# How do infants get their very first gut bacteria?

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First of all, I'm a complete layman in Biology. Recently I read "The evidence for evolution" by Richard Dawkins. Pondering about this matter, I wondered about the origin of human gut bacteria (could be more generalized to mammal gut bacteria).

Generally, I see two possibilities:

These bacteria are

• acquired later on through the environment (probably after birth) or
• they are produced by the organism itself

The former option seems to me a little bit too vulnerable to be true (different food, different bacteria due to geographical location, etc.)

The latter option seems baffling to me. Bacteria are, as far as I know, independent lifeforms. Is it possible, that our DNA contains the information for other lifeforms as well?

Well, the former is exclusively the answer. Humans (or other mammals, or any species in general) cannot give birth to new species (of bacteria, here); they have to be acquired from the environment at all costs. However, the environment does vary, leading to variance in gut microbiome, as will be discussed later.

First of all, gut microbiome is only a part of the total microbiome of humans (Sherwood et al, 2013). Now, usually, gut bacteria are acquired by a person within one to two years of birth (Sommer et al, 2013), while the gut of a fetus is considered as sterile. However, microbial colonization in a fetus is possible (Matamoros et al, 2013). For example, species of Lactobacillus and Bifidobacterium were found in biopsies of placenta in one study (Mueller et al, 2017).

During birth and rapidly thereafter, bacteria from the mother and the surrounding environment colonize the infant's gut. Infants born by caesarean section may also be exposed to their mothers' microflora, but the initial exposure is most likely to be from the surrounding environment such as the air, other infants, and the nursing staff, which serve as vectors for transfer. During the first year of life, the composition of the gut flora is generally simple and it changes a great deal with time and is not the same across individuals (Sommer et al, 2013).

Now lets talk about variation in gut microbiome among individuals. The gut microbiome of an individual may vary depend mainly due to:

• Age: the diversity of microbiota composition of fecal samples is significantly higher in adults than in children, although interpersonal differences are higher in children than in adults. Much of the maturation of microbiota into an adult-like configuration happens during the three first years of life.

As the microbiome composition changes, so does the composition of bacterial proteins produced in the gut. In adult microbiomes, a high prevalence of enzymes involved in fermentation, methanogenesis and the metabolism of arginine, glutamate, aspartate and lysine have been found. In contrast, in infant microbiomes the dominant enzymes are involved in cysteine metabolism and fermentation pathways (Yatsunenko et al, 2012).

• Diet: Gut microflora is mainly composed of Prevotella, Bacteroides, and Ruminococcus. There is an association between the concentration of each microbial community and diet. Prevotella is related to carbohydrates and simple sugars, while Bacteroides is associated with proteins, amino acids, and saturated fats. Specialist microbes that break down mucin, survive on their host's carbohydrate excretions. One enterotype will dominate depending on the diet. Altering the diet will result in a corresponding change in the numbers of species (Wu et al, 2011).

• Geography: In simple terms, gut microbiome composition depends on the geographic origin of populations. For example, the US population has a high representation of enzymes encoding the degradation of glutamine and enzymes involved in vitamin and lipoic acid biosynthesis; whereas Malawi and Amerindian populations have a high representation of enzymes encoding glutamate synthase and they also have an overrepresentation of $$alpha$$-amylase in their microbiomes. As the US population has a diet richer in fats than Amerindian or Malawian populations which have a corn-rich diet, the diet is probably a main determinant of gut bacterial composition (Yatsunenko et al, 2012).

Further studies have indicated a large difference in the composition of microbiota between European and rural African children. The fecal bacteria of children from Florence were compared to that of children from the small rural village of Boulpon in Burkina Faso. The diet of a typical child living in this village is largely lacking in fats and animal proteins and rich in polysaccharides and plant proteins. The fecal bacteria of European children was dominated by Firmicutes and showed a marked reduction in biodiversity, while the fecal bacteria of the Boulpon children was dominated by Bacteroidetes. The increased biodiversity and different composition of gut flora in African populations may aid in the digestion of normally indigestible plant polysaccharides and also may result in a reduced incidence of non-infectious colonic diseases (de Filippo et al, 2010).

Also, as @com.prehens.ible says in comments, there are various other modes through which an infant may acquire bacteria to give rise to their gut microbiome. For more information, you can also read the Wikipedia page on Gut Flora.

References:

1. Sherwood, Linda; Willey, Joanne; Woolverton, Christopher (2013). Prescott's Microbiology (9th ed.). New York: McGraw Hill. pp. 713-721. ISBN 9780073402406.

2. Sommer F, Bäckhed F (2013). "The gut microbiota-masters of host development and physiology". Nat Rev Microbiol. 11 (4): 227-38. PMID 23435359.

3. Matamoros S; et al. (2013). "Development of intestinal microbiota in infants and its impact on health.". Trends Microbiol. 21 (4): 167-73. PMID 23332725. doi:10.1016/j.tim.2012.12.001.

4. Mueller, Noel T.; Bakacs, Elizabeth; Combellick, Joan; Grigoryan, Zoya; Dominguez-Bello, Maria G. (2017-04-08). "The infant microbiome development: mom matters". Trends in molecular medicine. 21 (2): 109-117. ISSN 1471-4914. PMC 4464665 . PMID 25578246. doi:10.1016/j.molmed.2014.12.002.

5. Yatsunenko, T.; Rey, F. E.; Manary, M. J.; Trehan, I.; Dominguez-Bello, M. G.; Contreras, M.; Magris, M.; Hidalgo, G.; Baldassano, R. N.; Anokhin, A. P.; Heath, A. C.; Warner, B.; Reeder, J.; Kuczynski, J.; Caporaso, J. G.; Lozupone, C. A.; Lauber, C.; Clemente, J. C.; Knights, D.; Knight, R.; Gordon, J. I. (2012). "Human gut microbiome viewed across age and geography". Nature. 486 (7402): 222-227. Bibcode:2012Natur.486… 222Y. PMC 3376388 . PMID 22699611. doi:10.1038/nature11053.

6. Wu, G. D.; Chen, J.; Hoffmann, C.; Bittinger, K.; Chen, Y.-Y.; Keilbaugh, S. A.; Bewtra, M.; Knights, D.; Walters, W. A.; Knight, R.; Sinha, R.; Gilroy, E.; Gupta, K.; Baldassano, R.; Nessel, L.; Li, H.; Bushman, F. D.; Lewis, J. D. (2011). "Linking Long-Term Dietary Patterns with Gut Microbial Enterotypes". Science. 334 (6052): 105-8. Bibcode:2011Sci… 334… 105W. PMC 3368382 . PMID 21885731. doi:10.1126/science.1208344.

7. De Filippo, C.; Cavalieri, D.; Di Paola, M.; Ramazzotti, M.; Poullet, J. B.; Massart, S.; Collini, S.; Pieraccini, G.; Lionetti, P. (2010). "Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa". Proc. Natl. Acad. Sci. U.S.A. 107 (33): 14691-14696. Bibcode:2010PNAS… 10714691D. PMC 2930426 . PMID 20679230. doi:10.1073/pnas.1005963107.

There is also evidence that exposure of the newborn to the vaginal microbiota of the mother during delivery might be important for the establishment of its own gut, oral and skin microbiota (Dominguez-Bello et al. 2016):

Exposure of newborns to the maternal vaginal microbiota is interrupted with cesarean birthing. Babies delivered by Cesarean section (C-section) acquire a microbiota that differs from that of vaginally delivered infants, and C-section delivery has been associated with increased risk for immune and metabolic disorders

## How baby’s first microbes could be crucial to future health

Sarah DeWeerdt is a freelance science writer in Seattle, Washington.

You can also search for this author in PubMed Google Scholar

Ingredients in breast milk can help to establish a healthy community of microorganisms in the infant gut. Credit: Peter Menzel/SPL

Within a few weeks of being born, a baby is host to a community of billions of bacteria, viruses and fungi — most of which are found in the gut — that can shape many aspects of health. How that community, or microbiota, assembles is a matter of debate: some researchers have begun to question the dogma that the womb is a sterile environment. Yet it’s clear that birth sets off a radical transformation of the infant gut.

“It’s an incredible ecological event,” says Phillip Tarr, a paediatric gastroenterologist at Washington University in St. Louis, Missouri. Colonization of the gut begins in earnest when a baby encounters microorganisms from its mother’s vagina during birth. As the baby suckles at the breast, it picks up more microbes from its mother’s skin. It also consumes microbes from its mother’s gut that have infiltrated her breast milk.

Part of Nature Outlook: The future of medicine

Later, microbes are picked up from adoring visitors or a lick from the family dog, as well as what Nicholas Embleton, a neonatologist at Newcastle University, UK, refers to as “living in a normal, dirty home environment”. By the age of two or three, the composition of a child’s gut microbiota is very similar to that of an adult’s.

Should the assembly process be derailed, the consequences can be deadly. A considerably altered microbiota has been linked to a form of gut inflammation that is a leading cause of death in infants who are born prematurely. Less extreme changes to the microbiota in otherwise healthy babies might have long-term consequences for health, perhaps playing a part in conditions such as asthma and diabetes.

Researchers are looking for ways to rebalance the microbiota in premature infants. And some are wondering whether it might be possible to reshape the microbial community of the healthy infant gut to help prevent chronic diseases in adulthood.

Premature infants are especially vulnerable to disruption of the microbiota. Many are delivered by caesarean section, and therefore do not come into contact with the microbes that live in the birth canal. Such babies are also often given courses of powerful antibiotics and housed in sterile plastic incubators where they have minimal contact with human skin. Given that these interventions separate babies from their environment, it’s not surprising that the gut microbiota of premature infants is markedly different from that of babies born at full term. It tends to have a lower proportion of microbes that are beneficial to gut health, such as Bifidobacterium and Lactobacillus, as well as a greater abundance of disease-causing bacteria and a lower diversity of bacteria in general. And the bacterial community is often chaotic, with dramatic shifts in composition over a matter of days.

The abnormal gut microbiota of premature infants is thought to have a role in their vulnerability to necrotizing enterocolitis, a severe form of gut inflammation that strikes suddenly in the first few weeks of life and can cause permanent damage to the intestine. Although full-term babies can develop the condition, at least three-quarters of cases occur in infants born prematurely. In the past two decades, as doctors have learnt to manage the respiratory problems of premature infants more effectively, necrotizing enterocolitis has become a main threat to such babies.

The cause of necrotizing enterocolitis isn’t a particular microbe, but rather a dysfunction of the gut microbiota as a whole. As well as its role in digestion, the gut is an immune organ, says Barbara Warner, a neonatologist at Washington University in St. Louis. Early interactions of the gut with microbes are therefore powerful shapers of a child’s immune system.

Necrotizing enterocolitis could be the consequence of this process going awry — perhaps representing “the baby’s immune system struggling to work out what’s the right thing to do”, Embleton says. “Probably, this disease we see is a sort of exaggerated inflammatory condition challenging a very immature and naive gut immune system.”

Treatment for the condition “is very, very crude and basic”, Embleton adds. Some babies with necrotizing enterocolitis can be treated with antibiotics and a temporary switch to intravenous feeding to give the intestine time to heal. More-severe cases require surgery to remove the damaged portion of intestine. The loss of a large part of the intestine can lead to lifelong difficulties with feeding or absorbing nutrients. About one-quarter of babies who develop the condition will die.

But now, researchers are looking to the gut microbiota for ways to stop the condition taking hold. Some are searching for clues that could help to predict the development of necrotizing enterocolitis, enabling earlier medical intervention. For example, an overgrowth of bacteria from the phylum Proteobacteria can precede the condition. But these microbes are also found in healthy infants, so it’s not always clear when to sound the alarm. And such changes in microbiota composition might not be the true cause of the illness.

Breast milk might hold a solution. Since the 1990s, several studies have shown that breastfed babies are less vulnerable to necrotizing enterocolitis than are those fed with formula milk. A subsequent flurry of research into the relationship between breast milk and gut microbes found that breast milk contains ingredients that promote the establishment of a healthy gut microbiota.

One example is short chains of sugar molecules known as human-milk oligosaccharides. “They’re the second-most-abundant carbohydrate source in human milk after lactose, but they’re not for nutrition of the babies,” says Victoria Niklas, a neonatologist at the University of California, Los Angeles. Instead, these oligosaccharides provide food for helpful microbes such as Bifidobacterium. They also coat the lining of the gut and bind to pathogenic bacteria, making it more difficult for disease-causing microbes to invade.

Another component of breast milk, the protein lactoferrin, has a number of antimicrobial properties. It suppresses the growth of bacteria and can even trigger the death of certain harmful microbes by binding to inflammatory molecules called lipopolysaccharides.

Offering support to mothers of premature infants who wish to breastfeed might therefore help to promote a healthy gut microbiota and prevent necrotizing enterocolitis. A further potential strategy is to supplement the diets of early babies with human-milk oligosaccharides or lactoferrin. Several trials of such supplements have been completed and more are under way. Biotechnology companies are also developing supplements that contain key components of breast milk. (Niklas is chief medical and scientific officer of one such company, Prolacta Bioscience in Duarte, California.)

Another approach to fighting necrotizing enterocolitis is to feed beneficial bacteria, or probiotics, to premature infants. The goal “is to try and mimic what happens in healthy, full-term, breastfed babies”, says neonatologist and researcher Keith Barrington at the University of Montreal in Canada.

In 2011, the neonatal intensive-care unit at Sainte-Justine University Hospital Center, where Barrington works, began to routinely feed probiotics to babies born before 32 weeks’ gestation. The infants received a cocktail of four species of Bifidobacterium and one of Lactobacillus, and the incidence of necrotizing enterocolitis fell by around 50%. More than half of the neonatal intensive-care units in Canada have followed suit in providing probiotics, with similar results. However, it’s not a perfect solution. Barrington’s team has shown that the probiotic strains are present in stools of the premature babies, which indicates that the microbes are able to grow in the infant gut. But these babies still have fewer beneficial bacteria and more pathogenic bacteria in their gut than do healthy, full-term breastfed babies. Combining probiotics with molecules such as human-milk oligosaccharides or lactoferrin might help to improve the picture, Barrington says. He plans to compare the effects on the gut microbiota of the combination of probiotics and lactoferrin with those of the probiotic treatment alone.

The neonatology community is divided on the role of probiotics in preventing necrotizing enterocolitis. “Half of us think that they’re probably a good idea and half think that the case isn’t proven yet,” says Embleton. “And even if we were to use probiotics, we really don’t know which ones we should be using and how much we give,” he says.

As the debate continues, researchers are investigating whether having the correct gut microbes might also be crucial to enabling healthy infants to thrive. For example, children delivered by caesarean section have a different gut microbiota from those born vaginally. Breastfed and formula-fed babies also have distinct microbiotas in their gut. Epidemiological studies suggest that caesarean delivery and formula feeding are associated with an increased risk of obesity and asthma, as well as other conditions, and many researchers think that these effects might be shaped by the gut microbiota. Could the infant gut microbiota therefore hold the key to preventing such conditions in later life?

The links are not straightforward. “These are complex problems and I think, to be honest, the microbiota is just one piece of it,” says Warner. However, she adds, the microbiota is an attractive target for intervention because it might be easier to modify than other risk factors for certain conditions.

Some doctors have advocated, for example, that babies born by caesarean section be swabbed with a sample of their mother’s vaginal microbiota. But if that microbiota helps to promote a condition such as obesity, the intervention could have a downside. And if the mother harbours disease-causing bacteria, it could even be dangerous.

Few studies have been able to demonstrate the ability of probiotics to make a lasting change to the infant gut microbiota. “It’s extremely difficult to engineer microbial populations that will stick and benefit the host,” says Tarr. When the probiotics are discontinued, the gut microbiota usually reverts to its previous state with a matter of days.

More from Nature Outlooks

But there could be progress on that front. In a 2017 study (S. A. Frese et al. mSphere 2, e00501-17 2017), researchers from the University of California, Davis, and biotechnology company Evolve BioSystems of Davis, California, reported that breastfed infants who were given strain EVC001 of Bifidobacterium longum infantis still had the microbes in their guts 30 days after treatment with the probiotic had been stopped. This strain, which was developed as a probiotic supplement to breastmilk for babies by Evolve BioSystems, is extremely efficient at consuming human-milk oligosaccharides, says neonatologist Mark Underwood, who led the study. (Underwood has no financial interest in the company.)

“We thought, maybe we can make a big difference in this [microbial] community by — instead of keeping them on probiotics forever — treating them for a short period of time with probiotics, but then giving these beneficial bacteria a food source that they are uniquely capable of consuming,” Underwood says.

The babies seeded with B. infantis also had fewer pathogenic bacteria and more beneficial metabolites in their gut than did breastfed babies who did not receive the probiotic. This suggests that the microbiotas of healthy breastfed infants, used as a benchmark for studies in premature babies, are also ripe for improvement.

How far such improvement could go is uncertain. The B. infantis study is only a first step, and researchers are unsure about what an ideal neonatal gut microbiota would look like. Yet the growing importance of the microbiota is changing the approach of the doctors who care for the youngest patients. Among the medical specialities, “neonatology has never been at the top of the food chain”, Niklas says. “But it has now become abundantly clear that our practices and our interventions really hold the seed of future health.”

Nature 555, S18-S19 (2018)

## Birth of a hypothesis

Four years after finishing her graduate work, Nagler started running a lab at Harvard Medical School. She was studying inflammatory bowel disease, not food allergies, back then. But as research in the 1990s showed that inflammatory bowel disease was primarily caused by immune reactions against gut bacteria, she shifted her attention to the microbiome.

Then, in 2000, she came across an intriguing publication. It described a mouse model for peanut allergy that mimics key symptoms experienced by people. The mice scratch relentlessly. Their eyes and mouths get puffy. Some struggle to breathe&mdasha life-threatening allergic response called anaphylaxis.

Credit: Getty Images

All of this happens after researchers feed the mice peanut powder. &ldquoThat caught my eye,&rdquo Nagler says. It ran counter to her earlier findings with the arthritic mice, where feeding collagen calmed the immune reaction. Why the difference?

The peanut-allergy mice, another report showed, had a genetic glitch that damages a receptor called TLR4 that sits in the membranes of immune cells and recognizes microbes. It looked as though the peanut-allergy mice lacked the normal cross talk that takes place between gut microbes and immune cells.

&ldquoThat was my lightbulb moment,&rdquo Nagler says. Perhaps the trillions of microbes that live in us suppress immune responses to food by stimulating the TLR4 receptor. And perhaps perturbations in that teeming microbiome alter the suppression and cause a rise in allergies.

The idea meshes with historical trends. As societies modernized, people moved to urban areas, had more babies by cesarean section, took more antibiotics and ate more processed, low-fiber foods&mdashall of which shake up microbiomes. The timing of these lifestyle shifts parallels the observed increase in food and other types of allergies, whose steep rise over a generation points to some environmental cause.

In 2004, Nagler and her coworkers published a report showing that peanuts provoked anaphylaxis only in mice with a mutated TLR4 receptor, not in genetically related strains with a normal TLR4. The difference disappeared when the scientists wiped out populations of gut bacteria with antibiotics. Then, even normal mice became susceptible to food allergies, implying that bacteria are at the heart of the protection.

Nagler&rsquos lab has been working ever since to identify which bacteria are helpful, and to understand how they regulate allergic responses.

## Gut reaction: the surprising power of microbes

I was standing in a lift at Washington University in St Louis, with Professor Jeff Gordon and two of his students, one of whom was holding a metal canister.

“Just some faecal pellets in tubes,” she said.

“They’re microbes from healthy children, and also from some who are malnourished. We transplanted them into mice,” explained Gordon, as if this was the most normal thing in the world.

The lift doors opened, and I followed Gordon, his students, and the thermos of frozen pellets into a large room. It was filled with rows of sealed chambers made of transparent plastic. Peering inside one of these chambers, I met the eyes of one of the strangest animals on the planet. It looked like just a mouse, and that is precisely why it was so weird. It was just a mouse, and nothing more.

Almost every other animal on Earth, whether centipede or crocodile, flatworm or flamingo, hippo or human, is a teeming mass of bacteria and other microbes. Each of these miniature communities is known as a microbiome. Every human hosts a microbiome consisting of some 39 trillion microbes, roughly one for each of their own cells. Every ant in a colony is a colony itself. Every resident in a zoo is a zoo in its own right. Even the simplest of animals such as sponges, whose static bodies are never more than a few cells thick, are home to thriving microbiomes.

But not the mice in Gordon’s lab. They spend their entire lives separated from the outside world, and from microbes. Their isolators contain everything they need: drinking water, brown nuggets of chow, straw chips for bedding, and a white styrofoam hutch for mating in privacy. Gordon’s team irradiates all of these items to sterilise them before piling them into loading cylinders. They sterilise the cylinders by steaming them at a high temperature and pressure, before hooking them to portholes in the back of the isolators, using connecting sleeves that they also sterilise.

It is laborious work, but it ensures that the mice are born into a world without microbes, and grow up without microbial contact. The term for this is “gnotobiosis”, from the Greek for “known life”. We know exactly what lives in these animals – which is nothing. Unlike every other mouse on the planet, each of these rodents is a mouse and nothing more. An empty vessel. A silhouette, unfilled. An ecosystem of one.

Each isolator had a pair of black rubber gloves affixed to two portholes, through which the researchers could manipulate what was inside. The gloves were thick. When I stuck my hands in, I quickly started sweating.

I awkwardly picked up one of the mice. It sat snugly on my palm, white-furred and pink-eyed. It was a strange feeling: I was holding this animal but only via two black protrusions into its hermetically sealed world. It was sitting on me and yet completely separated from me. When I had shaken hands with Gordon earlier, we had exchanged microbes. When I stroked this mouse, we exchanged nothing.

The mouse seemed normal, but it was not. Growing up without microbes, its gut had not developed properly – it had less surface area for absorbing nutrients, its walls were leakier, it renewed itself at a slower pace, and the blood vessels that supplied it with nutrients were sparse. The rest of its body hadn’t fared much better. Compared with its normal microbe-laden peers, its bones were weaker, its immune system was compromised, and it probably behaved differently too. It was, as microbiologist Theodor Rosebury once wrote, “a miserable creature, seeming at nearly every point to require an artificial substitute for the germs [it] lacks”.

The woes of the germ-free mouse vividly show just how invaluable the microbiome is. Most of us still see microbes as germs: unwanted bringers of pestilence that we must avoid at all costs. This stereotype is grossly unfair. Most microbes do not make us sick. At worst, they are passengers or hitchhikers. At best, they are invaluable parts of our bodies: not takers of life but its guardians. They help to digest our food, educate our immune systems, protect us from disease, sculpt our organs, guide our behaviour, and maintain our health. This wide-ranging influence explains why the microbiome has, over the last decade, become one of the hottest areas of biology, and why Gordon – arguably the most influential scientist in the field – is so fascinated by it.

By studying our microbial companions, he is trying to unpick exactly how the microbiome is connected to obesity and its polar opposite – malnutrition. He is studying which species of microbes influence these conditions, and how they in turn are influenced by our diets, our immune systems, and other aspects of our lives. Ultimately, he wants to use that knowledge to manipulate the microbial worlds within us to improve our health.

Jeff Gordon may be one of the most respected scholars of the human microbiome, but he is also one of the hardest to get in touch with. It took me six years of writing about his work to get him to answer my emails, so visiting his lab was a hard-won privilege. I arrived expecting someone gruff and remote. Instead, I found an endearing and affable man with crinkly eyes, a kindly smile, and a whimsical demeanour. As he walked around the lab, he called people “professor” – including his students. His aversion to the media comes not from aloofness, but from a distaste for self-promotion. He even refrains from attending scientific conferences, preferring to stay out of the limelight and in his laboratory.

Ensconced there, Gordon has done more than most to address how microbes affect our health. But whenever I asked Gordon about his influence, he tended to deflect credit on to students and collaborators past and present – a roster that includes many of the field’s biggest stars. Their status testifies to Gordon’s – he’s not just a king, but a king-maker, too. And his figurehead status is all the more remarkable because long before the microbiome crossed his mind, he was already a well-established scientist who had published hundreds of studies on how the gut develops in a growing human body.

Professor Jeff Gordon, one of the world’s leading experts on the human microbiome, talks to students at Washington University in St Louis. Photograph: Mark Katzman

In the 1990s, he started to suspect that bacteria influence this process, but he was also struck by how difficult it would be to test that idea. The gut contains thousands of species of microbes. Gordon aimed to isolate parts of this daunting whole and examine it under controlled conditions. He needed that critical resource that scientists demand but biology withholds: control. In short, he needed germ-free mice – and lots of them – so he bred them himself. He could load these rodents with specific microbes, feed them with pre-defined diets, and do so again and again in controlled and repeatable conditions. He could treat them as living bioreactors, in which he could strip down the baffling complexity of the microbiome into manageable components that he could systematically study.

In 2004, Fredrik Bäckhed, a member of Gordon’s team, used the sterile rodents to run an experiment that would set the entire lab on a focused path – one devoted to understanding the connections between the microbiome, nutrition, and health. They inoculated germ-free mice with microbes harvested from the guts of conventionally raised rodents. Normally, the sterile rodents can eat as much as they like without putting on weight, but this ability disappeared once their guts were colonised. They didn’t start eating any more food – if anything, they ate slightly less – but they converted more of that food into fat and so put on more pounds.

Mouse biology is similar enough to that of human beings for scientists to use them as stand-ins in everything from drug testing to brain research the same applies to their microbes. Gordon reasoned that if those early results apply to humans, our microbes must surely influence the nutrients that we extract from our food, and thus our body weight. That was a powerful insight. We typically think of weight as a simple balance between the calories we take in through food and those we burn through physical activity. By contrast, the idea that multitudes of organisms in our bodies could influence that balance was outlandish at the time. “People weren’t talking about it,” says Gordon.

And yet, in 2004, team member Ruth Ley found another connection between microbes and weight, when she showed that obese people (and mice) have different communities of microbes in their guts. The most obvious difference lay in the ratio of the two major groups of gut bacteria – the firmicutes and the bacteroidetes. Obese people had more firmicutes and fewer bacteroidetes than their leaner counterparts. This raised an obvious question: does extra body fat cause a relative increase in firmicutes – or, more tantalisingly, does the tilt make individuals fatter? Is the connection, as Gordon likes to put it, causal or casual? The team couldn’t answer that question by relying on simple comparisons. They needed experiments.

That’s where Peter Turnbaugh came in. Then a graduate student in the lab, he harvested microbes from fat and lean mice, and then fed them to germ-free rodents. Those that got microbes from lean donors put on 27% more fat, while those with obese donors packed on 47% more fat. It was a stunning result: Turnbaugh had effectively transferred obesity from one animal to another, simply by moving their microbes across. “It was an ‘Oh my God’ moment,” said Gordon. “We were thrilled and inspired.”

These results showed that the guts of obese individuals contain altered microbiomes that can indeed contribute to obesity, at least in some contexts. The microbes were perhaps harvesting more calories from the rodents’ food, or affecting how they stored fat. Either way, it was clear that microbes don’t just go along for the ride sometimes, they grab the wheel.

They can also turn it in both directions. While Turnbaugh showed that gut microbes can lead to weight gain, others have found that they can trigger weight loss. Akkermansia muciniphila, one of the more common species of gut bacteria, is over 3,000 times more common in lean mice than in those genetically predisposed to obesity. If obese mice eat it, they lose weight and show fewer signs of type 2 diabetes.

Gut microbes also partly explain the remarkable success of gastric bypass surgery – a radical operation that reduces the stomach to an egg-sized pouch and connects it directly to the small intestine. After this procedure, people tend to lose dozens of kilograms, a fact typically accredited to their shrunken stomachs. But as a side-effect, the operation also restructures the gut microbiome, increasing the numbers of various species, including Akkermansia. And if you transplant these restructured communities into germ-free mice, those rodents will also lose weight.

Experiments on mice using gut microbes could lead to a greater understanding of the causes of obesity. Photograph: Deco Images II/Alamy

The world’s media treated these discoveries as both salvation and absolution for anyone who struggles with their weight. Why bother adhering to strict dietary guidelines when a quick microbial fix is seemingly around the corner? “Fat? Blame the bugs in your guts,” wrote one newspaper. “Overweight? Microbes might be to blame,” echoed another. These headlines are wrong. The microbiome does not replace or contradict other long-understood causes of obesity it is thoroughly entangled with them.

Another of Gordon’s students, Vanessa Ridaura, demonstrated this in 2013 by using mice to stage battles between the gut microbes of lean and obese people. First, she loaded these human microbial communities into two different groups of germ-free rodents. Next, she housed the mice in the same cages. Mice readily eat each other’s droppings and so constantly fill their guts with their neighbours’ microbes. When this happened, Ridaura saw that the “lean” microbes invaded guts that were already colonised by “obese” communities, and stopped their new hosts from putting on weight. The opposite invasions never worked: the obese communities could never establish themselves in the gut when the lean ones were already there.

It’s not that the lean communities were inherently superior at taking hold in a mouse’s gut. Instead, Ridaura had tipped the battles in their favour by feeding her mice with plant-heavy chow. Plants contain a wide variety of complex fibres, and microbe communities from lean guts contain a wider range of fibre-busting species than those from obese guts. So, when the obese communities colonised lean guts, they found that every morsel of fibre was already being devoured.

By contrast, when the lean communities entered obese guts, they found a glut of uneaten fibre – and flourished. Their success only evaporated when Ridaura fed the mice with fatty, low-fibre chow, designed to represent the worst extremes of the western diet. Without fibre, the lean communities couldn’t establish themselves or stop the mice from putting on weight. They could only infiltrate the guts of mice that ate healthily. The old dietary advice still stands, over-enthusiastic headlines be damned.

An important lesson emerged: microbes matter but so do we, their hosts. Our guts, like all ecosystems, aren’t defined just by the species within them but also by the nutrients that flow through them. A rainforest isn’t just a rainforest because of the birds, insects, monkeys, and plants within it, but also because ample rain and sunlight fall from above, and bountiful nutrients lurk in the soil. If you threw the forest’s inhabitants into a desert, they would fare badly. Ridaura’s experiments emphasised that although the microbiome can help to explain what makes us fat or lean, it offers no simple solutions. And that’s something the team learned a second time, by studying a very different condition, in a very different part of the world.

M alawi has among the highest rates of child mortality in the world, and half of these deaths are due to malnourishment. One form of malnourishment, known as kwashiorkor, is especially severe and hard to treat. From an early age, a child’s fluids leaks from their blood vessels, leading to puffy swollen limbs, distended stomachs, and damaged skin.

Kwashiorkor has long been shrouded in mystery. It is said to be caused by protein-poor diets, but how can that be when children with kwashiorkor often don’t eat any less protein than those with marasmus, another form of severe malnutrition? For that matter, why do these children often fail to get better despite eating protein-rich food delivered by aid organisations? And why is it that one child might get kwashiorkor while their identical twin, who shares all the same genes, lives in the same village, and eats the same food, gets marasmus instead?

Gordon thinks that gut microbes are involved, and might explain the differences in health between children who, on paper, look identical. After his team carried out their groundbreaking obesity experiments, he started to wonder: if bacteria can influence obesity, could they also be involved in its polar opposite – malnutrition? Many of his colleagues thought it unlikely but, undeterred, Gordon launched an ambitious study. His team went to Malawi and collected regular stool samples from infants until the age of three some had kwashiorkor, while others were healthy.

The team found that babies with kwashiorkor don’t go through the same progression of gut microbes as their healthy counterparts. Typically, these microbial communities change in the first years of life, in dramatic but predictable ways. Just as new islands are first colonised by lichens, then shrubs, then trees, so too is the infant gut colonised by waves of species that arrive in standardised patterns. But in kwashiorkor infants, microbiomes fail to diversify and mature correctly. Their inner ecosystems become stagnant. Their microbiological age soon lags behind their biological age.

When Gordon’s team transplanted these immature communities from children with kwashiorkor into germ-free mice, the rodents lost weight – but only if they also ate chow that mirrored the nutrient-poor Malawian diet. If the mice ate standard rodent chow, they didn’t lose much weight, no matter whose bacteria they were carrying. It was the combination of poor food and the wrong microbes that mattered. The kwashiorkor microbes seemed to interfere with chemical chain reactions that fuel our cells, making it harder for children to harvest energy from their food – food that contains very little energy to begin with.

The standard treatment for malnutrition is an energy-rich, fortified blend of peanut paste, sugar, vegetable oil and milk. But Gordon’s team found that the paste only has a brief effect on the bacteria of children with kwashiorkor (which perhaps explains why it doesn’t always work). As soon as they reverted to their normal Malawian diet, their microbes also boomeranged back to their earlier impoverished state. Why?

All ecosystems have a certain resilience to change, which must be overcome to push them into a different state. That’s true for coral reefs, rainforests, grassland – and a child’s gut. A poor diet could change the microbes within the gut. The dietary deficiencies could also impair the child’s immune system, changing its ability to control the gut microbiome, and opening the door to harmful infections that alter the gut communities even further. These communities could themselves start to harm the gut, stopping it from absorbing nutrients efficiently and leading to even worse malnutrition, more severe immune problems, more distorted microbiomes, and so on.

This is what microbiome scientists call dysbiosis – a state where the entire microbial community shifts into a harmful configuration. None of its members causes disease in its own right instead, the entire community is at fault. It’s not clear exactly why the microbiomes of malnourished infants stall in their development in the first place. There are many possible reasons including antibiotic exposures, gut diseases, and poor diets, which vary from person to person. What’s clearer is that once microbiomes end up in a dysbiotic state, it can be hard to pull them back.

But Gordon is trying. His student Laura Blanton, the same woman who I met carrying that thermos of mouse droppings in the lift, recently implanted mice with microbes from either healthy infants or underweight ones. She then housed rodents from both groups in the same cages, allowing them to swap their microbiomes. When they did so, the normal communities from the healthy infants invaded and displaced the immature communities from the malnourished ones.

Blanton found that five species of bacteria from the healthy microbiomes were especially good at colonising the immature ones. When she fed this quintet to mice carrying the microbiomes of malnourished children, the rodents put on weight in a normal, healthy way. Rather than breaking down the amino acids in their diet for energy, they instead converted these nutrients into flesh and muscle.

This promising experiment suggests that the team might be able to create a probiotic cocktail of specially chosen bacteria that can turn a dysbiotic gut into a healthy one. But there’s reason to be cautious. Despite the hype that surrounds them, current probiotics – products that contain supposedly beneficial microbes – confer few big health benefits, because they contain small amounts of bacteria and consist of strains that are bad at taking hold in the gut. Gordon knows that if he wants to concoct better products, he must find ways of giving the incoming microbes a competitive advantage in their new homes. Maybe that means pairing the probiotics with foods that will nourish them. Maybe it means treating the human hosts as well as the microbes they carry, or training their immune systems to accept the newcomers.

Gordon is optimistic but cautious. As he sees it, studying the microbiome will ultimately help us to better treat conditions that are still mysterious and often intractable. But as he has said to me on more than one occasion, he’s wary of the intense hype that clouds the microbiome world. “I talk about the importance of sobriety and humility,” he says. “There’s lots of hope and expectation around this transcendent view of ourselves.” But he and other microbiome researchers still need to show that their discoveries can help people.

Bifidobacterium are used as a probiotic to promote good digestion, boost immune function, and increase resistance to infection. Photograph: Phototake/Alamy

Discoveries by Gordon and others have created the perception that the microbiome is the answer to everything. It has been linked to an absurdly long list of conditions that includes Crohn’s disease, ulcerative colitis, irritable bowel syndrome, colon cancer, type 1 diabetes, type 2 diabetes, coeliac disease, allergies, atherosclerosis, autism, asthma, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, depression, anxiety, rheumatoid arthritis, stroke, and many more.

Many of these proposed links are just correlations. Researchers often compare people with a particular disorder to healthy volunteers, find microbial differences, and stop. Those differences hint at a relationship but they don’t reveal its nature or its direction. Studies by Gordon and others go one step further. By showing that transplanted microbes can reproduce health problems in germ-free mice, they strongly hint at a causal effect.

Still, they provide more questions than answers. Did the microbes set symptoms in motion or just make a bad situation worse? Was one species responsible, or a group of them? Is it the presence of certain microbes that matters, or the absence of others, or both? And even if experiments show that microbes can cause diseases in mice and other animals, we still don’t know if they actually do so in people. Beyond the controlled settings of laboratories and the atypical bodies of lab rodents, are microbial changes really affecting our everyday health? When you enter the messy, multifaceted world of dysbiosis, the lines of cause and effect become much harder to untangle.

There is still a lot about the microbiome that we do not understand, and some of what we think we know is almost certainly wrong.

Remember how obese people and mice have more firmicutes and fewer bacteroidetes in their guts than their lean counterparts? This famous finding worked its way into the mainstream press and the scientific literature – and it’s a mirage. In 2014, two attempts to re-analyse past studies found that the F/B ratio is not consistently connected to obesity in humans. This doesn’t refute a connection between the microbiome and obesity. You can still fatten germ-free mice by loading them with microbes from an obese mouse (or person). Something about these communities affects body weight it’s just not the F/B ratio, or at least not consistently so.

It is humbling that, despite a decade of work, scientists are barely any closer to identifying microbes that are clearly linked to obesity, which has received more attention from microbiome researchers than any other. “I think that everybody is coming to the realisation that, unfortunately, a really compelling simple biomarker, like the percentage of a certain microbe, is not going to be enough to explain something as complicated as obesity,” said Katherine Pollard, who led one of the re-analyses.

These conflicting results naturally arise in the early days of a field because of tight budgets and imprecise technology. Researchers run small, exploratory studies comparing handfuls of people or animals in hundreds or thousands of ways. “The problem is that they end up being like the Tarot,” said Rob Knight, another leading microbiome scientist. “You can tell a good story with any arbitrary combination.”

Human geneticists faced the same problem. In the early 21st century, when technology hadn’t quite caught up with ambition, they identified many genetic variants that were linked to diseases, physical traits, and behaviours. But once sequencing technology became cheap and powerful enough to analyse millions of samples, rather than dozens or hundreds, many of these early results turned out to be false positives. The human microbiome field is going through the same teething problems.

It doesn’t help that the microbiome is so variable that the communities in lab mice can differ if they belong to different strains, come from different vendors, were born to different mothers, or were reared in different cages. These variations could account for phantom patterns or inconsistencies between studies. There are also problems with contamination. Microbes are everywhere. They get into everything, including the chemical reagents that scientists use in their experiments. But these problems are now being ironed out. Microbiome researchers are getting increasingly savvy about experimental quirks that bias their results, and they’re setting standards that will shore up the quality of future studies. They are calling for experiments that will show causality, and tell us how changes in the microbiome lead to disease. They are looking at the microbiome in even greater detail, moving towards techniques that can identify the strains within a community, rather than just the species.

They are also setting up longer studies. Rather than capturing a single screenshot of the microbiome, they are trying to watch the entire movie. How do these communities change with time? What makes them resilient or unstable? And does their degree of resilience predict a person’s risk of disease? One team is recruiting a group of 100 volunteers who will collect weekly stool and urine samples for nine months, while eating specific diets or taking antibiotics at fixed times. Others are leading similar projects with pregnant women (to see if microbes contribute to pre-term births) and people at risk of developing type 2 diabetes (to see if microbes affect their progression to full-blown disease).

And Gordon’s group has been charting the normal progression of microbes in healthy developing babies, and how it stalls in kids with kwashiorkor. Using stool samples collected from Bangladeshi and Malawian children over their first two years, the team has created a score that measures the maturity of their gut communities and will hopefully predict if symptomless infants are at risk of developing kwashiorkor. The ultimate goal of all of these projects is to spot the signs of disease as early as possible, before a body turns into the equivalent of an algal reef or a fallow field: a degraded ecosystem that is very hard to repair.

Children wait for water at a borehole near Malawi’s capital Lilongwe. Photograph: Mike Hutchings/Reuters

“Professor Planer!” said Jeff Gordon. “How are you?” He meant Joe Planer, one of his students, who was standing in front of a standard laboratory bench, complete with pipettes, test tubes and Petri dishes, all of which had been sealed in a transparent, plastic tent. It looked like one of the isolators from the germ-free facility but its purpose was to exclude oxygen rather than microbes. It allowed the team to grow the many gut bacteria that are extremely intolerant of the gas. “If you write the word oxygen on a piece of paper and show it to these bugs, they’ll die,” said Gordon.

Starting off with a stool sample from a Malawian child with kwashiorkor, Planer used the anaerobic chamber to culture as many of the microbes within it as possible. He then picked off single strains from these collections, and grew each one in its own compartment. He effectively turned the chaotic ecosystem within a child’s gut into an orderly library, dividing the teeming masses of microbes into neat rows and columns. “We know the identity of the bacteria in each well,” he said. “We’ll now tell the robot which bacteria to take and combine in a pool.”

He pointed to a machine inside the plastic, a mess of black cubes and steel rods. Planer can programme it to suck up the bacteria from specific wells and mix them into a cocktail. Grab all the Enterobacteriaceae, he might say, or all the Clostridia. He can then transplant these fractions back into germ-free mice to see if they alone can confer the symptoms of kwashiorkor. Is the whole community important? Will the culturable species do? A single family? A single strain? The approach is both reductionist and holistic. They’re breaking down the microbiome, but then recombining it. “We’re trying to work out which actors are responsible,” said Gordon.

A few months after I saw Planer working with the robot, the team had narrowed down the kwashiorkor community to just 11 microbes that replicate many of the disease’s symptoms in mice. None of these were harmful on their own. They only caused a problem when acting together – and even then, only when the mice were starved of nutrients. The team also created culture collections from healthy twins who didn’t develop kwashiorkor, and identified two bacteria that counteract the damage inflicted by the deadly 11. The first is Akkermansia, which is being studied as a way of reducing body weight, but seemingly guards against malnutrition too. The second is Clostridium scindens, which tamps down inflammation by stimulating certain branches of the immune system.

Opposite the tented bench, there was a blender that could take foods representative of different diets and pulverise them into rodent-friendly chow. (On a piece of sticky tape, affixed to the blender, someone had written “Chowbacca”.) Gordon’s lab could now explore the behaviour of Akkermansia and C scindens, either in test tubes or in the gnotobiotic mice, and work out which nutrients the microbes needed. This allowed the team to compare the effects of the same microbes when fed a Malawian diet, or an American one, or on sugars from breast milk that have specifically evolved to feed beneficial microbes. Which of these foods works best? And which genes do the microbes switch on? The team can take any one microbe and create a library of thousands of mutants, each of which contains a broken copy of a single gene. They can put these mutants in a mouse to see which genes are important for surviving in the gut, liaising with other microbes, and both causing or protecting against kwashiorkor.

What Gordon has built is a causality pipeline – a set of tools and techniques that, he hopes, will more conclusively tell us how our microbes affect our health, and take us from guesswork and speculation to actual answers. Kwashiorkor is just the start. The same techniques could work for any disease with a microbial influence.

It is the right time to be doing this work. Our planet has entered the Anthropocene – a new geological epoch when humanity’s influence is causing global climate change, a loss of wild spaces, and a drastic decline in the richness of life. Microbes are not exempt. Whether on coral reefs or human guts, we are disrupting the relationships between microbes and their hosts, often pulling apart species that have been together for millions of years. Gordon is working hard to understand these partnerships to better forestall their untimely end. He is not just a scholar of the microbiome he is one of its stewards.

Main photograph of faecal bacteria: Science Photo Library

## Healthy bacteria thrive in the gut before birth

Diversity of the environmental microbiome. Credit: Gene Drendel

Australian researchers have discovered the gut microbiome—a complex ecosystem of microorganisms made up of bacteria, viruses, fungi and other life forms—is selected and starts developing as early as five months in utero.

The gut microbiome is vital for survival and has been linked to digestion, brain and heart health, weight control, and reduced blood sugar.

Several diseases and autoimmune disorders are also thought to be influenced by processes in the gut microbiome including cancer, multiple sclerosis and autism spectrum disorder.

This discovery, led by scientists from La Trobe University, sheds new light on the developing fetal immune system, gut and brain.

Using extremely rigorous contamination controls, the researchers examined microbiome development along the gastrointestinal system in calves before birth. The innovative study, published in Scientific Reports, reveals the microbiome is distinct along different components of the fetal gastrointestinal tract.

It's the first study to completely eliminate the potential for microbial contamination, putting to rest the long-held theory that fetal development occurs in a sterile womb.

La Trobe University's Professor Ashley Franks said the research turns this area of science on its head.

"The gut microbiome plays a major role in human health," Professor Franks said.

"We know that from infancy to adulthood, trillions of microbial cells make up an essential part of our biology and physiological functions. They maintain the integrity of our gut lining and even protect us from disease and illness. Until now, the baby's first gut microbiome was thought to be collected from the mother's vagina or from the environment it's born into. Our findings confirm, without doubt, that bacteria colonize in the gut before birth, changing the future of fetal research and our understanding of how the microbiome influences our developing immune system, gut and brain."

The researchers identified 559 bacterial and 1736 discreet archaeal taxa through next generation sequencing of five components of the fetal gastrointestinal tract—ruminal fluid, ruminal tissue, caecal fluid, caecal tissue and meconium—and in the amniotic fluid.

Co-author Dr. Jennifer Wood, from La Trobe's Applied and Environmental Microbiology Lab, said the study showed distribution of these microorganisms across the gastrointestinal tract was not random, indicating the selection of our optimal microbiome is occurring in utero.

"We found tightly controlled microbial selection was occurring along the gastrointestinal tract and that this selection was the same in every calf we examined. We believe the reliability with which we observed the microbiome to develop shows gut microbiota are essential to fetal development," Dr. Wood said.

The findings are of major significance to humans because of the importance of the gut-brain axis, or gut microbiota signals to the brain, in development.

"These findings inform our ongoing research with Dr. Elisa Hill at RMIT University. Together, we are studying the connection between the nervous system and microbes in neurodevelopmental disorders such as autism. The development of the gut in very early life and the role of the prenatal gut-microbiome could have far reaching consequence for human health," Dr. Wood said.

## Could baby’s first bacteria take root before birth?

Cassandra Willyard is a freelance journalist based in Madison, Wisconsin.

You can also search for this author in PubMed Google Scholar

Most infants first come into contact with microbes during birth — or so researchers have assumed. Credit: Edgard Garrido/Reuters

Soon after conception, a human embryo begins to assemble a remarkable organ crucial to its survival. The placenta is both a lifeline and a guardian: it shuttles oxygen, nutrients and immune molecules from the mother’s bloodstream to her developing fetus, but it also serves as a barrier against infections. For more than a century, doctors have assumed that this ephemeral structure — like the fetus and the womb itself — is sterile, unless something goes wrong.

Starting around 2011, Indira Mysorekar began questioning this idea. She and her colleagues had sliced and stained samples from nearly 200 placentas collected from women giving birth at a hospital in St Louis, Missouri. When the researchers examined the samples under a microscope, they found bacteria in nearly one-third of them 1 . “They were actually inside cells there,” says Mysorekar, a microbiologist at Washington University in St Louis.

### Hear Kjersti Aagaard and Marcus de Goffau’s thoughts on a possible baby microbiome.

Bacteria often signal infection, and infections are a common cause of premature birth. But the microbes that Mysorekar observed didn’t seem to be pathogens. She didn’t see any immune cells near them nor did she see signs of inflammation. And bacteria weren’t present only in the placentas of women who gave birth early Mysorekar also found them in samples from women who had normal, healthy pregnancies. “That was our first hint that this may be like a normal microbiome,” she says.

Studies seeking to understand how microbes help to shape human health and development have become extremely popular over the past few decades, but some researchers are concerned that a crucial question — when bacteria first colonize the body — has not yet been answered. Doctors have assumed that the first contact with colonizing bacteria occurs in the birth canal. Clinicians are even looking to see whether babies born by caesarean section might benefit from a swab of their mother’s vaginal microbes. But Mysorekar and other scientists have found evidence of bacteria in the placenta, amniotic fluid and meconium — the tar-like first stool that forms in a fetus in utero. This has led some researchers to posit that the microbiome might be seeded before birth.

If that is true and bacteria are a normal — perhaps even crucial — part of pregnancy, they could have an important role in shaping the developing immune system. Scientists might be able to find ways to shift the microbial composition in the womb and possibly ward off allergies, asthma and other conditions. They might also be able to uncover microbial profiles associated with preterm birth or other complications during pregnancy, which could help to illuminate why they occur.

Bacterial culture from a belly button: there is some debate as to how different parts of the body are first seeded with microbes. Credit: Steve Gschmeissner/SPL

The scientists at the centre of these discoveries argue that the dogma of a sterile womb is on its way out. Perhaps humans, like species such as clams, tsetse flies and turtles, can inherit a mother’s microbes before they are even born 2 . “If we do not have microbes in utero, I think we would be the only species that has been interrogated that doesn’t,” says Susan Lynch, a microbiologist at the University of California, San Francisco.

But even as the number of papers supporting this idea grows, some scientists are pushing back. “I just don’t think that these microbiomes exist,” says Jens Walter, a microbiologist at the University of Alberta in Edmonton, Canada. Where some see an intriguing new avenue of research, others see biological implausibility, sloppy science and a spectre that has long haunted microbiome research — contamination. Now, studies are getting under way that could answer the question once and for all.

One paediatrician likens the controversy over the placental microbiome to a scientific “knife fight”. But if fetal microbiomes do exist, that could have far-reaching implications not only for medicine, but also for basic biology. “If we start thinking of the placenta as a conduit or facilitator of maternal-fetal communication and not as a barrier, then I think we open ourselves up to very interesting perspectives on how we’ve interpreted a lot of developmental biology today,” says Kjersti Aagaard, an obstetrician at Baylor College of Medicine in Houston, Texas.

The sterile-womb dogma goes back to French paediatrician Henry Tissier, who investigated the source of a baby’s first bacteria around the turn of the twentieth century. Researchers began to find bits of evidence against sterility more than three decades ago, but the idea that the placenta might harbour a fully fledged microbiome didn’t gain much attention until 2014, when a team of researchers led by Aagaard identified bacterial DNA in placental tissue 3 .

Bacteria found in healthy placentas

Aagaard, who was working on the Human Microbiome Project, noticed something odd. Babies were supposed to get the bacteria that will become their microbiome in the birth canal, but she saw a mismatch between the bacteria present in the vaginas of pregnant women and those present in infants in their first week of life. That might make sense, she thought, if the microbiome gets seeded before birth.

Aagaard reasoned that if mothers were passing bacteria to their babies in the womb, there might be evidence of that transfer in the placenta, the organ that connects the two. To investigate, she and her team harvested tiny bits of tissue under sterile conditions from the placentas of 320 women, including some who gave birth early and some who had infections during pregnancy. Bacteria can be difficult to culture. So, to identify what was there, they used gene sequencing. They took biopsies of the placentas in a sterile room within an hour of delivery, sliced off the surfaces to avoid contamination, and placed those samples into vials. They also analysed the contents of empty vials to rule out contamination from the environment or the DNA-extraction reagents.

Not every placenta contained detectable bacterial DNA, but many did 3 . To get a more in-depth picture of the capabilities of these microbes, the researchers performed whole-genome sequencing on a subset of the samples. In most, they found communities dominated by Escherichia coli and a few other groups. And when they compared the bacterial DNA from placentas with that from bacteria typically found in other areas of the body, the results best matched the kinds of microbe found in the mouth. How oral bacteria would have made their way to the placenta isn’t clear, but one possibility is that they travelled through the bloodstream. Even routine tooth brushing can allow bacteria access to the blood. What’s more, the microbial signature seemed to differ in women who had experienced a preterm birth or an earlier infection. Physicians have assumed that the mere existence of bacteria in the placenta signals infection, but to Aagaard it seemed clear that which bacteria are present is much more important than whether they are there at all.

The paper made a splash in the popular press, but critics argued that Aagaard was overreaching. “DNA is not bacteria,” says Mathias Hornef, head of the Institute of Medical Microbiology at the University Hospital RWTH Aachen in Germany. DNA can be used to characterize a microbiome, he says, but not to establish its existence.

The man who can map the chemicals all over your body

Aagaard’s findings weren’t an isolated event, however. Several other groups have found bacterial DNA and more in the placenta. Mysorekar, for example, saw the host of bacterial structures inside cells taken from the placenta 1 . And in 2016, a Finnish group managed to culture bacteria from placental tissues taken from women who had healthy pregnancies 4 .

Researchers have also found bacteria in amniotic fluid 4 , 5 , leading them to wonder whether the fetus might occasionally ingest microbes when it swallows some of that fluid. And some researchers, including Josef Neu, a neonatologist at the University of Florida in Gainesville, identified bacterial DNA in meconium 6 , a finding that suggests the fetus’s gut itself may harbour bacteria before birth. Some of the DNA came from the same genera found in amniotic fluid. And the results showed that the microbes in the stool of preterm infants were different from those in babies born at full term.

Neu hypothesized that some strains of bacteria might prompt the fetal gastrointestinal tract to produce inflammatory proteins that would trigger early labour. And indeed, some studies 7 have shown that amniotic fluid from premature babies does hold more of these proteins. That association doesn’t prove anything, but it does provide “some interesting pieces of the puzzle”, he says. “The fetal–maternal microbiome may be at least a partial explanation for some of these cases of preterm delivery.”

Lynch’s group is one of several that have been able to culture bacteria from meconium. But it’s not yet clear whether those bacteria are simply passing through the fetus, or whether they’re actually growing, dividing and taking up residence in the fetal gut, she says. Lynch is now looking at human fetal tissue to see whether she and her colleagues can find evidence of bacteria in the intestinal lining.

A handful of animal studies suggests that this kind of bacterial transfer from mother to fetus is possible. In the mid-2000s, a team of researchers led by microbiologist Juan Miguel Rodríguez at the Complutense University of Madrid inoculated pregnant mice with labelled bacteria, and delivered the pups by caesarean section. They found the labelled bacteria in both the amniotic fluid 8 and pups’ meconium 9 .

“What we’re seeing in these animal models and what we’re seeing in humans really seems to support this fetal–maternal microbiome,” says Neu. “I’m not 100% convinced, but I think the data is becoming very strong.”

Contamination questions

A number of researchers, however, remain deeply sceptical. The traces of placental microbes, they argue, are ‘kitome’ — contaminants from the DNA-extraction kits used in the research. There’s some evidence to support this. Samuel Parry, a perinatologist at the University of Pennsylvania’s Perelman School of Medicine in Philadelphia, was initially intrigued by Aagaard’s data. So he planned a study to examine differences between the placental microbiomes of preterm infants and those of babies born at term. As a first step, his team sought out trace amounts of DNA found on sterile swabs, reagents, DNA-purification kits and other equipment that they would routinely use. The bacterial DNA that they ultimately recovered from six placenta samples was indistinguishable from that found on the extraction kits 10 . They’ve since tested several dozen placentas, Parry says. “We just can’t find a microbiome.” Marcus de Goffau, a microbiome researcher at the Wellcome Sanger Institute in Hinxton, UK, says that he and his colleagues have similar unpublished results from “hundreds” of placentas.

One of the problems, he says, is that any bacterial signal in the placenta would be weak. In faeces or saliva, there are so many bacteria that it’s easy to distinguish the microbiome from background contamination. But when microbes are scarce, a true signal is much harder to pick up. The problem goes much further than studies on human fetuses, he adds: “The entire sequencing field is littered with nonsense.”

Aagaard stands by her results. “We are very cautious,” she says. “Could we be misinterpreting things? Of course. But we have put in the negative and positive controls every place we can.” And she points out that several other groups have found evidence of bacterial DNA in the placenta.

Parry and obstetrician Roberto Romero at the National Institute of Child Health and Human Development in Detroit, Michigan, are planning a multi-centre study to examine the question in even more placentas. They hope to hold a meeting to design the protocol in the next couple of months. If all goes well, they could have an answer as soon as next year, Romero says. They have invited Aagaard to participate, and she says she is willing. “Kjersti Aagaard is an outstanding investigator and she has put forth an idea that is interesting, is important and deserves to be tested,” Romero says. “This controversy can be solved.”

They aren’t the only ones looking for answers. de Goffau is part of a team that has received a £1.6-million (US\$2-million) grant from the UK Medical Research Council to examine placental tissue and blood for infectious agents that might be correlated with pregnancy complications. And last year, the US National Institutes of Health announced that it would offer funding for research into the early development of the immune system. The announcement specifically mentioned studies to examine how the fetal microbiome gets seeded and evolves, and how that might impact the brain.

If research fails to detect a microbiome in the womb, that doesn’t eliminate the possibility that the fetus might encounter microbes there. “There’s very little in and on the human body that could be considered sterile,” says Juliette Madan, a neonatologist at Dartmouth–Hitchcock Medical Center in Lebanon, New Hampshire. But a handful of microbes does not necessarily mean there’s a complex, thriving microbiome. Madan doesn’t expect researchers to find any meaningful sharing of bacteria between mother and fetus.

But de Goffau, one of the most vehement critics of the placenta papers, isn’t so sure. He has himself managed to detect bacteria in meconium. “It’s not completely sterile. That’s pretty clear,” he says. Although the evidence isn’t complete, he adds, a fetal microbiome is at least possible.

Maria Dominguez-Bello, a microbial ecologist at New York University, runs a study looking at the development of the infant microbiome and the potential benefits of putting babies in contact with their mothers’ vaginal microbes after a caesarean section. She doesn’t find the reports of bacteria in meconium all that convincing, however. She argues that sterility is broken when the amniotic sac breaks, which leaves plenty of time for bacteria to make their way into the infant’s gut. “Labour takes hours, during which the baby is swallowing and rubbing against the walls of the birth canal,” she adds. Even if a baby is born by caesarean section, it might take hours or even days for the infant to pass its first stool — a window during which it might acquire bacteria outside the womb.

The most compelling evidence that a fetal microbiome doesn’t exist, say Dominguez-Bello and others, is the existence of laboratory mice that are free of bacteria. To create these germ-free rodents, pups are surgically delivered from mothers with normal microbiomes and then raised under sterile conditions. “We’ve done these experiments, and we’ve done them for 70 years,” Walter says. If just one bacterium were present inside the pup, it would quickly colonize, and the protocol would fail. It would be impossible to complete such experiments.

“I would argue that if you talk with real microbiologists, they wouldn’t consider it controversial,” says Walter. The question, he adds, has already been answered.

Mysorekar, who is a microbiologist, disagrees. Some people are stuck on the idea that the placental microbiome is “fake news”, she says. That, she argues, is a shame. “There are some very exciting questions to address.” Humans start to develop a repertoire of immune cells while still in the womb, Mysorekar says, which suggests some sort of microbial exposure. She wonders where these microbes come from and how the exposure occurs. “There’s so much to learn,” she says. But she isn’t surprised by the scepticism. In any emerging field, she says, you’ll find “some naysayers, some dirty data, but also a lot of compelling new observations which together push the field forward”.

## Digging in Diapers for History of Gut Bacteria

The human gut teems with bacteria. There are 10 microbes in the body for every human cell thanks mainly to the profusion of colonies in the intestines. Yet babies are born without any such germ populations rather they develop them in fits and starts over time. Now researchers have mapped this development for the first time in 14 California babies, including a set of fraternal twins.

Researchers collected an average of 26 stool samples from each baby from their very first bowel movement to subsequent ones, including those following major events such as travel, illness or treatment with an antibiotic. Geneticist Chana Palmer, now a program director at the Canary Foundation in San Jose, Calif., which focuses on early cancer detection, and colleagues at Stanford University then tested each sample to glean what microbes lurked within. Palmer used a new tool&mdasha microarray designed to detect differences in the ribosomal RNA of different microbes&mdashto assess the whole panoply of microscopic critters inhabiting the babies' guts.

"The infant's gut is an exciting and rapidly evolving place," Palmer says. "Populations are quite unstable over the first few months but by a year of age they resemble each other and also resemble adult guts." Among the microbes that eventually dominate: Bacteroides, Eubacteriales, Clostridium, Ruminococcus and Faecalibacterium, as well as small amounts of fungi and archaea.

But these microbes did not start out at the top of the bottom. In fact, infants are born with no bacterial colonies at all but they subsequently develop a wide variety in their guts&mdashand how long that process takes. "Some were in 24 hours," Palmer notes. "The twins were both the latest," taking a full week to develop comparable numbers of bacteria.

She speculates that the single set of fraternal twins in the study may have developed colonies slowly because they were the only subjects delivered by planned caesarean section, meaning there was no contact with their mother's microbial community. But the twins also demonstrated that genetics plays a role in determining the gut's microbial makeup their intestinal flora were more similar to one another's than to those of any other baby, and even more so than to the intestines of parents or siblings and related infants, Palmer says.

The research results, published this week in PLoS Biology, indicate that all babies seem to acquire the same set of bacteria over time, though they start in radically different places and experience dramatically different shifts in the populations over the first year. Future experiments with the ribosomal RNA microarray will compare healthy and sick infants and the effects of antibiotics on adult gut populations. The Human Gut Microbiome Initiative is seeking to sequence all this genetic material. Yet, it remains unclear what kind of microbes are best for your&mdashor your baby's&mdashgut. "We really have no idea," Palmer admits, "what is ideal."

## How do I boost my baby’s microbiome?

### 1. Eat lots of fiber-rich foods while pregnant and nursing–

Fiber-rich diets increase the diversity of our gut bugs and shift the suite of species closer to those found in traditional hunter-gatherer societies.

As a byproduct of their metabolism, fiber-loving bugs produce acetate and butyrate, two compounds that help maintain the gut barrier and may therefore lower the risk of autoimmune diseases.

This is not just about how to feed your baby when he or she starts solids around four to six months of age. By upping your fiber intake while pregnant and nursing, you can pass these healthy, fiber-loving gut bugs to your baby during delivery and through your breast milk.

### 2. Before delivery, take steps to lower your risk of a C-section–

Let’s start by admitting that C-sections can be life-saving. Let me be clear, I am incredibly thankful that C-sections are both safe and widely available. Some of my closest friends are still with us, because of C-sections.

But as we all know, not all C-sections performed today are necessary. The C-section rate has climbed from under 20% in the 1970s to 32% today. While pinpointing the right rate is difficult , nearly everyone agrees that 32% is too high. Most experts believe the percentage of deliveries that need a C-section is closer to 15-20%.

C-sections entail not just longer recovery times and higher chances of complications, especially with repeat C-sections, but may disrupt your baby’s gut microbiome for months, if not years.

Vaginally-delivered babies arrive in a bacterially-laden splash, coated head to foot in their mother’s bacteria, whereas babies delivered via C-section enter the world under sterile surgical conditions through their mother’s lower abdomen. This difference has a profound impact on gut colonization.

C-sections may disrupt your baby’s gut microbiome for months, if not years.

Researchers now believe that these changes in gut colonization may explain why babies born via C-sections are at higher risk for obesity, allergies and Type 1 Diabetes.

Obviously, birth is unpredictable, and not all C-sections are avoidable. The best way to lower your chances of an unnecessary C-sections may be to pick your hospital carefully. In the U.S., C-section rates vary as much as 10-fold between hospitals, from 7% to 70%. Check out this website that lists hospital rates by state.

### 3. Swabbing–

Swabbing refers to dousing (or dabbing) your C-section baby with all the lovely bodily fluids he missed out on by bypassing your birth canal.

While swabbing is catching on among some parents , in the medical profession it still remains controversial .

Critics of the practice fear swabbing could expose your baby to nasty bacteria and viruses like HIV, Herpes, Chlamydia, and Group B Strep.

The other major criticism levied is that we do not know whether swabbing actually benefits long-term health. Proponents assume that swabbing will have health benefits. But they are making a leap of logic. Yes, C-sections are linked with higher rates of obesity, asthma, allergies, and autoimmune diseases. However, we do not know for certain whether this is because of changes to the microbiome.

C-sections are also more common in high-risk pregnancies, such as those affected by gestational diabetes, preeclampsia, and fetal distress. Perhaps it is problems during pregnancy and delivery, not lack of microbes, that raise one’s risk of metabolic and immune disorders.

Babies delivered via C-section also miss out on more than just microbes, they often miss out on the hormonal effects of delivery.

Would-be swabbers also face a practical problem, no one has determined the “right way” to swab. In a 2016 study , researchers soaked gauze inside laboring women’s vaginas. Shortly after birth, they rubbed the gauze over the newborn’s face, mouth, and bottom.

Newborns born via C-section but swabbed had oral and skin microbial communities that more closely resembled those of vaginally-delivered newborns, but this was less true when it came to their gut microbial communities. Perhaps because, as we all know but do not like to talk about, newborns are exposed to—ahem—more than just vaginal secretions during birth.

The controversy over swabbing has, at its heart, a question about what is the status quo. Swabbing mimics what occurs naturally in a vaginal birth. Yet, as soon as you take something away, you change its frame of reference and what is perceived as the status quo.

For C-section babies, this makes delivery in a sterile surgical environment the new normal.

In one sense, the science isn’t settled yet. In another sense, compared to other choices you might be making this is a very natural choice. Had you not delivered your baby by C-section there’s no way you could escape coating your baby in these bacteria.”- Rob Knight , a microbiologist at the University of California, San Diego .

### 4. Wait on the first bath–

there’s no need to dunk your newborn in a tub, and many hospitals have now updated their procedures so that the first bath is delayed at least 24 hours, as the World Health Organization recommends . Early bathing may not only increase the risk of low blood sugar in newborns, but could also interfere with early bacterial colonization by mom’s bacteria.

Health benefits aside, who wants to wash away that delightful vaguely vanilla-ish newborn baby smell. Savor it!

### 5. Probiotics–

Ah, the supplement du jour. The “live cultures” touted on your bottle of kefir. The slimy phlegm at the bottom of your kombucha bottle. By definition, any microorganisms that can survive the harrowing journey from your highly acidic stomach to your less acidic intestines.

So, marketing aside, does imbibing “healthy gut bugs” benefit your baby?

Surprisingly enough, the answer seems to be, at least in some cases…YES.

For preemies, probiotics cut the risk of necrotizing enterocolitis–a severe and often fatal condition common in preemies– in half . They also lower their odds of sepsis, a severe and potentially fatal blood infection, by 12% . For preventing these serious complications of prematurity, probiotics “cocktails” containing multiple bacterial strains, usually a mix of Lactobacillus and Bifidobacteria , appear to be the most effective .

For all babies, probiotics also lower the risk of childhood eczema, although not their risk of childhood allergies or asthma. Again, probiotics cocktails appear the most effective. Probiotics can also reduce newborn colic, according to a meta-analysis of five randomized trials.

For preemies, probiotics cut the risk of necrotizing enterocolitis in half.

For older children who receive a course of antibiotics, probiotics cuts their risk of antibiotic-associated diarrhea in half, with high dose probiotics (>5 billion CFUs/day) being most effective .

## Want to Fight Allergies? Get a Dirty Dog

A dog in the house is more than just good company. There’s increasing evidence that exposure to dogs and livestock early in life can lessen the chances of infants later developing allergies and asthma. Now, researchers have traced this beneficial health effect to a microbe living in the gut. Their study, in mice, suggests that supplementing an infant’s diet with the right mix of bacteria might help prevent allergies—even without a pet pooch.

"This paper elegantly illustrates how an environmental exposure protects against an allergic response by mediating the gut [bacteria]," says John Penders, a molecular epidemiologist at the Maastricht University Medical Center in the Netherlands, who was not involved with the work. "Studies like this provide new leads” about how one might manipulate the microbes in the gut to prevent or treat allergies.

More than a decade ago, U.S. researchers reviewing the health records of children with pets—dogs, and, to a lesser extent, cats—discovered that the kids were less likely to develop allergies and asthma than other children were. Other epidemiology studies in Europe have supported this connection, not just with pets, but with livestock as well. In 2010, Susan Lynch, a microbiologist at the University of California, San Francisco, showed that dogs who partly live outdoors shuttled environmental microbes into the house, some of which were also found in the human gut. She and others had already discovered that gut microbes affected immune responses, and so she wondered if the allergy protection provided by pooches happened via gut bacteria.

Lynch and her colleagues collected dust from a house with no animals and from a house with an indoor/outdoor dog. They fed that dust mixed with water to young mice and subsequently challenged the immune systems of the animals by giving them ground-up cockroaches or egg protein, two substances known to elicit allergic reactions in both rodents and people.

Mice receiving dust from the dog’s house weathered the challenge with little to no allergic reaction, but the other mice developed the mouse equivalent of a runny nose and revved up immune activity in their airways, the researchers report online today in the Proceedings of the National Academy of Sciences. In the dog dust-exposed mice, there were fewer allergy-associated immune cells and those that were present produced fewer immune system molecules that tend to lead to a strong reaction.

Lynch’s team surveyed the kinds of bacteria in the mouse guts before and after exposure to the dust. Mice with the dog’s dust—and a less allergenic immune system—had an unusually large amount of a microbe called Lactobacillus johnsonii, the team reports. When it fed that bacterium to mice, those mice had a dampened allergic reaction, even without being exposed to the dog’s dust. Those mice also got less sick when infected with a virus that in humans can cause infants to later become asthmatic. “Our studies suggest that [this bacteria] is a critical mediator of airway protection against environmental insults,” Lynch says.

The new work adds another piece of evidence to the long-debated hygiene hypothesis, which holds that a modern, cleaner lifestyle may make us more susceptible to allergies, asthma, and autoimmune disorders. "There are a lot of studies which show exposure to pets and/or livestock reduces prevalence of allergic disorders, so this is an exciting and provocative step in understanding the mechanism behind that," says Suzanne Havstad, a biostatistician at Henry Ford Hospital in Detroit, Michigan, who was not involved with the work.

While it’s possible dust from the dog’s household directly transfers extra L. johnsonii into a person’s gut, Lynch suspects that other bacteria in the environment get carried into the house on the dogs, become airborne, and are swallowed. Once in the gut, they force a change in that microbial community that favors an increase in L. johnsonii already present.

Before anyone starts thinking about a bacteria-laced dietary supplement for their kids or adopting a dog just to fight allergies, much more work, including clinical studies, would need to be done, Lynch notes. "One should be very careful about transferring results from mouse models to humans,” adds Markus Ege, an epidemiologist at the University of Munich in Germany. “The experimental setting in mice is very artificial.”

Still, Penders says, “[t]he potential of Lactobacillus johnsonii as a probiotic in the prevention of allergic diseases is definitely something that should be further explored.”

## Supplementing with quality synbiotics as a treatment option

We now have some evidence that there is indeed a connection between the health of our gut bacteria and our mental health. Can supplementation with quality probiotics offer an adjunct treatment option for people struggling with anxiety and depression?

Researchers looked at probiotics as a possible treatment option for individuals with major depressive disorder. The researchers suggested that stress can lower beneficial bacteria.

Bacteria in the GI tract can communicate with the central nervous system, even in the absence of an immune response. Probiotics have the potential to lower systemic inflammatory cytokines, decrease oxidative stress, improve nutritional status, and correct SIBO. The effect of probiotics on systemic inflammatory cytokines and oxidative stress may ultimately lead to increased brain derived neurotrophic factor (BDNF). It is our contention that probiotics may be an adjuvant to standard care in MDD. 7

In a recent study from Oxford University, 8 volunteers received either two prebiotics (fructooligosaccharides, FOS, or Bimuno-galactooligosaccharides, B-GOS) or a placebo (maltodextrin) daily for 3 weeks. Cortisol upon waking was significantly lower in those taking the prebiotic supplements. The researchers concluded that prebiotic bacteria may have an anti-anxiety effect on people.

Many probiotic supplements only contain probiotics. Prebiotics act as nourishment for good bacteria. With a synbiotic, the combination of prebiotics and probiotics may be beneficial in lowering stress and anxiety, as well as some symptoms of depression.

In conclusion, evidence is showing that there is a definitive connection between gut bacteria and mental health. What are some ways we can improve healthy bacteria in our gut? First, we can be more cognizant of birthing and breastfeeding practices, as we now know there is an impact on the health of the baby. We can also include fermented foods in our diet, such as raw sauerkraut, fermented beets, fermented carrots, and fermented teas, since fermented foods contain some beneficial bacteria. Finally, including a quality probiotic daily, to help reestablish healthy gut bacteria, can also be beneficial.

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