Microbes R Us

Duke Faculty Explore the Microbiome and its Role in Health and Disease

When we look in the mirror, what we see looks entirely human. But lurking both under and on the surface — in our arm pits, belly buttons and, of course, in our guts — is an absolutely incredible number of microbes. The microbial cells in and on our bodies outnumber human cells ten to one. In a contest for the most genes, we lose out even more impressively (or depressingly, depending on your point of view): for every one human gene we carry, we’ve got another 100 microbial genes. On top of all that, our organs are literally bathed in metabolic products that either originate from or are modified by many of those bugs. When it comes to understanding the health and disease susceptibility of an individual person, there is a very complicated and diverse internal ecosystem to consider.

“This is one of those environmental factors that is often ignored and that can affect everything from our response to drugs to our diet and mental health,” said Raphael Valdivia, director of Duke’s new Center for the Genomics of Microbial Systems (GeMS), a new hub of activity inspired by renewed interest in the roles that microbes play in human health, ecological balance, and biotechnology.

Some of that interest stems from The Human Microbiome Project, an effort akin to the Human Genome Project but aimed to catalog the microbial communities found in many parts of the human body — our noses and mouths, skin, guts and urogenital tracts — and to begin to unravel their roles in human health and disease. And if there could be any doubt about the power our microbiomes can have over us, those have also been dashed by recent evidence that fecal transplants (yes, that’s just what you think it is), designed to transfer intact microbial communities from one person to another, can be the best medicine for patients with serious Clostridium difficile infections and perhaps with other conditions. Emerging evidence suggests that a healthy microbiome might improve other, more run-of-the-mill problems like insulin resistance, obesity, and malnutrition too.

C. difficile infections can be life threatening,” said John Rawls, one of Duke’s newest faculty members in the Department of Molecular Genetics and Microbiology (MGM) and the IGSP. “That a fecal transplant can be the most effective treatment speaks volumes about the power of microbial ecology to solve important problems in human health, even though how it works remains a puzzle.”

Ancient History

Rawls is one of a growing contingent of Duke investigators whose labs are dedicated to the study of microbial systems in the context of human health, and he takes both a long and up-close, mechanistic view of the problem. Ultimately, he wants to understand how these very intimate associations with microbes work, why some microbes take up residence in the gut and others don’t, and what it is exactly that they do once they are there.

A transmission electron micrograph of the microbial community residing in the intestine of an adult zebrafish, with coloring added. – Credit: John Rawls

A transmission electron micrograph of the microbial community residing in the intestine of an adult zebrafish, with coloring added.
– Credit: John Rawls

“For as long as multi-cellular animals have been roaming the planet, there have been guts and gut microbiota,” Rawls said. “It’s a very ancient and fundamental part of our natural history.”

Because these associations have such a long history, Rawls can learn basic principles by studying host organisms that are more experimentally tractable than we ourselves are. Most study of the microbiome in non-human animals takes place in mice, but Rawls has dedicated his lab to establishing zebrafish as another powerful model system for the study of the microbiome.

The zebrafish microbiome has its own particular makeup that Rawls has shown to be surprisingly similar between animals caught in their natural habitat in India and those kept in the lab here in North Carolina. Zebrafish are optically transparent until adulthood, enabling Rawls and his team to visualize microbial community assembly and host-microbe interactions with incredible detail.

“We can image the intestinal bacteria and make movies of them swimming around in real time,” Rawls said. “We can label specific dietary nutrients — lipids, proteins and carbohydrates — and watch them be absorbed into intestinal cells. We can watch the development and behavior of immune cell populations as a function of an animal’s microbial colonization status. We can label any cell type or any process and watch it happen in live animals.”

Last year, Rawls’ team reported in Cell Host & Microbe evidence based on such live-imaging studies in the zebrafish that representatives of a microbial group known as Firmicutes that live in the fish guts stimulate the uptake of dietary fat into the animals’ intestines and livers. The more the fish ate, the more Firmicutes they had in their guts and the more fat they took in. In the context of the ongoing obesity epidemic, colleagues at Harvard said in a commentary that further illumination of the connections between gut microbes and fattiness could have “profound economic and social benefits.”

With his lab now up and running at Duke, Rawls is now partnering with Valdivia in an effort to better understand the genetic factors in those Firmicutes that enable their successful colonization in the fish and their influence on fat absorption. In collaboration with the IGSP’s Greg Crawford, Rawls is also exploring how the presence of microbiota regulate what intestinal cells do.

“As the Human Microbiome Project and related initiatives have revealed the staggering diversity and dynamics of our microbial residents, there is now increasing emphasis on identifying the molecular mechanisms that govern the assembly and activities of these microbial communities,” Rawls said. “Our experimental system is well-suited for this task.”

Take It Personally

Rawls isn’t the only newcomer to the Duke faculty focused on making sense out of human and animal microbiomes. Lawrence David, also in MGM and the IGSP, first began his study of microbes by focusing on abstract computational models to describe the evolution of microbes over billions of years. But a side project he began while at MIT set him on a much less abstract and decidedly more personal path.

David had a background in time-series analysis, and he decided to enlist two volunteers (himself being one of them) to collect daily samples of their own personal microbial communities. The only way to do that, he says, is to collect and store your own poop. As he told the story on a science story-telling podcast called The Story Collider, it sometimes led to interestingly humorous conversations at home, with his wife once remarking, “Serves you right for putting feces in our fridge!” (Listen to his story at storycollider.org.)

In addition to those sample collections, he and his fellow volunteer also recorded a list of about 300 things they did everyday with the help of an iPhone app. He anticipated they might be able to see effects on the microbiome of daily activities: a couple of beers with friends one evening or a particularly gnarly taco for lunch. During his year of study, he talked his advisor into a trip to Thailand during which he ate only street food.

The surprise that David finds absolutely fascinating: Very few things he or his co-participant did over the course of a year seemed to make any difference to the microbes.

“We have this incredibly diverse and complex ecosystem, and it is resistant to almost everything you throw at it,” David said. “This wasn’t an artificial, lab-based study. We did whatever we wanted and in spite of it all, this community persists.”

System Upsets

There are considerable differences between people in their microbial communities, David explained. It is likely that families may share each other’s microbes, but any two people you might select could have none of their two hundred or so types of microbes in common at all. In other words, these microbial communities are in some sense heritable. They are also part of what makes an individual unique and different from somebody else.

Lawrence David studied the makeup of his microbiome everyday over the course of a year.

Lawrence David studied the
makeup of his microbiome
everyday over the course of a year.

Despite the stability of these communities in most cases, there are some disturbances that really can have a major impact, however. At one point over the year, one subject got sick with food poisoning — three days with Salmonella. The experience knocked out half of the microbial species in his gut, and they didn’t come back for weeks.

“Sometimes there can be disturbances that really do upset the system,” David said. “It could potentially lead the gut community to a new stable state.”

That idea appeals to David, who is a tinkerer by nature. He likes to take things apart and figure out how they work. At the moment, there is no tool that enables scientists to remove microbes from the gut one by one. If someone ever figures out how to do that, the Duke researchers say, it will be a pivotal technology in the microbiome field.

For now, David plans to take advantage of naturally acquired illnesses to explore the succession of microbial communities. In this country, it’s not so easy to find people before they get sick and to watch what happens when they do. That’s why David teamed up with collaborators and study participants in Dhaka, Bangladesh, a place where cholera infection is rampant.

“People in this study have a one in five chance of getting cholera within one week of enrollment,” David said. “We can enroll 100 people, wait one month and see what happens to their communities.”

After the hit — the cholera infection itself and the antibiotics used to treat it — do those communities return to their original state? Or do they transition to a new one? How do individuals differ in their microbial communities and do those differences help to explain why one person gets sick and another doesn’t?

Bug Soup

While David waits for people to get sick with cholera overseas, Duke’s Pat Seed continues to explore similar questions about the composition and succession of microbial communities in the unique setting of Duke’s neonatal intensive care ward, where premature babies struggle to survive and often suffer from infections that would be rare in other groups. These babies are so different in every way from term babies that Seed says it’s almost like studying humans born on the moon.

The babies are much less able to connect with their family members. They can’t eat normally. Their skin is almost transparent and their organs are not yet fully developed. While premature birth remains poorly understood, it might sometimes be brought on by infection with certain kinds of microbes in the first place.

Everything about the way these babies are brought into the world differs. In a recently reported study, Seed and his colleagues found that the microbiomes of those extremely low-birthweight infants are also much less diverse, often including hard-to-treat Candida fungus, along with harmful bacteria and parasites.

“The babies’ guts were taken over by microbes we know are dangerous if they get into the blood,” Seed said of the study’s findings. “Even after the babies were no longer on antibiotics, healthier bacteria didn’t appear in the babies very quickly. This may be one reason why premature babies are so vulnerable to infections.”

It’s not clear if the newborns are picking up these early infections from their mother’s milk, blood, or in other ways, or if the pathogens are from the environment surrounding the infants. Certain bacteria and other microbes are helpful for growing babies and their immune systems, so a germ-free environment isn’t the answer.

“It’s a question of balance,” he said. “As vulnerable as these babies are, we still wouldn’t want to wipe out all of the bacteria, even all of the potentially harmful bacteria.”

His experience working with this very difficult premature population is leading him to other vulnerable patients, including those who have undergone bone marrow transplant. There is lots of work still to do.

“It’s kind of bug soup right now,” he says. In the end, he doesn’t think any simple microbial cocktail will do. As the C. difficile example suggests, there will likely be more to gain from the transfer of whole complex microbial communities than from single microbes. Maybe people could bank their own personal microbiomes so that they might be restored after a major upset.

A New Imperative

The interdependency of animals and their microbes is nothing new for the IGSP and Nicholas School’s Jennifer Wernegreen, who studies the intimate association of carpenter ants with specialized microbes that actually live inside some of their cells (see Two to Tango). She recently coauthored an article with Rawls and others calling the study of animal-bacterial interactions a “new imperative for the life sciences.” It’s yet another field in which the blurring of distinct fields will be absolutely key, and Valdivia’s new Center for the Genomics of Microbial Systems is determined to help lead the way.

“Advances in DNA sequencing have led to spectacular insights into the complexity and ubiquity of microbial communities throughout our planet, including on and within ourselves and other animals,” Rawls said. “This ongoing revelation has spurred further interest in understanding how microbial communities are assembled and how they influence and are influenced by the hosts they inhabit. This field of study is intrinsically interdisciplinary, bridging genomics, microbiology, physiology, immunology, nutrition, ecology, and evolution, for starters. GeMS is designed to facilitate this process by drawing from labs across campus in different departments that are studying microbes in a wide spectrum of ecosystems using genomic analysis as common ground. This is exactly what needs to happen.”

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