I Contain Multitudes: The Microbes Within Us and a Grander View of Life

by Ed Yong

The weird biology of germ-free animals is most obvious in the gut. A well-functioning gut needs a big surface area for absorbing nutrients, which is why its walls are densely lined with long, finger-like pillars. It needs to constantly regenerate the cells at its surface, which get sloughed off by the passing tide of food. It needs a rich network of underlying blood vessels to carry nutrients to and fro. And it needs to be sealed – its cells must stick tightly to each other to prevent foreign molecules (and microbes) from leaking into those blood vessels. All of these essential properties are compromised without microbes.

Gut. Technical.

Lora Hooper demonstrated this by infusing into germ-free mice a common gut bacterium called Bacteroides thetaiotaomicron – or B-theta to its friends.7 She found that the microbe activated a wide range of mouse genes that are involved in absorbing nutrients, building an impermeable barrier, breaking down toxins, creating blood vessels, and creating mature cells. In other words, the microbe told the mice how to use their own genes to make a healthy gut.

B-theta (Bacteroides thetaiotaomicron)

germ-free animals live in undemanding environments: climate-controlled bubbles with plentiful food and water, zero predators, and no infections of any kind. In the brutal wild, they wouldn’t last long. They could exist but probably wouldn’t persist. They can develop alone, but they’re better off with their microbial partners.

Remember that animals emerged in a world that had already been teeming with microbes for billions of years. They were the rulers of the planet long before we arrived. And when we did arrive, of course we evolved ways of interacting with the microbes around us. It would be absurd not to,

We know very little about what the first animals looked like because their soft bodies didn’t fossilise. They came and went like a winter breath, leaving no imprint upon the world. But we can make some educated guesses about them.

S. rosetta forms colonies only when it meets the right microbe. Alegado identified the culprit and named it Algoriphagus machipongonensis – a new species, but part of the Bacteroidetes lineage that dominates our guts.13 She also identified how the bacteria induce the rosettes: by releasing a fat-like molecule called RIF-1.

Alegado suspects that all these substances are a sign that food is near. The choanos are better at catching bacteria as a group than they are on their own, so if they sense bacteria nearby, they unite. ‘I think the choanos are eavesdropping,’ says Alegado. ‘They’re slow swimmers, and the Bacteroidetes are good indicators that they have entered an area with great resources and food. Then, they can commit to making a rosette.’

Interesting. Same idea with NMN.

‘In the oceans in which the first animals evolved, I think there’s no controversy that there was a ton of bacteria,’ says King. ‘They were diverse. They dominated the world, and animals had to accommodate to them. It’s not a stretch to think that some molecules produced by bacteria may have influenced the development of the first animals.’

You can see what happens to the ships by throwing a random scrap of metal into the water. Within hours, bacteria start growing on it. Algae might follow. There may be clams or barnacles. But eventually, within days, white tubes appear. They’re tiny – each just a few centimetres long and a few millimetres wide. But soon there are hundreds of them. Then thousands. Millions. Eventually, the entire surface looks like a frozen shag pile rug. These tubes get everywhere: on rocks, pilings, fishing cages, and ships. If an aircraft carrier sits in the harbour for a few months, the tubes will amass on its hull in layers several centimetres deep. The technical term is biofouling.

The truth behind the abrupt appearance of oysters and tube worms is more banal. These animals, like corals, sea urchins, mussels and lobsters, have larval stages that drift through the open ocean until they find somewhere to land. The larvae are microscopic, extraordinarily abundant (there might be 100 in a drop of seawater), and utterly unlike their adult counterparts.

At some point, the larvae settle down. They abandon their youthful wanderlust and remodel their bodies into sedentary adult shapes. This process – metamorphosis – is the most important moment in their lives. Scientists once suspected that it happened randomly, with the larvae settling in arbitrary places and surviving if they were lucky enough to hit a good location. In fact, they are purposeful and selective. They follow clues like chemical trails, temperature gradients, and even sounds, to find the best spots for metamorphosis.

Environment

The worms don’t respond to any old microbe. Of the many strains in Hawaiian waters, Hadfield found that only a few could induce metamorphosis, and only one did so strongly. Its gargled mouthful of a name is Pseudoalteromonas luteoviolacea. Mercifully, Hadfield just calls it P-luteo. More than any other microbe, this one excels at turning larval worms into adults. Without the bacteria, the worms would never reach adulthood.

Hadfield thinks that their value is simpler. The presence of a biofilm provides a larval animal with important information. It means that: (a) there’s a solid surface, (b) which has been around for a while, (c) isn’t too toxic, and (d) has enough nutrients to sustain microbes. Those reasons are as good as any to settle down. The better question would be: Why wouldn’t you rely on bacterial cues? Or better still: what choice do you have?

Without bacteria, the sociable choanos would forever be solitary, and the larval worms would forever be immature. These are beautiful examples of how thoroughly microbes can shape the bodies of animals (or animal cousins).

And yet, they aren’t symbioses in the classical sense. The worms don’t actually harbour P-luteo in their bodies, and they don’t seem to interact with the bacterium after they become adults. Their relationship is transient. They are like tourists asking passers-by for directions and then moving on. But other animals form more lasting and co-dependent relationships with microbes.

Paracatenula is a master of regeneration. Cut it in two, and both ends become fully functional animals. The back half will even re-grow a head and brain. ‘Chop them up and you can get ten,’ says Gruber-Vodicka. ‘That’s probably what they do in nature. They get longer and longer, and then one end breaks off and there are two.’

trophosome, the bacteria inside it, and the energy they lock away. As long as a fragment of flatworm contains enough symbionts, it can produce an entire animal. If the symbionts are too scarce, the fragment dies.

Counter-intuitively, this means that the only bit of the flatworm that can’t regenerate is the bacteria-free head. The tail will re-grow a brain but the brain alone will not produce a tail.

interesting

In the words of Oliver Sacks, ‘Nothing is more crucial to the survival and independence of organisms – be they elephants or protozoa – than the maintenance of a constant internal environment.’19 And in maintaining such constancy, microbes are crucial.

This tells us that an animal’s genome doesn’t provide everything it needs to create a mature immune system. It also needs input from a microbiome.22 Hundreds of scientific papers, on species as diverse as mice, tsetse flies and zebrafish, have shown that microbes help to shape the immune system in some way. They influence the creation of entire classes of immune cells, and the development of organs that make and store those cells.

Take inflammation: a defensive response, where immune cells rush to the site of an injury or infection, leading to swelling, redness, and heat. It’s important for protecting the body against threats; without it, we’d be riddled with infections. But it becomes a problem if it spreads throughout the body, lasts too long, or launches at the slightest provocation: that leads to asthma, arthritis, and other inflammatory and autoimmune diseases. So, inflammation must be triggered at the right time, and controlled appropriately. Suppressing it is as important as activating it. Microbes do both.

PSA is a bacterial molecule: exactly the type of substance that, according to common wisdom, the immune system should see as a threat. PSA ought to trigger inflammation. Instead, it does the opposite: it quells inflammation and calms the immune system. Mazmanian calls it a ‘symbiosis factor’ – a chemical message from microbe to host that says: I come in peace.27 This clearly shows that the immune system isn’t innately hard-wired to tell the difference between a harmless symbiont and a threatening pathogen. In this case, it’s the microbe that makes that distinction clear.

‘You can observe lions in the field, but they’ll just lie there, and you can work with wolves for years and just see scats or hear howls,’ says hyena aficionado Kevin Theis. ‘But hyenas . . . there are greetings, reintroductions, dominance and submissive signalling. You’ll have young cubs trying to learn their place within the clan, immigrant males doing a run-through to see who’s there. Their social lives are incredibly more complex.’

By sequencing the DNA of the resident microbes, he found more types of bacteria than all the previous surveys put together. He also showed that these bacteria, and the chemicals they produce, vary between spotted and striped hyenas, between spotted hyenas from different clans, between males and females, and between fertile and infertile ones.31 Based on these differences, the paste could act as chemical graffiti that reveal who their makers are, which species they’re from, how old they are, and whether they’re ready to mate. By impregnating grass stalks with their smelly microbes, hyenas spray their personal tags all over the savannah.

If those chemical cues reflect a trait that’s useful to know about – say, gender, strength, or fertility – the host animal might evolve scent organs to nourish and harbour those specific microbes. Eventually, the inadvertent cues turn into full-blown signals. So, by creating airborne messages, microbes could affect the behaviour of animals far outside their original hosts. And if that’s the case, it shouldn’t be surprising to learn that they can affect animal behaviour in more local ways.

Mazmanian had shown that gut microbes affect the immune system, and Patterson had found that the immune system affects the developing brain. And they realised that Patterson’s mice had gut problems in common with actual autistic children: both were more likely to have diarrhoea and other gastrointestinal disorders, and both harboured unusual communities of gut microbes.

Gut problem and autism

perhaps, they reasoned, fixing those gut problems might also lead to changes in behaviour? To test this idea, the duo fed B-frag to Patterson’s mice.34 The results were remarkable. The rodents became keener to explore, harder to startle, less prone to repetitive movements, and more communicative. They were still reluctant to approach other mice, but in every other respect B-frag had reversed the changes caused by their mothers’ immune responses.

How? And why? Here’s the best guess: By mimicking a viral infection in the pregnant mothers, the team triggered an immune response that landed their offspring with an excessively permeable gut, and one with an unusual collection of microbes. Those microbes produced chemicals that entered the bloodstream and travelled to the brain, where they triggered atypical behaviours. The top culprit is a toxin called 4-ethylphenylsulfate (4EPS), which can trigger anxiety in otherwise healthy animals. When the mice swallowed B-frag, this microbe sealed up their guts and stemmed the flow of 4EPS (and other substances) to their brain, reversing their atypical symptoms.

Interesting

Patterson died in 2014 but Mazmanian is now carrying on his friend’s work. His long-term goal is to develop a bacterium that people can swallow to control some of the more difficult symptoms of autism. That might be B-frag: it certainly worked well in the mice, and happens to be the most heavily depleted microbe in the guts of people with autism.

B-frag and autism

Mazmanian’s team recently did an experiment which hints that the two sets of behaviour are related. They transferred gut microbes from children with autism into mice, and found that the rodents developed the same quirks that Patterson saw, such as repetitive behaviour and social aversion.

At the very least, Patterson and Mazmanian showed that tweaking a mouse’s gut microbes – or even a single microbial molecule, 4EPS – could change its behaviour.

4Eps

We put such a premium on our free will that the prospect of losing independence to unseen forces informs many of our deepest societal fears. Our darkest fiction is full of Orwellian dystopias, shadowy cabals, and mind-controlling supervillains. But it turns out that the brainless, microscopic, single-celled organisms that live inside us have been pulling on our strings all along.

Our darkest fiction is full of Orwellian dystopias, shadowy cabals, and mind-controlling supervillains. But it turns out that the brainless, microscopic, single-celled organisms that live inside us have been pulling on our strings all along.

Among Beaumont’s many observations, he noticed that St Martin’s mood affected his stomach. When the man became angry or irritable – and it’s hard to imagine not getting irascible when a surgeon is dangling food through the hole in your side – his rate of digestion changed. That was the first clear sign that the brain affects the gut. Almost two centuries later, this maxim seems all too familiar. We lose our appetite when our mood changes, and our mood changes when we feel hungry. Psychiatric problems and digestive problems often go hand in hand.

We lose our appetite when our mood changes, and our mood changes when we feel hungry. Psychiatric problems and digestive problems often go hand in hand.

We now know that gut microbes are part of this axis, in both directions. Since the 1970s, a trickle of studies have shown that any kind of stress – starvation, sleeplessness, being separated from one’s mother, the sudden arrival of an aggressive individual, uncomfortable temperatures, overcrowding, even loud noises – can change a mouse’s gut microbiome. The opposite is also true: the microbiome can affect a host’s behaviour, including its social attitudes and its ability to deal with stress.

At Sweden’s Karolinska Institute, Sven Petterson found that germ-free mice were less anxious and took more risks than their microbe-laden cousins. But if these mice were colonised by microbes as pups, they grew up into adults that behaved in the usual cautious ways.

Interesting

It was as dramatic a result as Collins could have hoped for: by swapping the bacteria in the animals’ guts, he had also swapped part of their personalities.

They worked with the same strain of timid mice that Collins studied, and managed to change the animals’ behaviour by feeding them with a single strain of Lactobacillus rhamnosus – a bacterium commonly used in yoghurts and dairy products. After the mice ingested this strain, known as JB-1, they were better able to overcome anxiety: they spent more time in the exposed parts of a maze, or the centre of an open field.

JB-1

They were also better at resisting negative moods: when dropped into a bottle of water, they spent more time paddling away than floating aimlessly.41 These kinds of test are commonly used to test the effectiveness of psychiatric drugs, and JB-1 was behaving rather like substances with anti-anxiety and antidepressant properties.

JB-1

they also have many inconsistencies. Some studies found that microbes only affect the brains of young mice; others that adolescents and adults are also affected. Some found that bacteria make rodents less anxious; others, more so. Some show that the vagus nerve is vital; others emphasise that microbes can produce neurotransmitters like dopamine and serotonin, which carry messages from one neuron to another.43 These contradictions aren’t unexpected – when two things as fiendishly complex as the microbiome and the brain collide, it would be naïve to expect clean results.

Inconsistency

In one of the more promising studies (albeit, still a small one), Kirsten Tillisch found that women who ate twice-daily servings of a microbe-rich yoghurt showed less activity in parts of the brain involved in processing emotions, compared to women who ate microbe-free milk products. The meaning of these differences is open to debate, but they do at least show that bacteria can affect human brain activity.

for your gut bacteria, the choice is more important. Different microbes fare better on certain diets. Some are peerless at digesting plant fibres. Others thrive on fats. When you choose your meals, you are also choosing which bacteria get fed, and which get an advantage over their peers. But they don’t have to sit there and graciously await your decision. As we have seen, bacteria have ways of hacking into the nervous system. If they released dopamine, a chemical involved in feelings of pleasure and reward, when you ate the ‘right’ things, could they potentially train you to choose certain foods over others? Do they get a say in your menu picks?

There’s a reason why all of these strategies are bad news for males. Wolbachia can only pass to the next generation of hosts in eggs; sperm are too small to contain it. Females are its ticket to the future; males are an evolutionary dead end. So it has evolved many ways of screwing over male hosts to expand its pool of female ones.

Where Wolbachia does allow males to survive, it still manipulates them. It often changes their sperm so that they cannot successfully fertilise eggs unless the eggs are infected with the same strain of Wolbachia. From the females’ perspective, this incompatibility means that infected females (which can mate with whomever they like) gain a competitive advantage over uninfected females (which can only mate with uninfected males).

In many animals, Wolbachia is a reproductive parasite: an organism that manipulates the sex lives of its hosts to further its own ends. The hosts suffer. Some die, others become sterile, and even unaffected individuals must live in a skewed world with few potential mates. Wolbachia might seem like the archetypal ‘bad microbe’, but it has a beneficent side, too. It provides some unknown benefit to certain nematode worms, which cannot survive without it. It protects some flies and mosquitoes from viruses and other pathogens. The wasp Asobara tabida cannot make eggs without it.

The most striking use for Wolbachia becomes apparent if you walk through a European apple orchard in the autumn. Among the yellow and orange leaves, you might find some with small green islands, defiantly resisting the seasonal decay. These are the work of the spotted tentiform leaf miner, a moth whose caterpillars live inside the leaves of apple trees. Almost all of them carry Wolbachia. In these insects, the microbe releases hormones that stop the leaves from yellowing and dying. They are the means by which the caterpillar holds back the autumn, to give itself enough time to become an adult. If you cure Wolbachia the leaves will die and fall, as will the caterpillars inside.

Wolbachia

Here is a strange but critical sentiment to introduce in a book about the benefits of living with microbes: there is no such thing as a ‘good microbe’ or a ‘bad microbe’. These terms belong in children’s stories. They are ill-suited for describing the messy, fractious, contextual relationships of the natural world.

wow

Nichole Broderick found a great example of this when she was studying a soil-dwelling microbe called Bacillus thuringiensis, or Bt. It produces toxins that can kill insects by punching holes in their guts. Farmers have exploited this ability since the 1920s, by spraying Bt onto crops as a living pesticide. Even organic farmers do this. The bacterium’s effectiveness is undeniable, but for decades scientists had the wrong idea about how it kills. They assumed that its toxins inflict so much damage that their victims starve to death. But this couldn’t be the whole story. It takes more than a week for a caterpillar to starve, and Bt kills in half that time.

Bt.

Even symbionts as essential and long-standing as mitochondria, the energy-providing power plants that exist in all animals’ cells, can wreak havoc if they end up in the wrong place. A cut or a bruise can split some of your cells apart and spill fragments of mitochondria into your blood – fragments that still keep some of their ancient bacterial character. When your immune system spots them, it mistakenly assumes that an infection is under way and mounts a strong defence. If the injury is severe, and enough mitochondria are released, the resulting body-wide inflammation can build into a lethal condition called systemic inflammatory response syndrome (SIRS).10 SIRS can be worse than the original injury.

SIRS (systemic inflammatory response syndrome). Mitochondria.