Researchers in the rapidly expanding research field of microbiomics have shown that the human body is home to an entire ecosystem of bacteria, viruses, fungi, and other microbes. What’s more, they play an important role in regulating many physiological processes.
By Lisa J. Bain
Although it may sound weird, unappealing, even disgusting, fecal transplantation has piqued the interest of gastroenterologists and infectious disease specialists around the world. Meanwhile, patients suffering from severe diarrhea are demanding the procedure and the FDA has weighed in with restrictions on how this “unapproved therapy” can be delivered.
Why all the excitement? Fecal transplantation is not new: Ben Eiseman, a surgeon at the University of Colorado, described it more than 50 years ago to treat a life-threatening diarrheal disease caused by a bacterium called pseudomonas enterocolitis. But until a few years ago, Eiseman’s unconventional treatment was largely dismissed by the medical community, at least in the United States (it has been much more widely used in Australia). Then, in 2010, The New York Times ran a story about a doctor in Minnesota using fecal transplantation to successfully treat a patient with a severe infection caused by a bacterium called Clostridium difficile, or “C. diff.” And this year, a randomized controlled trial of the treatment was stopped early when an interim review of the data showed not only that it worked, but that it was far superior to the standard treatment with powerful antibiotics. Fecal transplantation, also known as fecal microbiota transplantation (FMT) or bacteriotherapy, had arrived.
The acceptance of FMT for the treatment of diarrheal disease caused by C. diff exemplifies a paradigm shift in how many diseases are viewed, as well as a translational application of the science of microbiomics -- a rapidly expanding research field that Science magazine dubbed “The Germ Theory of Everything.” Microbiomics researchers have shown that the human body is home to an entire ecosystem of bacteria, viruses, fungi, and other microbes, and that these bugs play important roles in keeping us healthy and regulating all sorts of physiologic processes. When the gut microbiota (the population of microbes) is disrupted, for example by overuse of antibiotics, the consequences can be lethal, as is the case with C. diff. It infects as many as 3 million people worldwide each year and in recent years these infections have become less and less responsive to antibiotic treatment. In the U.S. alone, medical costs to treat C. diff infections exceed $1 billion per year, and some 14,000 Americans die.
Big Science and the Microbiome
Following on the heels of the massive Human Genome Project, which identified about 22,000 protein-coding genes in humans, the National Institutes of Health launched the Human Microbiome Project (HMP) in 2007 to map the collective genomes of the human microbiota. HMP researchers at nearly 80 institutions, including Penn, analyzed tissue from 242 healthy individuals, sampling 15 body sites in men and 18 in women. The findings made the genome project look modest in comparison: the human gut microbiome alone is home to 100 trillion bacteria -- ten times the number of cells in the human body -- with somewhere around 8 million protein-coding genes, 360 times as many as in the human genome.
The same gene sequencing technology that fueled the genome project also made mapping the microbiome possible. “You literally can get more than a hundred billion bases of sequence information from a single instrument run these days,” says Frederic Bushman, Ph.D., professor of microbiology. “It’s astounding. We’re analyzing a dataset of over a trillion bases of sequence information. When I was a student, it would be a few days’ work to get a few reads of a hundred bases each. Today, I have a little machine in my lab that will do a hundred thousand sequence reads of about 250 bases each in a day.” One of the changes the new technology has brought about in Bushman’s research is the makeup of his laboratory personnel: “We now have four programmers in the lab and lots of collaborating statisticians to work with that kind of data. That’s all new in the last ten years.”
Indeed, gene sequencing has brought about a transformation in the entire field of microbiology, as it has in many other areas of biomedical research. David Artis, Ph.D., associate professor of microbiology, says that when he completed his training in immunology back in the mid-’90s, he was advised not to be concerned about the microbiota or what was then referred to as the commensal bacteria (bacteria that live harmoniously with the host) that colonized the intestine. “We were told, ‘don’t worry about it -- it’s too complicated, you can’t culture the bugs, and you can’t phenotype what they are.’ What has been an incredible journey for us has been the rapidity with which we’ve been able to engage and interrogate the role of the microbiome in the last decade or so.”
Artis explains that before the advent of gene sequencing, the only way to study bacteria was to grow them in the laboratory, but perhaps as many as 90 percent of the commensal bacteria in the human intestine cannot be cultured. “We took a quantum leap from those culture-based assays to sequencing-based approaches,” he says. With Bushman as one of the leaders in this effort, these new technologies have enabled scientists to profile complex microbial communities and map the evolutionary relationships of diverse species of commensal bacteria. According to Artis, “What we’ve learned is that disorganization or change in the composition of the commensal bacteria can be associated with many human diseases, from inflammatory bowel disease to asthma, to arthritis, to multiple sclerosis.” One of the fundamental questions has been: are those changes in bacterial diversity a consequence of the disease, or could they have perhaps participated at some level in the development of these diseases?
Gene sequencing is not the only new tool that has fueled progress in answering some of these questions about the microbiome. “In addition to profiling microbial communities in human health and disease, we have been able to adopt animal model systems -- flies, fish, and mice -- where we can deliberately manipulate the diversity of commensal bacteria and ask if that influences the immune system, the physiology of the heart, liver function, the enteric nervous system, or whatever organ system that we might be interested in,” explains Artis. He directs the Penn Gnotobiotic Mouse Facility, where mice that have never been exposed to live microbes are housed in germ-free conditions, enabling scientists to introduce a single organism or group of organisms to the mice to study the colonization of different body sites and how it affects disease resistance or susceptibility. “We’re finding remarkable effects indicating that changes you see in human diseases may not just be a consequence of the disease; the microbes may actually participate in the development or progression of these diseases,” Artis continues. He notes that Penn’s germ-free mouse facility was one of the first of its kind in the United States. Germ-free mice, for example, have been used at Penn to study the role of commensal bacteria in the development of the immune system and other tissue systems, as well as the role of specific organisms in a range of diseases that affect organs from the skin to the gut.
“All Diseases Begin in the Gut”
Back around 460 B.C., Hippocrates himself identified the human gut as the gateway to the rest of the body, yet some 25 centuries later, scientists are still only beginning to understand what commensal bacteria do in the healthy human gut, much less in a disease state. Evidence suggests that this ecosystem within our bodies plays an important role in keeping us healthy by producing vitamins, enzymes, and other compounds that help us digest and metabolize food and regulate the immune system. In addition, many of the advances in hygiene over the past decades -- including improved refrigeration, sanitation, vaccination, increased antibiotic use, and food processing -- are thought to have upset the mutual balance between the microbes and their human hosts.
Bushman, an internationally recognized expert on the microbiome, is working with Gary Wu, M.D., and James Lewis, M.D., M.S.C.E, both professors in gastroenterology, on one of 15 demonstration projects for the Human Microbiome Project. They are examining how diet influences the gut microbiome in people with Crohn’s disease, a particularly insidious type of intestinal bowel disease (IBD). Like all microbiome research, this work is very multidisciplinary by virtue of its complexity, so Penn is an ideal place to study it: Wu investigates the microbiome in mouse models; Lewis does the human translational work; and Bushman performs the detailed gene sequencing that produces the reams and reams of data to be analyzed by bioinformaticians and computational biologists.
The HMP project followed earlier studies in which the Penn team assessed the effect of diet on the gut microbiome. The project comprised an observational component, where the microbiomes from 98 healthy volunteers were analyzed in relation to their diets as reported on a questionnaire. The second component compared two dietary interventions -- high fat, low fiber vs. low fat, high fiber -- in 10 volunteers sequestered for 10 days at Penn’s Clinical Trial Research Center.
As Wu puts it, “We found a couple of interesting things. First, there was a significant impact of a change of diet on the gut microbiome within 24 hours.” They also found that individuals change in different ways, and that even if they are forced to eat the same thing, the composition of the microbiome does not converge. “Our hypothesis going into the study was that the intersubject variability was in part due to the fact that we are all eating different things. But we didn’t observe that at all. Short-term diet is not the explanation for why people have different compositions of the microbiota.” But the observation that diet affects the human microbiome led the HMP to examine the impact on the microbiome of defined formula diets, which are used as therapy for IBD.
“We got into this because we were particularly interested in inflammatory bowel disease, where we know that for children in Europe, a nutrition-based therapy is actually one of the first-line treatments for Crohn’s disease,” said Lewis. “It’s been known for decades that this approach worked, but nobody knew how.” Known as enteral therapy, the procedure involves delivering a defined formula diet through a naso-gastric tube. It is used very little in the United States, with the possible exception of the Children’s Hospital of Philadelphia.
One reason for the limited use of these diets, as Wu points out, is that they are unpalatable. “You can’t really drink them, but even if you could -- and there are some palatable forms of the diet -- it’s very difficult to be compliant with just drinking those diets and not eating anything else.”
Lewis, Wu, and Bushman started a collaboration with Robert N. Baldassano, M.D., director of the Center for Pediatric Inflammatory Bowel Disease at CHOP, and the other members of his team, as a unique opportunity to understand how nutritional therapy works. “One of our hypotheses has always been that it had an impact on the gut microbiome,” says Lewis. The researchers designed a study that compared the gut microbiome in children receiving enteral therapy to those receiving a completely different but often-used type of therapy that suppresses the immune system with injections of monoclonal antibodies against a protein called tumor necrosis factor (TNF). Anti-TNF therapy is highly effective as a treatment for Crohn’s disease but is associated with an increased risk of infection. The phase of the study for collecting data is complete; however, results are not yet available.
“The question is, ‘What’s the mechanism?’ and that’s what we’re trying to find out,” says Wu. “We don’t know if it’s the bacteria, or what’s responsible. We just know that diet does influence bacteria, and we know that bacteria are important for the development of IBD. And we know that the composition of the microbiota is abnormal in patients with IBD.”
One way that the gut microbiome may influence the development of intestinal bowel disease is through the immune system. “A significant portion of our total immune system is associated with the gastrointestinal tract, so it’s an interaction between commensal microbes and the mammalian immune system that plays a critical role in balancing whether there is a state of health or a state of disease in the GI tract,” says Gregory Sonnenberg, Ph.D., research associate in the Division of Gastroenterology and the Institute for Immunology. And it’s not only the gut that is affected, he continues. “What’s going on in the gut also plays a key role in the health of the cardiovascular system, the liver, spleen, central nervous system, and other body systems.” Sonnenberg’s lab, in collaboration with the Artis lab, is studying how balance is maintained between commensal bacteria and the immune system. When this balance is disrupted, notes Sonnenberg, the immune system attacks commensal bacteria with an inflammatory response that may underlie many chronic diseases such as IBD, diabetes, cancer, and cardiovascular disease. His lab recently identified a previously unrecognized innate immune cell that appears to play a central role in regulating the balance between commensal bacteria and the host. He believes that a better understanding of how this cell functions could lead to new therapies for chronic diseases.
Beyond bacteria in the gut
Bacteria are not, of course, the only commensal organisms in the human gut. Viruses, fungi, and archaea (a distinct type of microorganism similar in some ways to bacteria) also inhabit the intestinal ecosystem. Bushman is especially interested in studying the predators that eat gut bacteria -- a group of viruses called bacteriophage. Bacteriophage (or “phage”) are perhaps the most abundant organisms on the planet, far exceeding the number of bacteria. While humans carry around huge and diverse populations of bacteria in their guts, the number and diversity of gut bacteriophage particles is potentially even greater. Researchers like Bushman are just beginning to clarify the importance of phage in human disease. They are known to carry genes for toxins and antibiotics -- and probably many other important genes involved in physiological processes, including metabolism. And because they move easily between different strains of bacteria, they are likely to have an enormous influence on the composition of the microbiota as well as the pathogenicity of various organisms.
In addition, it’s not just the gut microbiome that is important in human disease. Microbes colonize every barrier surface in the body. Even the lung, which was thought to be a sterile environment in healthy people, harbors low levels of bacteria, according to Ronald Collman, M.D. Collman, professor of medicine and co-director of the Penn Center for AIDS Research, became interested in the microbiome as a means of better understanding how HIV triggers lung complications in infected people. New technologies developed for studying the gut microbiome enabled Collman and colleagues to ask whether the immunodeficiency in AIDS patients may lead to changes in bacterial or fungal populations that could contribute to lung disease. But to answer these questions, the researchers first had to develop another new set of sampling and bioanalytic techniques to ensure that the gene sequences being analyzed came from the lung and not from cross-contamination from the upper respiratory tract or reagents.
“These techniques enabled us to open up this set of questions and apply it not only to HIV infection but also the impact of bacteria in lung transplantation,” says Collman. “And we’re also using it to look for potential novel pathogens in diseases of unknown origin,” such as sarcoidosis, an inflammatory disease that can affect almost any organ but most commonly the lungs. Although research on the lung microbiome lags far behind that of the gut microbiome, the field is evolving quickly. As he says, “There’s a growing sense that even in diseases such as asthma, there may be changes in the microbiome with consequences for patients, but we aren’t there yet in terms of an overarching new vision.”
Studies of the skin microbiome also lag behind the gut but are beginning to catch up, according to Elizabeth Grice, Ph.D., assistant professor of dermatology. “I think people are beginning to realize that the microbiome may be involved in a lot of different disease processes,” she says. “There is an increased understanding in our field that the diversity of what is on the skin is much greater than we ever would have thought. And with that comes a realization that perhaps even those diseases that do not have overt infectious types of pathologies may, in part, be modulated, triggered, or influenced by the microbiome.”
For example, Grice recently showed, in collaboration with John Lambris, Ph.D., the Dr. Ralph and Allie Weaver Professor of Research Medicine in the Department of Pathology and Laboratory Medicine, that the skin microbiome is regulated in part by the complement system, an evolutionarily ancient part of the immune system that works with antibodies to destroy bacteria. The proteins that make up the complement cascade have many other functions as well, including inducing inflammation. Because complement activation is known to be involved in several different diseases of the skin, such as psoriasis, a better understanding of the relationship between the skin microbiome and complement may lead to new therapeutic strategies to combat these diseases.
Grice envisions changes in how skin diseases are treated to emerge from this research. “Can we transplant a skin microbiome in the same way that they’re doing fecal microbiome? To me, that would be much less disgusting and easier. It’s very real in the future that those types of things will be part of our arsenal.”
The Germ Theory of Everything?
The popular press has embraced the microbiome in a big way, with prominent features in The New York Times, The New Yorker, The Economist, Forbes, and Mother Jones, among others. Pharmaceutical and biotech companies are also climbing aboard the bandwagon. So was Science magazine right when it suggested that “the microbiomic theory of life” may be true? Changes in the microbiome have been linked not only to obesity and many of the major diseases of humankind -- heart disease, diabetes, and cancer -- but also to autoimmune diseases and complex brain diseases such as Alzheimer's and autism.
“In a fairly short space of time, there has really been a revolution in biomedical research, encompassing not just the inflammatory and metabolic diseases, but cancer and even behavioral diseases,” says Artis. “We have to go beyond identifying that organism A regulates processes B, C, and D, or these groups of organisms are associated and can cause this disease. I think the challenge of molecular medicine is to define the molecules that control these relationships. That knowledge is going to allow us to develop drugs or intervention strategies that will allow us to either mimic a healthy microbiome or limit the signals being generated by an unhealthy microbiome. And I think that is where the field is shifting.”
In Collman’s view, Penn is ideally situated to lead this new field. “This is a really unique place in that we have great interactions and collaborations between clinical/translational microbiologists like me, and more basic molecular/computational microbiologists like Rick Bushman. It’s a very synergistic collaboration.”
That view is shared by Glen Gaulton, Ph.D., executive vice dean and chief scientific officer at the School of Medicine. He notes that Penn scientists are ideally positioned to develop a better mechanistic understanding of the microbiome and to tie observations about different bacterial populations back to what is happening in patients. “We have the ability to do that because we are so closely linked between researchers in the school and physicians who interact with patients in the hospital and outpatient clinics,” he said. “We’re working now to determine how this key initiative might benefit from formation of a center that would coalesce these teams and strategically guide project development.”
As Bushman puts it, “I think the sky’s the limit.” There are technical challenges to solve, “but it gets better with each passing year,” with increased knowledge, better methods, and more rigorous statistics.