therapy of acute bacterial diarrhea with two coliphage preparations: a randomized trial in children from Bangladesh.
      Clinical case reportPhage cocktail (A. baumannii)5x109 PFU

      Intracavitary and Intravenous
      68-year-old male with necrotising pancreatitis complicated by pancreatic pseudocystPatient completely recovered
      • Schooley R.T.
      • Biswas B.
      • Gill J.J.
      • Hernandez-Morales A.
      • Lancaster J.
      • Lessor L.
      • et al.
      Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection.
      Clinical trialPhage cocktail PreforPro (E. coli)One 15 mg capsule,

      Oral administration
      32 healthy individuals with mild to moderate gastrointestinal distressSafe and tolerable, but no difference from placebo
      • Gindin M.
      • Febvre H.P.
      • Rao S.
      • Wallace T.C.
      • Weir T.L.
      PHAGE Study: Bacteriophages as Novel Prebiotics.
      Clinical trialPhage cocktail PreforPro (E. coli), together with probiotics Bifidobacterium animalis subspecies lactis strain BL04One 15 mg capsule,

      Oral administration
      68 healthy individuals with mild to moderate gastrointestinal distressSafe and tolerable, but no compelling evidence of efficacy
      • Grubb D.S.
      • Wrigley S.D.
      • Freedman K.E.
      • Wei Y.
      • Vazquez A.R.
      • Trotter R.E.
      • et al.
      PHAGE-2 study: supplemental bacteriophages extend Bifidobacterium animalis subsp. lactis BL04 benefits on gut health and microbiota in healthy adults.
      Preclinical studyPhage cocktail (adherent-invasive E. coli)3x107 PFU

      Oral administration
      Wild-type mice colonised with 108 CFU of adherent-invasive E. coli,

      Dextran sodium sulphate-induced colitis
      Faecal E. coli level decreased; dextran sodium sulphate-induced colitis ameliorated
      • Galtier M.
      • Sordi L.D.
      • Sivignon A.
      • de Vallée A.
      • Maura D.
      • Neut C.
      • et al.
      Bacteriophages targeting adherent invasive Escherichia coli strains as a promising new treatment for Crohn’s disease.
      Preclinical studyPhage cocktail (E. faecalis)1010 PFU

      Oral administration
      Gnotobiotic mice colonised with stool samples from cytolysin-positive patients with alcoholic hepatitis,

      Ethanol-induced liver disease
      Faecal E. faecalis level decreased, ethanol-induced liver disease ameliorated
      • Duan Y.
      • Llorente C.
      • Lang S.
      • Brandl K.
      • Chu H.
      • Jiang L.
      • et al.
      Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.
      A. baumannii, Acinetobacter baumannii; CFU, colony-forming unit; E. coli, Escherichia coli; E. faecalis, Enterococcus faecalis; PFU, plaque-forming unit.
      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 multidrug-resistant Acinetobacter baumannii.
      • Schooley R.T.
      • Biswas B.
      • Gill J.J.
      • Hernandez-Morales A.
      • Lancaster J.
      • Lessor L.
      • et al.
      Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection.
      Phages were applied by intracavitary and intravenous routes, and the patient completely recovered after 5 months.
      • Schooley R.T.
      • Biswas B.
      • Gill J.J.
      • Hernandez-Morales A.
      • Lancaster J.
      • Lessor L.
      • et al.
      Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection.
      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 multidrug-resistant 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.
      • Rolhion N.
      • Darfeuille-Michaud A.
      Adherent-invasive Escherichia coli in inflammatory bowel disease.
      ,
      • Palmela C.
      • Chevarin C.
      • Xu Z.
      • Torres J.
      • Sevrin G.
      • Hirten R.
      • et al.
      Adherent-invasive Escherichia coli in inflammatory bowel disease.
      Administration of a phage cocktail against these E. coli strains reduced intestinal AIEC colonisation in transgenic mice expressing the human AIEC receptor.
      • Galtier M.
      • Sordi L.D.
      • Sivignon A.
      • de Vallée A.
      • Maura D.
      • Neut C.
      • et al.
      Bacteriophages targeting adherent invasive Escherichia coli strains as a promising new treatment for Crohn’s disease.
      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.
      • Galtier M.
      • Sordi L.D.
      • Sivignon A.
      • de Vallée A.
      • Maura D.
      • Neut C.
      • et al.
      Bacteriophages targeting adherent invasive Escherichia coli strains as a promising new treatment for Crohn’s disease.
      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.
      • Galtier M.
      • Sordi L.D.
      • Sivignon A.
      • de Vallée A.
      • Maura D.
      • Neut C.
      • et al.
      Bacteriophages targeting adherent invasive Escherichia coli strains as a promising new treatment for Crohn’s disease.
      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).
      • Use of phage therapy for the treatment of multidrug resistant infections shows promise in case reports, and several clinical trials involving phage therapy are underway.

      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).
      Table 2Bacterial genera and species with known correlations to different aetiologies of liver disease.
      IncreasedDecreased
      Non-alcoholic fatty liver disease
      • Bacteroides
        • Loomba R.
        • Seguritan V.
        • Li W.
        • Long T.
        • Klitgord N.
        • Bhatt A.
        • et al.
        Gut microbiome-based metagenomic signature for non-invasive detection of advanced fibrosis in human nonalcoholic fatty liver disease.
        ,
        • Boursier J.
        • Mueller O.
        • Barret M.
        • Machado M.
        • Fizanne L.
        • Araujo-Perez F.
        • et al.
        The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota.
        ,
        • Ponziani F.R.
        • Bhoori S.
        • Castelli C.
        • Putignani L.
        • Rivoltini L.
        • Del Chierico F.
        • et al.
        Hepatocellular carcinoma is associated with gut microbiota profile and inflammation in nonalcoholic fatty liver disease.
      • Blautia
        • Del Chierico F.
        • Nobili V.
        • Vernocchi P.
        • Russo A.
        • De Stefanis C.
        • Gnani D.
        • et al.
        Gut microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients unveiled by an integrated meta-omics-based approach.
        ,
        • Ponziani F.R.
        • Bhoori S.
        • Castelli C.
        • Putignani L.
        • Rivoltini L.
        • Del Chierico F.
        • et al.
        Hepatocellular carcinoma is associated with gut microbiota profile and inflammation in nonalcoholic fatty liver disease.
        ,
        • Shen F.
        • Zheng R.D.
        • Sun X.Q.
        • Ding W.J.
        • Wang X.Y.
        • Fan J.G.
        Gut microbiota dysbiosis in patients with non-alcoholic fatty liver disease.
      • Dorea
        • Del Chierico F.
        • Nobili V.
        • Vernocchi P.
        • Russo A.
        • De Stefanis C.
        • Gnani D.
        • et al.
        Gut microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients unveiled by an integrated meta-omics-based approach.
        ,
        • Raman M.
        • Ahmed I.
        • Gillevet P.M.
        • Probert C.S.
        • Ratcliffe N.M.
        • Smith S.
        • et al.
        Fecal microbiome and volatile organic compound metabolome in obese humans with nonalcoholic fatty liver disease.
      • Escherichia
        • Duarte S.M.B.
        • Stefano J.T.
        • Miele L.
        • Ponziani F.R.
        • Souza-Basqueira M.
        • Okada L.
        • et al.
        Gut microbiome composition in lean patients with NASH is associated with liver damage independent of caloric intake: a prospective pilot study.
        ,
        • Loomba R.
        • Seguritan V.
        • Li W.
        • Long T.
        • Klitgord N.
        • Bhatt A.
        • et al.
        Gut microbiome-based metagenomic signature for non-invasive detection of advanced fibrosis in human nonalcoholic fatty liver disease.
        ,
        • Caussy C.
        • Hsu C.
        • Lo M.T.
        • Liu A.
        • Bettencourt R.
        • Ajmera V.H.
        • et al.
        Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD.
        ,
        • Shen F.
        • Zheng R.D.
        • Sun X.Q.
        • Ding W.J.
        • Wang X.Y.
        • Fan J.G.
        Gut microbiota dysbiosis in patients with non-alcoholic fatty liver disease.
        ,
        • Zhu L.
        • Baker S.S.
        • Gill C.
        • Liu W.
        • Alkhouri R.
        • Baker R.D.
        • et al.
        Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH.
        • Escherichia coli
          • Loomba R.
          • Seguritan V.
          • Li W.
          • Long T.
          • Klitgord N.
          • Bhatt A.
          • et al.
          Gut microbiome-based metagenomic signature for non-invasive detection of advanced fibrosis in human nonalcoholic fatty liver disease.
          ,
          • Caussy C.
          • Hsu C.
          • Lo M.T.
          • Liu A.
          • Bettencourt R.
          • Ajmera V.H.
          • et al.
          Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD.
      • Lactobacillus
        • Da Silva H.E.
        • Teterina A.
        • Comelli E.M.
        • Taibi A.
        • Arendt B.M.
        • Fischer S.E.
        • et al.
        Nonalcoholic fatty liver disease is associated with dysbiosis independent of body mass index and insulin resistance.
        ,
        • Duarte S.M.B.
        • Stefano J.T.
        • Miele L.
        • Ponziani F.R.
        • Souza-Basqueira M.
        • Okada L.
        • et al.
        Gut microbiome composition in lean patients with NASH is associated with liver damage independent of caloric intake: a prospective pilot study.
        ,
        • Raman M.
        • Ahmed I.
        • Gillevet P.M.
        • Probert C.S.
        • Ratcliffe N.M.
        • Smith S.
        • et al.
        Fecal microbiome and volatile organic compound metabolome in obese humans with nonalcoholic fatty liver disease.
      • Klebsiella
        • Yuan J.
        • Chen C.
        • Cui J.
        • Lu J.
        • Yan C.
        • Wei X.
        • et al.
        Fatty liver disease caused by high-alcohol-producing Klebsiella pneumoniae.
      • Roseburia
        • Raman M.
        • Ahmed I.
        • Gillevet P.M.
        • Probert C.S.
        • Ratcliffe N.M.
        • Smith S.
        • et al.
        Fecal microbiome and volatile organic compound metabolome in obese humans with nonalcoholic fatty liver disease.
      • Ruminococcus
        • Del Chierico F.
        • Nobili V.
        • Vernocchi P.
        • Russo A.
        • De Stefanis C.
        • Gnani D.
        • et al.
        Gut microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients unveiled by an integrated meta-omics-based approach.
        ,
        • Ponziani F.R.
        • Bhoori S.
        • Castelli C.
        • Putignani L.
        • Rivoltini L.
        • Del Chierico F.
        • et al.
        Hepatocellular carcinoma is associated with gut microbiota profile and inflammation in nonalcoholic fatty liver disease.
      • Bacteroides
        • Caussy C.
        • Hsu C.
        • Lo M.T.
        • Liu A.
        • Bettencourt R.
        • Ajmera V.H.
        • et al.
        Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD.
        ,
        • Shen F.
        • Zheng R.D.
        • Sun X.Q.
        • Ding W.J.
        • Wang X.Y.
        • Fan J.G.
        Gut microbiota dysbiosis in patients with non-alcoholic fatty liver disease.
        • Bacteroides caccae
          • Caussy C.
          • Hsu C.
          • Lo M.T.
          • Liu A.
          • Bettencourt R.
          • Ajmera V.H.
          • et al.
          Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD.
      • Bifidobacterium
        • Duarte S.M.B.
        • Stefano J.T.
        • Miele L.
        • Ponziani F.R.
        • Souza-Basqueira M.
        • Okada L.
        • et al.
        Gut microbiome composition in lean patients with NASH is associated with liver damage independent of caloric intake: a prospective pilot study.
        ,
        • Zhu L.
        • Baker S.S.
        • Gill C.
        • Liu W.
        • Alkhouri R.
        • Baker R.D.
        • et al.
        Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH.
      • Coprococcus
        • Da Silva H.E.
        • Teterina A.
        • Comelli E.M.
        • Taibi A.
        • Arendt B.M.
        • Fischer S.E.
        • et al.
        Nonalcoholic fatty liver disease is associated with dysbiosis independent of body mass index and insulin resistance.
        ,
        • Zhu L.
        • Baker S.S.
        • Gill C.
        • Liu W.
        • Alkhouri R.
        • Baker R.D.
        • et al.
        Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH.
        ,
        • Wang B.
        • Jiang X.
        • Cao M.
        • Ge J.
        • Bao Q.
        • Tang L.
        • et al.
        Altered fecal microbiota correlates with liver biochemistry in nonobese patients with non-alcoholic fatty liver disease.
      • Faecalibacterium
        • Wong V.W.
        • Tse C.H.
        • Lam T.T.
        • Wong G.L.
        • Chim A.M.
        • Chu W.C.
        • et al.
        Molecular characterization of the fecal microbiota in patients with nonalcoholic steatohepatitis--a longitudinal study.
        • Da Silva H.E.
        • Teterina A.
        • Comelli E.M.
        • Taibi A.
        • Arendt B.M.
        • Fischer S.E.
        • et al.
        Nonalcoholic fatty liver disease is associated with dysbiosis independent of body mass index and insulin resistance.
        • Duarte S.M.B.
        • Stefano J.T.
        • Miele L.
        • Ponziani F.R.
        • Souza-Basqueira M.
        • Okada L.
        • et al.
        Gut microbiome composition in lean patients with NASH is associated with liver damage independent of caloric intake: a prospective pilot study.
        ,
        • Chen Y.
        • Yang F.
        • Lu H.
        • Wang B.
        • Chen Y.
        • Lei D.
        • et al.
        Characterization of fecal microbial communities in patients with liver cirrhosis.
        ,
        • Zhu L.
        • Baker S.S.
        • Gill C.
        • Liu W.
        • Alkhouri R.
        • Baker R.D.
        • et al.
        Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH.
        • Faecalibacterium prausnitzii
          • Da Silva H.E.
          • Teterina A.
          • Comelli E.M.
          • Taibi A.
          • Arendt B.M.
          • Fischer S.E.
          • et al.
          Nonalcoholic fatty liver disease is associated with dysbiosis independent of body mass index and insulin resistance.
          ,
          • Duarte S.M.B.
          • Stefano J.T.
          • Miele L.
          • Ponziani F.R.
          • Souza-Basqueira M.
          • Okada L.
          • et al.
          Gut microbiome composition in lean patients with NASH is associated with liver damage independent of caloric intake: a prospective pilot study.
      • Lactobacillus
        • Wang B.
        • Jiang X.
        • Cao M.
        • Ge J.
        • Bao Q.
        • Tang L.
        • et al.
        Altered fecal microbiota correlates with liver biochemistry in nonobese patients with non-alcoholic fatty liver disease.
      • Oscillospira
        • Del Chierico F.
        • Nobili V.
        • Vernocchi P.
        • Russo A.
        • De Stefanis C.
        • Gnani D.
        • et al.
        Gut microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients unveiled by an integrated meta-omics-based approach.
      • Roseburia
        • Wang B.
        • Jiang X.
        • Cao M.
        • Ge J.
        • Bao Q.
        • Tang L.
        • et al.
        Altered fecal microbiota correlates with liver biochemistry in nonobese patients with non-alcoholic fatty liver disease.
      • Ruminococcus
        • Da Silva H.E.
        • Teterina A.
        • Comelli E.M.
        • Taibi A.
        • Arendt B.M.
        • Fischer S.E.
        • et al.
        Nonalcoholic fatty liver disease is associated with dysbiosis independent of body mass index and insulin resistance.
        ,
        • Duarte S.M.B.
        • Stefano J.T.
        • Miele L.
        • Ponziani F.R.
        • Souza-Basqueira M.
        • Okada L.
        • et al.
        Gut microbiome composition in lean patients with NASH is associated with liver damage independent of caloric intake: a prospective pilot study.
        ,
        • Loomba R.
        • Seguritan V.
        • Li W.
        • Long T.
        • Klitgord N.
        • Bhatt A.
        • et al.
        Gut microbiome-based metagenomic signature for non-invasive detection of advanced fibrosis in human nonalcoholic fatty liver disease.
        ,
        • Wang B.
        • Jiang X.
        • Cao M.
        • Ge J.
        • Bao Q.
        • Tang L.
        • et al.
        Altered fecal microbiota correlates with liver biochemistry in nonobese patients with non-alcoholic fatty liver disease.
      Alcohol-associated liver disease
      • Blautia
        • Leclercq S.
        • Matamoros S.
        • Cani P.D.
        • Neyrinck A.M.
        • Jamar F.
        • Starkel P.
        • et al.
        Intestinal permeability, gut-bacterial dysbiosis, and behavioral markers of alcohol-dependence severity.
      • Dorea
        • Leclercq S.
        • Matamoros S.
        • Cani P.D.
        • Neyrinck A.M.
        • Jamar F.
        • Starkel P.
        • et al.
        Intestinal permeability, gut-bacterial dysbiosis, and behavioral markers of alcohol-dependence severity.
      • Enterococcus
        • Duan Y.
        • Llorente C.
        • Lang S.
        • Brandl K.
        • Chu H.
        • Jiang L.
        • et al.
        Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.
        ,
        • Lang S.
        • Fairfied B.
        • Gao B.
        • Duan Y.
        • Zhang X.
        • Fouts D.E.
        • et al.
        Changes in the fecal bacterial microbiota associated with disease severity in alcoholic hepatitis patients.
        • Enterococcus faecalis
          • Duan Y.
          • Llorente C.
          • Lang S.
          • Brandl K.
          • Chu H.
          • Jiang L.
          • et al.
          Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.
      • Prevotella
        • Mutlu E.A.
        • Gillevet P.M.
        • Rangwala H.
        • Sikaroodi M.
        • Naqvi A.
        • Engen P.A.
        • et al.
        Colonic microbiome is altered in alcoholism.
        ,
        • Chen Y.
        • Yang F.
        • Lu H.
        • Wang B.
        • Chen Y.
        • Lei D.
        • et al.
        Characterization of fecal microbial communities in patients with liver cirrhosis.
      • Veillonella
        • Lang S.
        • Fairfied B.
        • Gao B.
        • Duan Y.
        • Zhang X.
        • Fouts D.E.
        • et al.
        Changes in the fecal bacterial microbiota associated with disease severity in alcoholic hepatitis patients.
      • Akkermansia
        • Grander C.
        • Adolph T.E.
        • Wieser V.
        • Lowe P.
        • Wrzosek L.
        • Gyongyosi B.
        • et al.
        Recovery of ethanol-induced Akkermansia muciniphila depletion ameliorates alcoholic liver disease.
        ,
        • Lang S.
        • Fairfied B.
        • Gao B.
        • Duan Y.
        • Zhang X.
        • Fouts D.E.
        • et al.
        Changes in the fecal bacterial microbiota associated with disease severity in alcoholic hepatitis patients.
      • Faecalibacterium
        • Leclercq S.
        • Matamoros S.
        • Cani P.D.
        • Neyrinck A.M.
        • Jamar F.
        • Starkel P.
        • et al.
        Intestinal permeability, gut-bacterial dysbiosis, and behavioral markers of alcohol-dependence severity.
        • Faecalibacterium prausnitzii
          • Leclercq S.
          • Matamoros S.
          • Cani P.D.
          • Neyrinck A.M.
          • Jamar F.
          • Starkel P.
          • et al.
          Intestinal permeability, gut-bacterial dysbiosis, and behavioral markers of alcohol-dependence severity.
      • Ruminococcus
        • Leclercq S.
        • Matamoros S.
        • Cani P.D.
        • Neyrinck A.M.
        • Jamar F.
        • Starkel P.
        • et al.
        Intestinal permeability, gut-bacterial dysbiosis, and behavioral markers of alcohol-dependence severity.
      Autoimmune hepatitis
      • Enterococcus
        • Manfredo Vieira S.
        • Hiltensperger M.
        • Kumar V.
        • Zegarra-Ruiz D.
        • Dehner C.
        • Khan N.
        • et al.
        Translocation of a gut pathobiont drives autoimmunity in mice and humans.
        • Enterococcus gallinarum
          • Manfredo Vieira S.
          • Hiltensperger M.
          • Kumar V.
          • Zegarra-Ruiz D.
          • Dehner C.
          • Khan N.
          • et al.
          Translocation of a gut pathobiont drives autoimmunity in mice and humans.
      • Veillonella
        • Wei Y.
        • Li Y.
        • Yan L.
        • Sun C.
        • Miao Q.
        • Wang Q.
        • et al.
        Alterations of gut microbiome in autoimmune hepatitis.
        ,
        • Elsherbiny N.M.
        • Rammadan M.
        • Hassan E.A.
        • Ali M.E.
        • El-Rehim A.S.A.
        • Abbas W.A.
        • et al.
        Autoimmune hepatitis: shifts in gut microbiota and metabolic pathways among Egyptian patients.
      • Prevotella
        • Elsherbiny N.M.
        • Rammadan M.
        • Hassan E.A.
        • Ali M.E.
        • El-Rehim A.S.A.
        • Abbas W.A.
        • et al.
        Autoimmune hepatitis: shifts in gut microbiota and metabolic pathways among Egyptian patients.
      Primary sclerosing cholangitis
      • Enterococcus
        • Ruhlemann M.
        • Liwinski T.
        • Heinsen F.A.
        • Bang C.
        • Zenouzi R.
        • Kummen M.
        • et al.
        Consistent alterations in faecal microbiomes of patients with primary sclerosing cholangitis independent of associated colitis.
        ,
        • Bajer L.
        • Kverka M.
        • Kostovcik M.
        • Macinga P.
        • Dvorak J.
        • Stehlikova Z.
        • et al.
        Distinct gut microbiota profiles in patients with primary sclerosing cholangitis and ulcerative colitis.
        ,
        • Sabino J.
        • Vieira-Silva S.
        • Machiels K.
        • Joossens M.
        • Falony G.
        • Ballet V.
        • et al.
        Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD.
      • Lactobacillus
        • Ruhlemann M.
        • Liwinski T.
        • Heinsen F.A.
        • Bang C.
        • Zenouzi R.
        • Kummen M.
        • et al.
        Consistent alterations in faecal microbiomes of patients with primary sclerosing cholangitis independent of associated colitis.
        ,
        • Bajer L.
        • Kverka M.
        • Kostovcik M.
        • Macinga P.
        • Dvorak J.
        • Stehlikova Z.
        • et al.
        Distinct gut microbiota profiles in patients with primary sclerosing cholangitis and ulcerative colitis.
        ,
        • Sabino J.
        • Vieira-Silva S.
        • Machiels K.
        • Joossens M.
        • Falony G.
        • Ballet V.
        • et al.
        Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD.
      • Streptococcus
        • Ruhlemann M.
        • Liwinski T.
        • Heinsen F.A.
        • Bang C.
        • Zenouzi R.
        • Kummen M.
        • et al.
        Consistent alterations in faecal microbiomes of patients with primary sclerosing cholangitis independent of associated colitis.
        ,
        • Bajer L.
        • Kverka M.
        • Kostovcik M.
        • Macinga P.
        • Dvorak J.
        • Stehlikova Z.
        • et al.
        Distinct gut microbiota profiles in patients with primary sclerosing cholangitis and ulcerative colitis.
        ,
        • Sabino J.
        • Vieira-Silva S.
        • Machiels K.
        • Joossens M.
        • Falony G.
        • Ballet V.
        • et al.
        Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD.
      • Veillonella
        • Ruhlemann M.
        • Liwinski T.
        • Heinsen F.A.
        • Bang C.
        • Zenouzi R.
        • Kummen M.
        • et al.
        Consistent alterations in faecal microbiomes of patients with primary sclerosing cholangitis independent of associated colitis.
        • Ruhlemann M.C.
        • Heinsen F.A.
        • Zenouzi R.
        • Lieb W.
        • Franke A.
        • Schramm C.
        Faecal microbiota profiles as diagnostic biomarkers in primary sclerosing cholangitis.
        • Kummen M.
        • Holm K.
        • Anmarkrud J.A.
        • Nygard S.
        • Vesterhus M.
        • Hoivik M.L.
        • et al.
        The gut microbial profile in patients with primary sclerosing cholangitis is distinct from patients with ulcerative colitis without biliary disease and healthy controls.
        • Bajer L.
        • Kverka M.
        • Kostovcik M.
        • Macinga P.
        • Dvorak J.
        • Stehlikova Z.
        • et al.
        Distinct gut microbiota profiles in patients with primary sclerosing cholangitis and ulcerative colitis.
        • Sabino J.
        • Vieira-Silva S.
        • Machiels K.
        • Joossens M.
        • Falony G.
        • Ballet V.
        • et al.
        Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD.
        • Lemoinne S.
        • Kemgang A.
        • Ben Belkacem K.
        • Straube M.
        • Jegou S.
        • Corpechot C.
        • et al.
        Fungi participate in the dysbiosis of gut microbiota in patients with primary sclerosing cholangitis.
      • Clostridium
        • Ruhlemann M.C.
        • Heinsen F.A.
        • Zenouzi R.
        • Lieb W.
        • Franke A.
        • Schramm C.
        Faecal microbiota profiles as diagnostic biomarkers in primary sclerosing cholangitis.
        • Kummen M.
        • Holm K.
        • Anmarkrud J.A.
        • Nygard S.
        • Vesterhus M.
        • Hoivik M.L.
        • et al.
        The gut microbial profile in patients with primary sclerosing cholangitis is distinct from patients with ulcerative colitis without biliary disease and healthy controls.
        • Bajer L.
        • Kverka M.
        • Kostovcik M.
        • Macinga P.
        • Dvorak J.
        • Stehlikova Z.
        • et al.
        Distinct gut microbiota profiles in patients with primary sclerosing cholangitis and ulcerative colitis.
      • Coprococcus
        • Ruhlemann M.
        • Liwinski T.
        • Heinsen F.A.
        • Bang C.
        • Zenouzi R.
        • Kummen M.
        • et al.
        Consistent alterations in faecal microbiomes of patients with primary sclerosing cholangitis independent of associated colitis.
        ,
        • Kummen M.
        • Holm K.
        • Anmarkrud J.A.
        • Nygard S.
        • Vesterhus M.
        • Hoivik M.L.
        • et al.
        The gut microbial profile in patients with primary sclerosing cholangitis is distinct from patients with ulcerative colitis without biliary disease and healthy controls.
        ,
        • Bajer L.
        • Kverka M.
        • Kostovcik M.
        • Macinga P.
        • Dvorak J.
        • Stehlikova Z.
        • et al.
        Distinct gut microbiota profiles in patients with primary sclerosing cholangitis and ulcerative colitis.
      • Faecalibacterium
        • Ruhlemann M.
        • Liwinski T.
        • Heinsen F.A.
        • Bang C.
        • Zenouzi R.
        • Kummen M.
        • et al.
        Consistent alterations in faecal microbiomes of patients with primary sclerosing cholangitis independent of associated colitis.
        ,
        • Bajer L.
        • Kverka M.
        • Kostovcik M.
        • Macinga P.
        • Dvorak J.
        • Stehlikova Z.
        • et al.
        Distinct gut microbiota profiles in patients with primary sclerosing cholangitis and ulcerative colitis.
        ,
        • Lemoinne S.
        • Kemgang A.
        • Ben Belkacem K.
        • Straube M.
        • Jegou S.
        • Corpechot C.
        • et al.
        Fungi participate in the dysbiosis of gut microbiota in patients with primary sclerosing cholangitis.
      • Ruminococcus
        • Bajer L.
        • Kverka M.
        • Kostovcik M.
        • Macinga P.
        • Dvorak J.
        • Stehlikova Z.
        • et al.
        Distinct gut microbiota profiles in patients with primary sclerosing cholangitis and ulcerative colitis.
        ,
        • Lemoinne S.
        • Kemgang A.
        • Ben Belkacem K.
        • Straube M.
        • Jegou S.
        • Corpechot C.
        • et al.
        Fungi participate in the dysbiosis of gut microbiota in patients with primary sclerosing cholangitis.
      Cirrhosis

      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.
      • Rinella M.E.
      Nonalcoholic fatty liver disease: a systematic review.
      Several studies have found a decreased faecal abundance of Faecalibacterium and specifically Faecalibacterium prausnitzii in both obese and non-obese patients with NASH.
      • Wong V.W.
      • Tse C.H.
      • Lam T.T.
      • Wong G.L.
      • Chim A.M.
      • Chu W.C.
      • et al.
      Molecular characterization of the fecal microbiota in patients with nonalcoholic steatohepatitis--a longitudinal study.
      • Da Silva H.E.
      • Teterina A.
      • Comelli E.M.
      • Taibi A.
      • Arendt B.M.
      • Fischer S.E.
      • et al.
      Nonalcoholic fatty liver disease is associated with dysbiosis independent of body mass index and insulin resistance.
      • Duarte S.M.B.
      • Stefano J.T.
      • Miele L.
      • Ponziani F.R.
      • Souza-Basqueira M.
      • Okada L.
      • et al.
      Gut microbiome composition in lean patients with NASH is associated with liver damage independent of caloric intake: a prospective pilot study.
      Ruminococcus was enriched in obese patients with NASH in 1 study,
      • Del Chierico F.
      • Nobili V.
      • Vernocchi P.
      • Russo A.
      • De Stefanis C.
      • Gnani D.
      • et al.
      Gut microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients unveiled by an integrated meta-omics-based approach.
      but reduced in other studies of obese
      • Da Silva H.E.
      • Teterina A.
      • Comelli E.M.
      • Taibi A.
      • Arendt B.M.
      • Fischer S.E.
      • et al.
      Nonalcoholic fatty liver disease is associated with dysbiosis independent of body mass index and insulin resistance.
      and non-obese patients.
      • Duarte S.M.B.
      • Stefano J.T.
      • Miele L.
      • Ponziani F.R.
      • Souza-Basqueira M.
      • Okada L.
      • et al.
      Gut microbiome composition in lean patients with NASH is associated with liver damage independent of caloric intake: a prospective pilot study.
      Ruminococcus obeum was specifically found to be reduced in patients with NAFLD in 1 study.
      • Loomba R.
      • Seguritan V.
      • Li W.
      • Long T.
      • Klitgord N.
      • Bhatt A.
      • et al.
      Gut microbiome-based metagenomic signature for non-invasive detection of advanced fibrosis in human nonalcoholic fatty liver disease.
      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.
      • Loomba R.
      • Seguritan V.
      • Li W.
      • Long T.
      • Klitgord N.
      • Bhatt A.
      • et al.
      Gut microbiome-based metagenomic signature for non-invasive detection of advanced fibrosis in human nonalcoholic fatty liver disease.
      • Boursier J.
      • Mueller O.
      • Barret M.
      • Machado M.
      • Fizanne L.
      • Araujo-Perez F.
      • et al.
      The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota.
      • Caussy C.
      • Hsu C.
      • Lo M.T.
      • Liu A.
      • Bettencourt R.
      • Ajmera V.H.
      • et al.
      Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD.
      • Ponziani F.R.
      • Bhoori S.
      • Castelli C.
      • Putignani L.
      • Rivoltini L.
      • Del Chierico F.
      • et al.
      Hepatocellular carcinoma is associated with gut microbiota profile and inflammation in nonalcoholic fatty liver disease.
      • Shen F.
      • Zheng R.D.
      • Sun X.Q.
      • Ding W.J.
      • Wang X.Y.
      • Fan J.G.
      Gut microbiota dysbiosis in patients with non-alcoholic fatty liver disease.
      Although no causative role of these bacterial strains for steatohepatitis has been demonstrated in preclinical models, Yuan et al. demonstrated that an ethanol-producing 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.
      • Yuan J.
      • Chen C.
      • Cui J.
      • Lu J.
      • Yan C.
      • Wei X.
      • et al.
      Fatty liver disease caused by high-alcohol-producing Klebsiella pneumoniae.

      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.
      • Lieber C.S.
      Alcoholic fatty liver: its pathogenesis and mechanism of progression to inflammation and fibrosis.
      Studies of the intestinal bacterial microbiome in patients with alcohol-associated liver disease have revealed enrichment of Enterobacteriaceae
      • Mutlu E.A.
      • Gillevet P.M.
      • Rangwala H.
      • Sikaroodi M.
      • Naqvi A.
      • Engen P.A.
      • et al.
      Colonic microbiome is altered in alcoholism.
      ,
      • Tuomisto S.
      • Pessi T.
      • Collin P.
      • Vuento R.
      • Aittoniemi J.
      • Karhunen P.J.
      Changes in gut bacterial populations and their translocation into liver and ascites in alcoholic liver cirrhotics.
      and a reduction of Lactobacillus,
      • Tuomisto S.
      • Pessi T.
      • Collin P.
      • Vuento R.
      • Aittoniemi J.
      • Karhunen P.J.
      Changes in gut bacterial populations and their translocation into liver and ascites in alcoholic liver cirrhotics.
      Bacteroidetes
      • Mutlu E.A.
      • Gillevet P.M.
      • Rangwala H.
      • Sikaroodi M.
      • Naqvi A.
      • Engen P.A.
      • et al.
      Colonic microbiome is altered in alcoholism.
      ,
      • Chen Y.
      • Yang F.
      • Lu H.
      • Wang B.
      • Chen Y.
      • Lei D.
      • et al.
      Characterization of fecal microbial communities in patients with liver cirrhosis.
      , and Akkermansia.
      • Grander C.
      • Adolph T.E.
      • Wieser V.
      • Lowe P.
      • Wrzosek L.
      • Gyongyosi B.
      • et al.
      Recovery of ethanol-induced Akkermansia muciniphila depletion ameliorates alcoholic liver disease.
      A recent study by Duan et al. demonstrated that patients with alcoholic hepatitis have an increased relative abundance of Enterococcus faecalis (E. faecalis) and specifically a strain that secrets the exotoxin cytolysin. The presence of cytolysin-secreting E. faecalis 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.
      • Duan Y.
      • Llorente C.
      • Lang S.
      • Brandl K.
      • Chu H.
      • Jiang L.
      • et al.
      Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.

      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.
      • Mieli-Vergani G.
      • Vergani D.
      • Czaja A.J.
      • Manns M.P.
      • Krawitt E.L.
      • Vierling J.M.
      • et al.
      Autoimmune hepatitis.
      Patients with autoimmune hepatitis have an overrepresentation of potential pathobionts, including Veillonella species such as Veillonella dispar, in their faecal microbiomes.
      • Wei Y.
      • Li Y.
      • Yan L.
      • Sun C.
      • Miao Q.
      • Wang Q.
      • et al.
      Alterations of gut microbiome in autoimmune hepatitis.
      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.
      • Manfredo Vieira S.
      • Hiltensperger M.
      • Kumar V.
      • Zegarra-Ruiz D.
      • Dehner C.
      • Khan N.
      • et al.
      Translocation of a gut pathobiont drives autoimmunity in mice and humans.
      E. gallinarum DNA was detected in the livers of most patients with autoimmune hepatitis but in none of the healthy control livers.
      • Manfredo Vieira S.
      • Hiltensperger M.
      • Kumar V.
      • Zegarra-Ruiz D.
      • Dehner C.
      • Khan N.
      • et al.
      Translocation of a gut pathobiont drives autoimmunity in mice and humans.

      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.
      • Little R.
      • Wine E.
      • Kamath B.M.
      • Griffiths A.M.
      • Ricciuto A.
      Gut microbiome in primary sclerosing cholangitis: a review.
      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.
      • Ruhlemann M.
      • Liwinski T.
      • Heinsen F.A.
      • Bang C.
      • Zenouzi R.
      • Kummen M.
      • et al.
      Consistent alterations in faecal microbiomes of patients with primary sclerosing cholangitis independent of associated colitis.
      • Ruhlemann M.C.
      • Heinsen F.A.
      • Zenouzi R.
      • Lieb W.
      • Franke A.
      • Schramm C.
      Faecal microbiota profiles as diagnostic biomarkers in primary sclerosing cholangitis.
      • Kummen M.
      • Holm K.
      • Anmarkrud J.A.
      • Nygard S.
      • Vesterhus M.
      • Hoivik M.L.
      • et al.
      The gut microbial profile in patients with primary sclerosing cholangitis is distinct from patients with ulcerative colitis without biliary disease and healthy controls.
      • Bajer L.
      • Kverka M.
      • Kostovcik M.
      • Macinga P.
      • Dvorak J.
      • Stehlikova Z.
      • et al.
      Distinct gut microbiota profiles in patients with primary sclerosing cholangitis and ulcerative colitis.
      • Sabino J.
      • Vieira-Silva S.
      • Machiels K.
      • Joossens M.
      • Falony G.
      • Ballet V.
      • et al.
      Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD.
      • Lemoinne S.
      • Kemgang A.
      • Ben Belkacem K.
      • Straube M.
      • Jegou S.
      • Corpechot C.
      • et al.
      Fungi participate in the dysbiosis of gut microbiota in patients with primary sclerosing cholangitis.
      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.
      • Nakamoto N.
      • Sasaki N.
      • Aoki R.
      • Miyamoto K.
      • Suda W.
      • Teratani T.
      • et al.
      Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis.
      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.
      • Nakamoto N.
      • Sasaki N.
      • Aoki R.
      • Miyamoto K.
      • Suda W.
      • Teratani T.
      • et al.
      Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis.

      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.
      • Chen Y.
      • Yang F.
      • Lu H.
      • Wang B.
      • Chen Y.
      • Lei D.
      • et al.
      Characterization of fecal microbial communities in patients with liver cirrhosis.
      ,
      • Bajaj J.S.
      • Hylemon P.B.
      • Ridlon J.M.
      • Heuman D.M.
      • Daita K.
      • White M.B.
      • et al.
      Colonic mucosal microbiome differs from stool microbiome in cirrhosis and hepatic encephalopathy and is linked to cognition and inflammation.
      ,
      • Bajaj J.S.
      • Ridlon J.M.
      • Hylemon P.B.
      • Thacker L.R.
      • Heuman D.M.
      • Smith S.
      • et al.
      Linkage of gut microbiome with cognition in hepatic encephalopathy.
      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.
      • Qin N.
      • Yang F.
      • Li A.
      • Prifti E.
      • Chen Y.
      • Shao L.
      • et al.
      Alterations of the human gut microbiome in liver cirrhosis.
      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.
      • Bajaj J.S.
      • Heuman D.M.
      • Hylemon P.B.
      • Sanyal A.J.
      • White M.B.
      • Monteith P.
      • et al.
      Altered profile of human gut microbiome is associated with cirrhosis and its complications.
      Moreover, an increased relative abundance of pathogenic bacteria was associated with the development of complications such as hepatic encephalopathy.
      • Our knowledge of taxonomic differences in the bacterial microbiomes of patients with different aetiologies of liver disease could help identify potential targets for phage therapy.
      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.
      • Wiest R.
      • Rath H.C.
      Gastrointestinal disorders of the critically ill. Bacterial translocation in the gut.
      Aerobic Gram-negative bacteria, such as E. coli, K. pneumoniae, Pseudomonas aeruginosa, and other Enterobacteriaceae, translocate much more readily than anaerobic bacteria.
      • Steffen E.K.
      • Berg R.D.
      • Deitch E.A.
      Comparison of translocation rates of various indigenous bacteria from the gastrointestinal tract to the mesenteric lymph node.
      Notably, these species have also been implicated in decompensation of cirrhosis.
      • Bert F.
      • Johnson J.R.
      • Ouattara B.
      • Leflon-Guibout V.
      • Johnston B.
      • Marcon E.
      • et al.
      Genetic diversity and virulence profiles of Escherichia coli isolates causing spontaneous bacterial peritonitis and bacteremia in patients with cirrhosis.
      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 data suggest that selective targeting of bacterial strains such as Enterococcus faecalis or Klebsiella pneumoniae by phage therapy can modify liver disease progression, such as in alcoholic hepatitis or steatohepatitis, respectively.

      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.
      • Duan Y.
      • Llorente C.
      • Lang S.
      • Brandl K.
      • Chu H.
      • Jiang L.
      • et al.
      Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.
      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.
      • Yuan J.
      • Chen C.
      • Cui J.
      • Lu J.
      • Yan C.
      • Wei X.
      • et al.
      Fatty liver disease caused by high-alcohol-producing Klebsiella pneumoniae.
      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.
      • Tetart F.
      • Repoila F.
      • Monod C.
      • Krisch H.M.
      Bacteriophage T4 host range is expanded by duplications of a small domain of the tail fiber adhesin.
      ,
      • Yoichi M.
      • Abe M.
      • Miyanaga K.
      • Unno H.
      • Tanji Y.
      Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7.
      ,
      • Ando H.
      • Lemire S.
      • Pires D.P.
      • Lu T.K.
      Engineering modular viral scaffolds for targeted bacterial population editing.
      • Dunne M.
      • Rupf B.
      • Tala M.
      • Qabrati X.
      • Ernst P.
      • Shen Y.
      • et al.
      Reprogramming bacteriophage host range through structure-guided design of chimeric receptor binding proteins.
      • Yehl K.
      • Lemire S.
      • Yang A.C.
      • Ando H.
      • Mimee M.
      • Torres M.T.
      • et al.
      Engineering phage host-range and suppressing bacterial resistance through phage tail fiber mutagenesis.
      ,
      • Scholl D.
      • Rogers S.
      • Adhya S.
      • Merril C.R.
      Bacteriophage K1-5 encodes two different tail fiber proteins, allowing it to infect and replicate on both K1 and K5 strains of Escherichia coli.
      ,
      • Duplessis M.
      • Moineau S.
      Identification of a genetic determinant responsible for host specificity in Streptococcus thermophilus bacteriophages.
      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.
      • Dunne M.
      • Rupf B.
      • Tala M.
      • Qabrati X.
      • Ernst P.
      • Shen Y.
      • et al.
      Reprogramming bacteriophage host range through structure-guided design of chimeric receptor binding proteins.
      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 polysaccharide-degrading enzyme in the phage.
      • Lu T.K.
      • Collins J.J.
      Dispersing biofilms with engineered enzymatic bacteriophage.
      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.
      • Merril C.R.
      • Scholl D.
      • Adhya S.L.
      The prospect for bacteriophage therapy in Western medicine.
      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 phage-based therapies powerful new tools for the treatment of many diseases, including those of the liver.

      Abbreviations

      AIEC, adherent-invasive E. coli; A. baumannii, Acinetobacter baumannii; EBV, Epstein-Barr virus; E. coli, Escherichia coli; E. faecalis, Enterococcus faecalis; E. gallinarum, Enterococcus gallinarum; K. pneumoniae, Klebsiella pneumoniae; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PSC, primary sclerosing cholangitis.

      Financial support

      This review was supported in part by NIH grants R01 AA24726 , R01 AA020703 , U01 AA026939 , by Award Number BX004594 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development, and a Biocodex Microbiota Foundation Grant (to B.S.) and services provided by NIH centers P30 DK120515 and P50 AA011999. C.H. is supported by T32 DK007202.

      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.

      Conflicts of interest

      B.S. has been consulting for Ferring Research Institute, HOST Therabiomics, Intercept Pharmaceuticals, Mabwell Therapeutics, Patara Pharmaceuticals and Takeda. B.S.’s institution UC San Diego has received research support from Axial Biotherapeutics, BiomX, CymaBay Therapeutics, NGM Biopharmaceuticals, Prodigy Biotech and Synlogic Operating Company. B.S. is founder of Nterica Bio. UC San Diego has filed several patents with B.S. as inventor related to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is the supplementary data to this article:

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