Bacteriophages are viruses that target and kill specific bacteria, and phage therapy is being investigated as a potential strategy for treating serious bacterial infections. An international team of researchers headed by scientists at the University of California (UC) San Diego School of Medicine has now, for the first time, successfully applied phage therapy in mice to treat alcohol-related liver disease, a condition that’s not usually considered a classic bacterial infection. The researchers’ studies showed that human patients with severe alcoholic hepatitis exhibited high levels of a liver toxin-producing gut bacterium, Enterococcus faecalis. They were then able to use a specific cocktail of phages to target and kill the bacteria in mouse models of alcoholic hepatitis, which effectively eliminated the disease.
“We not only linked a specific bacterial toxin to worse clinical outcomes in patients with alcoholic liver disease, we found a way to break that link by precisely editing gut microbiota with phages,” said senior author Bernd Schnabl, MD, professor of medicine and gastroenterology at UC San Diego School of Medicine and director of the National Institutes of Health-funded San Diego Digestive Diseases Research Center.
“This ground-breaking study has evaluated the potential role of bacteriophages—viruses that specifically kill populations of bacteria in the gut—to beneficially change the gut microbiome in alcohol-related disease, added Debbie Shawcross, PhD, professor of hepatology and chronic liver failure at King’s College London, who is co-author of the team’s published paper in Nature. “The study team has shown that bacteriophages can specifically target cytolytic E. faecalis, providing a method to precisely edit the gut microbiome and offering new treatment for patients with severe alcoholic hepatitis. This novel approach now needs to be expanded to be tested in human clinical trials.” The researchers’ published paper is titled, “Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.”
Alcoholic hepatitis is the most severe form of alcohol-related liver disease, for which mortality rates range from 20% to 40% at 1–6 months, the authors wrote. Chronic liver disease is the third biggest cause of premature mortality and lost working life behind heart disease and self-harm, Shawcross added. “In the U.K., most people die from alcohol-related liver disease at a young age, with 90% under 70 years old and more than 1 in 10 in their 40s.” The condition is treated with corticosteroids, but therapy isn’t highly effective. An early liver transplant is the only cure, but this is only offered at a select number of medical centers, and to a limited number of patients.
While alcohol can directly damage liver cells, the Schnabl team had also previously discovered that alcohol diminishes natural gut antibiotics, leaving mice more prone to bacterial growth in the liver and exacerbating alcohol-induced liver disease. Interestingly, the investigators noted, alcohol-related liver disease can be transmitted by fecal microbiota; “The gut microbiota promotes ethanol-induced liver disease in mice.” What isn’t known, is how: “… little is known about the microbial factors that are responsible for this process.”
For their research reported in Nature, Schnabl and collaborators established a series of studies to address two questions. How do gut bacteria contribute to liver damage? And, can phages be used to reduce any relevant bacteria and thus alleviate alcoholic liver disease?
The team set out to investigate which microorganisms and microbial factors can transmit alcohol-related liver disease. They first carried out 16s ribosomal RNA (rRNA) gene sequencing, to find out whether chronic alcohol use and alcoholic hepatitis is linked to altered gut microbiome composition in human patients. The sequencing studies highlighted a substantial increase in the proportion of Enterococcus species bacteria in patients with the disease. Enterococcus bacteria made up 5.9% of fecal bacteria in people with alcoholic liver disease, compared with “almost none” (0.023%) in healthy people or in people with alcoholic use disorder. In particular, they wrote, “… fecal samples from patients with alcoholic hepatitis had about 2,700-fold more E. faecalis than samples from controls.” Tests showed that about 80% of patients with alcoholic hepatitis were positive for E. faecalis in their feces. Interestingly, however, it wasn’t the overall level of E. faecalis that correlated with disease severity, rather, it was the level of E. faecalis that specifically secreted the liver-damaging toxin cytolysin. “ … the presence of cytolysin-producing E. faecalis rather than the total amount or presence of E. faecalis per se determines the severity of alcoholic hepatitis and mortality.”
The researchers found that nearly 90% of cytolysin-positive individuals with alcoholic hepatitis died within 180 days of hospital admission, compared with approximately 4% of cytolysin-negative patients. “The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis,” they wrote.
The team next transferred fecal samples from cytolysin-positive and cytolysin-negative individuals with alcoholic hepatitis into germ-free mice that had no pre-existing gut micobiome. Animals with the cytolysin-positive humanized gut microbiomes developed more severe alcohol-induced liver disease and had shorter survival times than mice without cytolysin in their humanized microbiomes.
To investigate the potential for phage therapy, the researchers isolated from sewage water four different phages that specifically target cytolysin-producing E. faecalis. When they treated the mice with this cocktail of phages, the bacteria were eradicated and alcohol-induced liver disease was eliminated. Treatment using control phages that targeted other types of bacteria or non-cytolytic E. faecalis had no effect.
The researchers say their results indicate that phage therapy could represent a promising strategy for treating alcoholic hepatitis, although much more research will be needed. “Further work is required to determine whether phages that target cytolytic E. faecalis might be used to treat patients with alcoholic hepatitis, a life-threatening disease that at present has no effective treatment,” they wrote. “Eradication of this specific bacterial strain might produce better outcomes than current treatments, and environmental sources can be used to easily isolate phages that target cytolysin-positive E. faecalis.”
Levels of cytolysin could also feasibly be used to predict severity of disease. “Our data also suggest that cytolysin may be used as a predictive biomarker of severe alcoholic hepatitis; an independent, prospective cohort is therefore needed to validate cytolysin as a biomarker, and to extend the phage findings in mice to humans.”
“This phage therapy has only so far been tested in mice, and a clinical trial will be required to test the safety of this approach, and validate our findings in patients with alcoholic hepatitis,” Schnabl acknowledged. “Based on this finding, we believe detection of the cytolysin-gene in feces from patients with alcoholic hepatitis could be a very good biomarker for liver disease severity and risk of death. One day we might be able to select patients for tailored therapies based on their cytolysin status.”
“This novel avenue of research now needs to be expanded to test the safety and effectiveness of phage therapy in human clinical trials in patients with alcohol-related disease, Shawcross added. “It is also likely that other forms of chronic liver disease associated with changes in the gut microbiome will also benefit from this novel approach, such as fatty liver disease.”