By Bruce Goldman
Illustration by David Plunkert
The guy was David Relman, MD, and the year was 1998. Following a postdoctoral stint in the lab of renowned Stanford bacteriologist Stanley Falkow, PhD, Relman was undergoing a crucial transition, from focusing on individual bacteria as bad guys to thinking about bugs as they really live inside us in vast, complex, close-knit communities.
Relman — now a professor of medicine and of microbiology and immunology at Stanford’s School of Medicine and chief of infectious diseases at the Veterans Affairs Palo Alto Health Care System — has adopted the jargon of ecology, commandeered state-of-the-art biotechnology tools and altered his course to investigate our inner inhabitants. He’s hell-bent on learning who they are, what they’re up to and how we should treat them in sickness and in health. His methods have unearthed thousands of species whose existence couldn’t have been shown with standard techniques.
This newer, nuanced understanding of the bugs in and on our bodies is spurring a federal push to categorize and enumerate them. This effort is also producing a heightened caution regarding the use of antibiotics as well as a corresponding rise in interest in the possibilities for probiotics: reseeding gut-bacteria populations perturbed by drugs, diet or disease. Add in the press attention garnered by Relman’s studies — and, in recent weeks, other researchers’ parallel findings of the rugs of bugs that cover our skin — and it suggests that the general public seems to be getting beyond the “ew!” factor.
There’s been a paradigm shift in microbiology — from “us versus them” to “us is them.” Those microbes inside of us are mostly just doing their job.
The communities of micro-organisms lining or swimming around in our body cavities — our commensal microbes — work hard for their living. They synthesize biomolecules that manipulate us in ways that are helpful to both them and us. They produce vitamins, repel pathogens, trigger key aspects of our physiological development, educate our immune system, help us digest our food and for the most part get along so well with us and with one other that we forget they’re there. It’s often when those communities are disrupted by antibiotics or by changes in diet that we feel the effects.
“The planet just crawls with microbes,” says Falkow, a professor of medicine and microbiology. “Every inhabitable crevice is full of micro-organisms of one kind or another, from tops of mountains down to the thermal vents in the sea.”
They also colonize our bodies, big time. A coat of microbes paints our hands, our mouths, every orifice and outer or mucosal surface. Actually, they outnumber us: For every human cell in your body, there are 10 bacterial cells. Wouldn’t it be nice to know what they’re all doing in there?
Medical researchers have been following bacteria around for a good century, typically intent on isolating one or another type believed to cause a particular disease. But recent discoveries are highlighting their surprising diversity and interactions. At some point, you want to ask yourself whether the workings of the internal ecosystems are as attention-worthy as the occasional aberrant invader who violates them.
That’s what happened to Relman. Back in the 1990s, after several years in Falkow’s lab, he joined the Stanford faculty. A few years earlier, having caught a lecture by Falkow’s wife, Lucy Tompkins, MD, PhD, professor of medicine and of microbiology and immunology, about a bacterial species strongly suspected to cause a disease called bacillary angiomatosis but impossible to cultivate in a petri dish, Relman decided to adopt a molecular-detection technique to nail it. Up to then, the technique (“for lack of a better term, it’s called ‘broad-range polymerase chain reaction,’” he says) had been used largely by environmental microbiologists to conduct censuses of microbial populations in soil and seawater. In a nutshell, the technique takes advantage of the fact that certain snippets of genetic material found in all forms of cellular life can be copied, counted and distinguished from one species to the next. Each variation in the molecular sequence of these snippets can be logged separately, allowing for a remarkably complete census of the entire bacterial population in a sample.
Relman began using this technique for analyzing biopsies from various tissues to see if he could find molecular traces of the mysterious pathogen’s genome. He quickly showed that the bacterial species Tompkins had been talking about was related to the germ that causes a disease called trench fever; not long afterward, he used the same method to identify another micro-organism responsible for a fairly rare intestinal malady called Whipple’s disease.
But the new technique’s exquisite sensitivity to the presence of even the slightest amounts of bacterial genetic material soon translated into a royal pain. “The downside of the approach, I learned, was that it’s sensitive to anything and everything in the sample,” says Relman. “As I began to look in places in the human body where you don’t normally expect to find microbial life, I started finding it. There were microbial sequences everywhere — in lymph nodes, in the liver, in blood samples.”
Relman tried to deal with this problem — “at least, I thought it was a problem” — until it occurred to him that the thing he was calling a problem was also an interesting area for deliberate exploration. The noise was, for Relman, becoming the signal.
So he put his money — or more accurately, his career — where his mouth was. “I brought some collection materials with me to the dentist’s office, and asked him to hand me the stuff he was scraping out of my mouth instead of throwing it away.” He published his findings, a survey of bacterial diversity in the gums, in 1999. That’s how it all began.
Scientists’ fascination with dental plaque per se is nothing new. Antony van Leeuwenhoek, who perfected the microscope more than 350 years ago, was intrigued by what he saw in the crevices in his own teeth, says Falkow. “He once wondered aloud what the impact would be if people knew that these ‘animalcules,’ as he called them, swimming around in their mouths outnumbered the population of all of Rotterdam.”
What is new is the way Relman has co-opted the language of ecologists and applied emerging technology to longstanding problems in human health and medical microbiology. “In ecology,” Relman says, “there is an underlying emphasis on the importance of the community as the unit of study, as an entity that itself is composed of multiple participating members, but whose net effect is more than the sum of its parts. Almost all microbes normally live among their brothers and sisters as part of a community. It’s not natural for a microbe to be operating on its own.”
Furthermore, the majority of bacteriological study over the decades has been heavily dependent on the traditional approach, in vitro cultivation. Says Falkow: “The mantra of microbiology for many years was, ‘I’m gonna study this micro-organism in my laboratory, in pure culture, with no other organisms around to screw me up.’”
That approach not only flies straight into the fallacy of composition — the whole is more than the sum of its parts — but all too often ends in failure. First, microbes are often hard to tell apart just by looking at them, even under magnification. Second, even with some foolproof means of identifying similar but separate species, you’ll still never see what you can’t grow. And the vast majority of our internal microbes simply won’t multiply to any extent in a dish or a flask or a test tube. They don’t thrive in our artificial environments.
Although Relman’s search for internal microbial communities started in the mouth, he soon moved on to the ultimate microbial megalopolis: the human large intestine, where well over 90 percent of all our microsymbionts — tens of trillions of them — reside. The mammalian colon harbors one of the densest microbial communities found on our planet.
Instead of identifying bugs through culturing them, Relman and his colleagues extract and purify DNA from fecal samples. Then they use a workhorse molecular-biology technique to multiply gene sequences of interest until they have millions of billions of copies. These sequences are stretches of genetic material from absolutely mission-critical protein-generating factories called ribosomes, which are found in every living cell. (Bacterial ribosomes are different from those of higher organisms and can be easily distinguished.) The team sends the amplified gene-snippet extracts out for industrial-scale sequencing. What comes back a few days later are reports on the telltale gene sequence in the form of long strings of the four letters representing the four different chemicals that link together to form DNA.
These strings of letters, thousands of strings for each individual sample studied, appear on Les Dethlefsen’s computer screen, stacked one below the next to facilitate comparison. Dethlefsen, PhD, is one of the postdoctoral scholars in the Relman lab who have been doing much of the heavy lifting on this project.
Between long stretches of letters that are identical or nearly identical appear occasional letters that differ from one string to another, meaning that the DNA sequences the strings of letters represent are different. Because the gene in question is absolutely essential to all cellular life, its sequence is very slow to diverge as species drift apart over evolutionary time. Thus each variation can be presumed to represent a separate bacterial variant. Counting bacterial strains and species this way, rather than the haphazard culture-me-if-you-can technique, misses only the rarest variants.
In 2005, in the journal Science, Relman published the first comprehensive census of the microbial inhabitants of the far end of the human digestive tract. Whereas about 500 distinct microbial variants had been found through cultivation, Relman’s method identified thousands.
That first “Who’s Who” of gut bacteria was a major advance, says Jeffrey Gordon, MD, director of the Center for Genome Sciences at Washington University School of Medicine in St. Louis. Gordon is, like Relman, a leader in the field of human microbial ecology. His work focuses on the impact of diet on the microbial population and, conversely, the microbes’ influence on the host. In a January 2009 Nature paper, he and his colleagues showed that obesity might be partly related to differences in the collection of microbes present in our gut communities.
“David has been a pivotal contributor to our understanding of the microbial world in our bodies,” Gordon says. “He wandered into a place that could be branded terra incognita, and made it more cognita.”
Relman’s experiments and ideas were key to establishing the Human Microbiome Project, a $115 million, five-year government-funded effort, says Gordon. The National Institutes of Health launched this project in 2007 to explore the microbial communities occupying different body habitats (specifically, the digestive tract, mouth, skin, nose and female urogenital tract). Relman was one of 11 initial grant recipients; his project is to find a more efficient way to analyze DNA from single bacterial cells.
Meanwhile, the supersensitive counting approach makes it possible to fill in more knowledge gaps. The technique allows researchers to follow virtually the entire microbial ecosystem of a particular organ in detail to see how it changes over time. Now there is a way to track responses to perturbations — by drugs, diet, disease and changes in lifestyle.
In a study reported in 2008, the Relman team examined the effects of a common antibiotic, ciprofloxacin, which had been assumed by the medical community to go easy on intestinal microbes because users typically notice no gastrointestinal symptoms. But in fact, the drug caused significant changes in the relative abundance of variants in each study subject’s lower gut — albeit largely with no overt symptoms. Some rarer variants seem to have disappeared altogether.
The significance of these changes is not yet known. But now at least there is a way to track responses that were once invisible. Relman’s group is following up on the ciprofloxacin story with new experiments, and has initiated another study with the commonly prescribed antibiotic tetracycline.
Some think the appendix is not a vestigial organ at all, but exists as a kind of backwater providing a safe place for “starter cultures” of bugs to hide.
The mother of all perturbations is birth. We come into this world germ-free, Relman says. He’s looked, so he should know. “But as soon as the amniotic sac breaks, as soon as the baby either is plucked from the uterus in an operating room or sticks its first little protrusion out into the birth canal, everything changes.”
He found out what happens next through a study with Stanford biochemistry professor Pat Brown, MD, PhD, in which they charted the self-assembly of infants’ gut-bacterial communities over their first year. In the earliest days, the communities appear to be almost chaotic in their evolution, with each newborn’s bacterial population experiencing wild oscillations over the first several months of life. “They’re coming and going like crazy,” Relman says. But later in that first year after birth, the bacteria form more or less stable communities — “a typical adultlike picture, albeit somewhat distinctive from one infant to the next. And all these communities, although they look a little different, seem to be doing the same kinds of things.”
The rapid assembly of an internal microbiome sets off developmental transitions within that individual. The finishing touches in the gastrointestinal tract’s development, for example, occur only after its first contact with micro-organisms, whose presence in some way induces the maturation of epithelial cells lining the gut. That may seem a bit self-serving, as the bacteria responsible figure to be the guests of honor at the tubular dinner table. But they also sing for their supper. They manufacture vitamins for us, and convert complex polysaccharides — which are indigestible without bacterial assistance — into easily absorbed products so abundant they account for as much as 10 percent of our caloric intake.
Even in a passive way, our friendly bacterial communities repel pathogens simply by claiming turf. “These communities provide resistance against infection,” Relman says. “If you knock out major components, you render an animal more susceptible to invasion.” A nasty condition known as Clostridium difficile colitis can occur when this pathogen overruns a gastrointestinal tract that has been depleted of its indigenous microbes by antibiotic use.
But the bugs’ role in our response to disease is probably more pervasive than that. Like our digestive tracts, our immune systems don’t fully mature until they come in contact with microbes. The so-called hygiene hypothesis advanced by many allergists and immunologists holds that exposure to a healthy internal microbial ecosystem fine-tunes our immune system. Lower the exposure to these agents, as has widely been achieved in modern industrial societies, and the incidence of allergies — asthma, eczema, hay fever, food allergies — and some autoimmune diseases goes up. “There are a whole bunch of diseases, like inflammatory bowel disease or gum disease, where a disturbed community structure may be one of the main causes,” says Relman.
Some think the appendix, a finger-sized closed tube dangling from the upper large bowel, is not a vestigial organ at all, but exists as a kind of backwater providing a safe place for “starter cultures” of bugs to hide, says Dethlefsen. So if the intestinal communities are radically reduced due to, say, famine (a not uncommon ancestral event) these bacteria are ready to reseed the gut when food again becomes plentiful.
Intriguingly, countries undergoing rapid technological and cultural transitions are now experiencing an upwelling of diseases that were formerly reserved for heavily industrialized countries. The relatively recent advent of refrigeration, soap, international travel and new drugs mean fewer or, sometimes, newer bugs. Says Falkow: “With all the successes we’ve had in eliminating organisms from humans, by using antibiotics and vaccines and purifying water and so on, we may have removed some things that were there for a very long time.” We’ve also added a few, via cohabitation with farm animals or, more commonly in modernized countries, with pets.
When our diet was much more plant-based, says Dethlefsen, the human internal bacterial community itself may have been different. “Within this past century there’ve been quite significant changes in our diet: more highly processed foods, greater salt intake, an increase in red meat and simple sugars, and so forth. These changes may or may not be healthy. Go back, say, 10,000 years to pre-agriculture and the changes are even more significant. If you’re foraging on leafy greens and twigs and bark, there’s much higher fiber content.”
If changes in the composition of our internal microbial ecosystems can perhaps be for the worse, might these communities also be manipulated for the better? That’s the idea behind adding intact microbes to our diets — the probiotic approach epitomized by live cultures in yogurt. Some studies suggest that consuming certain microbes can affect the course or likelihood of disease, says Justin Sonnenburg, PhD, assistant professor of microbiology and immunology. But, he cautions, “a lot of this is driven by pseudoscience, by companies selling products. “Many non-resident microbes can exact biological effects in the intestine, and some of those effects are for the better,” he says. But we still need to learn more about exactly what various bacterial species in our gut do for us to be fully confident in our ability to get the right bug for the job.
Sonnenburg is working on that. He’s studying germ-free mice into which he can introduce bacteria of interest one at a time and then see which products these bacteria make and how they get along with one another and with us.
As we learn more about precisely what bacteria do, we may find — ironically — that they and the substances they produce are often less menacing than they are medicinal. “If we were to open a 21st-century medicine cabinet, microbes may be there,” says Gordon. “These master physiologic chemists have learned to manufacture chemical entities that may become part of our armamentarium of medicinal compounds.”
It’s a bit of a paradox, Dethlefsen says. “We don’t want to have E. coli in our spinach or Salmonella in our peanut butter. But not all bacteria are bad. We don’t want to completely eliminate them.”