Withdrawn and anxious, the mice in John Cryanâs lab were behaving like you or I might if we had experienced workplace bullying and thought we might encounter the bully again.
The good news, believe it or not, was that some of these rodents were also being fed a slurry of microbes derived from their own faeces. Unpalatable as this sounds, it had a surprisingly positive effect on their behaviour. âIt was phenomenal,â says Cryan, a neurobiologist at University College Cork in Ireland. âWe found that these stress-induced changes in behaviour normalised â they started to behave just like normal animals.â
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Even more surprising, this mental transformation wasnât brought about by changing the bacteria in their guts, but by tinkering with another crucial facet of the microbiome whose importance is only now being recognised: viruses.
It turns out that we are riddled with these. Not the ones that make us unwell, but trillions of stowaways that play a crucial role in cultivating a beneficial microbiome and making us healthier in turn. Recent research shows that the influence of this âviromeâ can be found across the body, from the blood to the brain. The hope is that by tweaking it, we could find new ways of treating various ailments, from inflammatory bowel disease and obesity to anxiety.
Microbiome diversity
The past decade has seen a surge of interest in the microbiome â all the really tiny things that live on and in us â but this has largely focused on bacteria. The assumption until recently was that viruses were mainly transitory visitors, using us to propagate themselves, making us ill in the process. In the early to mid-2000s, however, just as efforts to characterise the numerous bacteria in our bodies were taking off, some researchers began wondering if viruses might also constitute a longer-term component of our microbiomes.
Among them was , then a young microbiologist whose first foray into this hidden world was âquite frankly, the stupidest experiment weâd ever doneâ, he says. âWe literally took a bunch of tubes, spat in them, and then brought them down to the electron microscope to see what we could find.â
This proved a revelation. âThere were so many viruses, we couldnât miss them,â says Pride, now at the University of California in San Diego. Buoyed by this finding, he and his colleagues began scrutinising other body fluids and found viruses everywhere, from blood to the fluid surrounding the brain. âOur hypothesis went from âgee, we donât know if there are any viruses anywhereâ to âviruses are everywhere, and thereâs probably no place where there arenât any virusesâ, â says Pride. It is estimated that each of us is 380 trillion viruses â with these nanoscopic entities outnumbering bacteria by a ratio of 10:1.
But finding viruses is one thing; figuring out what they are and what they are doing is quite another. Due to technical challenges the in the human gut havenât yet been identified, meaning that research on the virome has lagged considerably behind that on the bacterial component of our bodies. Even so, genetic sampling of the gut virome has thrown up surprises.
âWe started with the idea that we were going to be sequencing viruses that infect human cells, but it turned out that 97 per cent of what we were sequencing were bacteriophages â viruses that infect bacteria,â says Pride.
It was, he says, âan extraordinarily disappointing moment. But I brushed it off and said, âwell then, letâs figure out how bacteriophages are important to humansâ.â
Phages, as they are known, have bulbous heads consisting of genetic material encased in a protein shell and leg-like tail fibres, which are used to penetrate the cell walls of bacteria. All of which means they bear an uncanny resemblance to ticks â parasites that suck blood from animals. In fact, some researchers think phages , as their life cycle involves attaching to susceptible bacteria and hijacking the cellular machinery to produce new copies of themselves. Sometimes they kill their hosts once this goal has been achieved â the word âphageâ derives from the Greek âphageinâ meaning âto devourâ.
Other types of phages, known as temperate phages, integrate their genomes into those of their bacterial hosts, remaining dormant until some external event triggers them to start replicating, or peacefully co-existing alongside the bacterium, shuttling their offspring out of their host without harming it.
Phages are thought to outnumber any other biological entity on the planet and can be found wherever bacteria are present, from the deepest ocean and soil to our guts.
âWhat is interesting about bacteriophages and other viruses in the gut is that every person has their own unique set, with almost no overlap between different people,â says at the Quadram Institute in Norwich, UK. One of her interests is understanding where these viruses come from, which she is investigating by analysing stool samples from women and their newborn infants and following them over time. âWhat we see is that healthy infants are born without a noticeable virome, then, , together with the bacteria and all of the other components of the microbiome,â says Adriaenssens.
Human virome
First to arrive are the temperate phages, most likely piggybacking on the genomes of the earliest bacteria that colonise a babyâs gut. Over the next few years, these phages are gradually replaced with varieties more in line with those of the adult gut, possibly linked to changes in intestinal bacteria as children grow older. Once established, this virome .
Despite their ubiquity, phages are extremely picky about which bacteria they infect. âEven within [bacterial] species, they only infect certain strains, so they are exquisitely selective,â says , who is also at University College Cork.
However, just because the phage population follows changes in the bacteria that gradually colonise our bodies, this doesnât mean they are inert passengers. Once established, phages may play an important role in maintaining the structure and function of bacterial communities. Although phages prey on bacteria, it wouldnât be in their interest to destroy all the individuals in a community. In certain situations, phages may even benefit their host bacteria. For instance, Pride has found that have genes that may help the bacteria they infect to evade the immune system â genes presumably acquired from bacteria that they infected previously.
Bacteria have also evolved strategies to evade or respond to phage attack, and phages have done likewise with countermeasures. We increasingly suspect that this ongoing arms race may help to keep bacterial numbers in check and boost the health of surviving bacteria. âTheyâre partners and need each other to co-evolve and to stay fit,â says Hill.
Another way to view phages is as bacterial gardeners. By weeding out weak bacteria, they create space for others to grow and flourish. âIn a system where you have a combination of bacteria and bacteriophages, you usually find a larger diversity of bacteria,â says Adriaenssens.
It is widely agreed that a more diverse microbiome is a healthier one â and this includes viruses too. Indeed, a 2023 suggests that longevity and a healthy lifespan may be linked to having a greater diversity of viruses in the gut.
The viromeâs impact might also be felt in the brain. Our gut microbes produce some of the same neurotransmitters that are made in the brain, and may also affect mood by triggering inflammation. But while most research on this âgut-brain axisâ has focused on the role of bacteria, there are tantalising hints that phages may be implicated as well.
In a , at the University of Girona in Spain and his team analysed stool samples from more than 1000 volunteers to check whether the types of phage in their guts correlated with an ability to learn and retain new information. They found that people with higher levels of a class of phages known as Caudovirales did better in cognitive tests, whereas those with lots of phages in the Microviridae family took longer to complete the tests and retained less information.
Next, they transplanted faecal samples from volunteers with high Caudovirales levels into the guts of mice to see if this altered their cognition. âWe were very surprised,â says FernĂĄndez-Real. Not only did the test scores of the animals improve following the transplant, but genes relevant to learning, memory and neuronal development became more active in the prefrontal cortex â an area associated with planning, decision-making and memory. Although the team couldnât be certain that these improvements were purely down to changes in the virome, further experiments in fruit flies fed a diet rich in Caudovirales phages suggested that their memory also improved.
Stress effects
âs work is also shining a light on the role of phages in brain function. About 15 years ago, he discovered that stress could affect the composition of microbial communities in the gut. suggested that boosting the levels of certain gut bacteria in mice could buffer them against some of the negative effects of stress â something that appeared to be mediated through the vagus nerve, which ferries messages between the brain and the rest of the body.
âIt was a defining moment,â says Cryan. âWe showed that the microbiome is regulating the stress response and you can target the microbiome to alleviate the stress response.â
To investigate whether phages might provide a means of doing this, Cryan and his colleagues subjected mice to a scenario designed to induce social stress by placing them in a cage with an aggressive peer. The mice are separated before any physical harm occurs and the victim is then housed within sight and smelling distance of the bully. Usually, mice exposed to such treatment become anxious and socially withdrawn. However, if at this stage their microbiomes are , they spend more time out in the open, travel more and, if placed in a cage with a different mouse, they interact with it more.
Further work revealed this supplementation buffered the effects of stress on the miceâs immune cells, while gene activity in the hippocampus and the amygdala â brain areas associated with memory, fear and social behaviour â was normalised. âWe think the viruses are affecting the bacteria, which are affecting the brain. But the viruses are a key regulator of the bacteria,â says Cryan.
He believes that virome transplants could someday provide an alternative means of treating stress-related disorders. âWeâre looking at whether we could generate some form of intervention that could be given to someone who knows theyâre going into a stressful situation that could help them manage that stress better.â One attraction of using phages to do this is their specificity. âIf you can identify specific bacteria that you want to target in specific situations, bacteriophages are a great tool to help you do that,â says Cryan. âThe problem that we have in neuroscience right now is that we donât yet know what are the specific bacteria that we want to shut out.â
However, there are other scenarios where we do know this. One is when tackling infections caused by antibiotic-resistant bacteria. âIn the textbooks, phage therapy is a dream solution because if youâve been infected with a particular pathogen and you have a phage that kills it, you can wipe out that pathogen without affecting the other bacteria in its environment, whereas an antibiotic kills a lot of other things as well,â says Hill.
Phage therapy
Earlier this year, the UKâs Science, Innovation and Technology Committee calling on the government and regulators to do more to advance the use of phages to treat antibiotic-resistant bacteria. Phage therapy has been used in parts of eastern Europe and Russia for decades. For instance, the George Eliava Institute of Bacteriophages, Microbiology and Virology in Tbilisi, Georgia, has been using phages to treat bacterial infections since the 1920s and today treats patients from across the world at its . âWe have six ready-to-use phage preparations, and if those donât work against a clinical sample, we will prepare a specific phage for a patient,â says , the instituteâs director.
However, medical systems elsewhere have been slow to embrace phage therapy â in part because large clinical trials havenât been published to demonstrate its efficacy. Regulators also tend to want to know the precise dose and mechanism through which interventions work, which are difficult to map for cocktails of phages, says Hill.
There may be an alternative: Hill and his colleagues are that phages of Clostridioides difficile bacteria use to break open their hostsâ cell walls and escape. C. difficile is a leading cause of healthcare-associated infections, and antibiotic resistance is a growing problem, but if these enzymes could be purified and delivered to the bacteria, they could kill them without damaging the wider microbiome. In the long term we could develop cocktails of phages or their enzymes to tweak the composition of our microbiomes and treat mental health conditions such as anxiety, or other conditions associated with an unbalanced microbiome, and .
For now though, our best bet for looking after our gut virome seems to be via what we eat. Though it is too soon to say precisely which phages are associated with good health and how to boost their growth, recent research has provided some clues. Phages tend to hitch a ride with their bacterial hosts; they are abundant in bacteria-rich foods, such as sauerkraut, kefir, kimchi, wine and fermented dairy products, including cheeses. FernĂĄndez-Real, for instance, found that participants in his study who had high levels of beneficial Caudovirales phages reported regular consumption of dairy products. Certain foods may also be able to change the abundance of gut bacteria by altering the behaviour of dormant phages. For example, a 2020 study found that the sugar substitute stevia and start killing their hosts.
Although it is early days, one thing is clear: viruses are far from one-dimensional villains. âViruses have historically had a really bad rap, and covid only made that worse. But as our data clearly show, we cannot forget about the role of viruses in maintaining overall health and how we manage stress,â says Cryan. âIt is time they came into the spotlight.â
