The Microbiome: Looking at Microbes as More Than Pathogens

The body is shared by a complex community of symbiotic bacteria, archaea, fungi and viruses. Although trillions of these microbes are housed in multiple body sites, including the gastrointestinal tract, urogenital tract, oral cavity and skin, their full impact on pathogenic infection and other areas of clinical health remained undefined for decades.

In recent years, however, a growing body of research has uncovered evidence suggesting the microbiome is more essential to healthy body functions than previously believed.

“In general, the feeling was that the microbiome contains pathogens, and we needed to control those pathogens — that was the prevailing concept for the last hundred years,” Robert J. Genco, DDS, PhD, State University of New York distinguished professor and director of the University of Buffalo Microbiome Center, told Healio Gastroenterology. “What we didn’t know was the extent to which the microbiome contributed to many of the normal functions of the body. I think that has been the realization: We are not just having to deal with pathogens in the microbiome, but having to look at the microbiome as a healthy part of our being that contributes to our normal physiology.”

As new data have recently come to light, Healio Gastroenterology spoke with experts about the microbiome and ongoing multidisciplinary efforts to define its role in human health.

Sequencing Technologies

Early efforts to study the specific composition and diversity of the microbiome were challenged by difficulties culturing the majority of sampled microbes, Genco explained. While this allowed a good understanding of the microbiome and its composition, the major breakthrough came with sequencing techniques involving the 16S ribosomal RNA gene.

“It was sensitive enough to detect virtually all of the bacteria present, down to those organisms that were present at very, very low percentages,” Genco said. “This has now allowed us to look at the totality of organisms in any one site — the gut, the mouth, the skin — and, to our surprise, we found that in many of these sites we were only cultivating with the previous technique a very small percentage.”

In 2008, 16S sequencing and an increasing interest in describing the microbiome led the NIH to initiate the Human Microbiome Project (HMP), according to Ryan T. Ranallo, PhD, program officer in the Enteric and Hepatic Diseases Branch of the National Institute of Allergy and Infectious Diseases. The $170 million project — which culminated in a series of papers in 2012 and 2013 — sampled more than 200 healthy participants to characterize the microbial composition of 15 anatomical sites. Ranallo said the effort also provided investments in tool and database development, paving the way for future microbiome research.

“The data demonstrated that there are distinct bacterial communities throughout the human body, that those communities can be altered, and that those alterations have been associated with disease states in general,” Ranallo told Healio Gastroenterology. “HMP really catalyzed what we see as the microbiome research field today, in terms of outgrowth and investigations.”

Ranallo said that while the association between antibiotic treatment and certain infections, such as Clostridium difficile, had been understood for years, data collected through improved sequencing techniques revealed these outcomes to be the product of poor microbiota composition.

“One of the things that they’re finding is that the microbial populations within the gut can be disturbed or altered after antibiotic treatment,” Ranallo said. “As we uncover these features of the microbiome ... we’re now trying to modify or come up with new therapeutics that don’t have such a dramatic impact on the gut microbiota.”

FMT for Damaged Gut

According to Colleen S. Kraft, MD, associate professor of pathology and laboratory medicine, and associate professor of medicine at the infectious disease division of Emory University School of Medicine, the most promising clinical research heralded by these advancements has focused on fecal microbiota transplants for recurrent C. difficile infection.

“I view it like a garden: antibiotics kill the bacteria, [C. difficile] grows up like a weed, other antibiotics such as vancomycin and [metronidazole] are like weedkillers,” Kraft told Healio Gastroenterology. “Fecal transplant replants your garden. That’s a really easy thing to explain, and in the clinical setting it also seems to work really well.”

The first randomized controlled study of FMT was published in 2013 by Els van Nood, MD, and colleagues, and was terminated early when an interim analysis of its 43 participants found higher rates of recurrence among those who were not receiving the intervention. More recently, another trial published in August by Colleen R. Kelly, MD, gastroenterologist at the Women’s Medicine Collaborative and assistant professor of medicine at the Warren Alpert Medical School of Brown University, and colleagues demonstrated a 90.9% (95% CI, 69.2-97.8) cure rate among 22 recurrent CDI patients receiving donated colonic fecal samples.

However, many of the concepts surrounding C. difficile colonization also can be applied to multidrug-resistant (MDR), health care-associated gastrointestinal infections, such as vancomycin-resistant enterococci and carbapenem-resistant Enterobacteriaceae.

“A lot of the recent research has looked at the features of the microbiota and risk of colonization with some of these bacteria,” Ranallo said. “What they’re finding is that the microbiota might be a good place to search for diagnostics looking at susceptibility to colonization with these bacteria, because one of the features of multidrug-resistant organisms in the gut is ... monodomination,” — or excess of a single species. “This situation is associated with poorer health outcomes and bacteremia.”

There is evidence of FMT’s potential to treat these illnesses as well. Case studies published in 2015 by Joshua T. Stripling, MD, internal medicine resident at the University of Alabama at Birmingham, and Nancy F. Crum-Cianflone, MD, MPH, infectious disease physician at Scripps Mercy Hospital, describe patients with multiple MDR organisms and C. difficile colonization whose conditions were resolved following FMT. Widespread adoption of FMT could potentially replace the need for traditional broad-spectrum antibiotic treatments, which Kraft said is especially appealing considering the current crisis of antimicrobial resistance.

“The antibiotic pipeline is drying up, and [some of] these medications used for MDR bacteria have a lot of toxicity,” Kraft said. “Where I think that it’s really exciting, from an infectious disease standpoint, is that we could be working on people and restoring their microbiome in order to prevent them from having drug-resistant infections or needing treatment with toxic antibiotics.”

Gut Dysbiosis

Gut microbes are also thought to play a role in intestinal conditions as well as chronic diseases affecting other areas of the body, but these data tend to be less conclusive than those supporting C. difficile and MDR colonization.

“There is information that is starting to come out that some of the metabolites from bacteria in our gut make it into our bloodstream, and that may have more far-reaching implications about chronic illness,” Kraft said. “We can learn a lot about our patients by knowing how disrupted their microbiome is.”

In a review published last year, Andrew B. Shreiner, MD, gastroenterologist in the department of internal medicine at the University of Michigan Medical School, and colleagues discussed data regarding irritable bowel syndrome and inflammatory bowel disease. They concluded that microbiota-altering therapies for IBS employed in these studies “have shown encouraging, though inconsistent, results,” noting that the investigation of potential therapies for the condition is complicated by the lack of specific diagnostics and potentially heterogeneous etiologies. For IBD, they wrote that an association between inflammatory responses and microbiota composition is well-established, “though it is not clear if microbial changes contribute to disease pathogenesis or develop as a result of local inflammation.” The complexity of the disease will require further characterization of microbiota populations at various points in IBD development, they continued.

Gut dysbiosis could be a factor in illnesses outside of the gastrointestinal tract as well. Shreiner and colleagues’ review included studies linking gut microbes and production of the proatherosclerotic metabolite trimethylamine-N-oxide (TMAO), which has been associated with increased risk for cardiovascular events. A separate metabolomics-focused review published in 2015 also identified microbe-produced TMAO as a potential cardiovascular risk, but warned of dietary impacts and other potential confounders.

Similarly, numerous studies are investigating the relationship between the gut microbiome and neuroendrocrinal system, according to Emeran A. Mayer, MD, PhD, professor of medicine and psychiatry in the division of digestive diseases at the David Geffen School of Medicine, University of California, Los Angeles.

“This concept that benign microbes in the gut would be able to influence the brain behavior didn’t really exist before 10 years ago,” Mayer told Healio Gastroenterology. “There was already information and very intriguing findings about how parasites from the gut can get into the brain and change behavior, with toxoplasmosis being the most spectacular example, but that idea was not applied to the vast number of microbes that live in us in synergy with our gut and the host.”

To demonstrate the link between gut microbiota and brain responses, Mayer and colleagues randomly assigned female participants to receive fermented milk with probiotic, unfermented milk with no probiotic or no intervention. They compared functional MRI scans of the participants, who also were given a standardized emotional faces recognition task, and found altered responses associated with changes in midbrain connectivity among those given the fermented milk.

“In terms of proof-of-principle study, [we showed] there is a connection between gut microbiota and brain response to an emotional stimulus,” he said.

Mayer noted that this was among the few published nonanimal studies offering clear evidence of the gut-microbiome-brain axis, and that no well designed studies have examined microbiome-based interventions in psychiatric disorders. In contrast, he continued, more conclusive data is available in the realm of obesity and weight loss.

“It’s certainly not known why people lose so much weight from [bariatric] surgery and are able to maintain the reduced weight but pretty much fail every other attempt for weight reduction,” Mayer said. “But it has been speculated that the significant change in gut microbial composition following the surgery is related to the rapid postprandial change in food preferences and brain changes.”

This theory was at the forefront of a 2014 data review by Joe Alcock, MD, MSCR, associate professor of emergency medicine at the University of New Mexico, and colleagues, who suggested that interventions to restructure gut microbiota could reduce the microbes’ influence over the eating behaviors of their hosts. A 2015 study by Fredrik Bäckhed, PhD, professor at the University of Gothenburg, and colleagues found that changes to the gut microbiome due to bariatric weight loss surgery could last up to a decade, and that transferring the microbiota after surgery to another host could lead to weight loss.

Mayer said cases involving weight loss and obesity outcomes could be the first to see new microbiome-based interventions because “you don’t have to just rely on subjective symptoms as outcome parameters. You can use weight loss and metabolic parameters as objective parameters in obese subjects,” as opposed to subjective symptoms reported by patients with psychiatric disorders or IBS, for example.

Alternative Anatomical Sites

Ranallo said that although many of the ongoing microbiome studies are centered in the gut — partially due to the ease of obtaining a representative sample from stool — there are clear compositional differences in microbiota collected from other anatomical sites that are the focus of research, including the skin, oral cavity and vaginal tract.

“The vaginal microbiota harbors a less diverse [microbial community] than the gut, but nonetheless, this anatomical site has been very well-studied,” he said.

A review of 63 vaginal microbiome composition studies found that communities dominated by lactobacilli are associated with healthy microenvironments; bacterial vaginosis is most often polybacterial; and vaginal dysbiosis is frequently connected to HIV, HPV and Trichomonas vaginalis infection. Although clinicians are often advised to treat vaginal dysbiosis only when symptomatic, “this might have to be re-evaluated in specific at-risk population groups if dysbiosis is identified as a strong risk factor for adverse outcomes in sufficiently powered longitudinal studies,” Janneke H.H.M. van de Wijgert, PhD, MPH, professor of infection and global health at the University of Liverpool, and colleagues wrote.

Efforts to describe the relationships between microbiome composition changes and various cutaneous diseases also are ongoing, according to Elizabeth A. Grice, PhD, assistant professor of dermatology at the University of Pennsylvania’s Institute for Immunology. Studies like these, she wrote, could potentially fine-tune diagnoses of ambiguous dermatologic conditions.

“These applications are particularly promising in light of emerging antibiotic resistance across medically relevant bacterial strains,” Grice wrote in Seminars in Cutaneous Medicine and Surgery. “Further, microbiome therapeutics and diagnostics are highly applicable to precision and personalized medicine, and may in the future transform management and treatment of dermatological disease.”

Although previously thought to be sterile, the lower respiratory tract also houses microbial colonies that may interact with respiratory infections such as tuberculosis, according to Jorge Cervantes, MD, PhD, assistant professor at Texas Tech University Health Sciences Center El Paso, and colleagues. Their recent review of six studies comparing the respiratory microbiota of TB patients and controls revealed frequent discrepancies in treatment outcomes — potentially due to weak sampling and substantial inconsistencies in specimen collection and analysis — but nonetheless suggested compositional changes at this site related to TB.

“The microbiome, through microbial products and immunomodulators released upon recognition of commensals and pathogens by immune cells, could have an impact [on] the inflammatory response in the lung,” Cervantes told Healio Gastroenterology. “Clinicians should be aware that both the lung and intestinal microbiota may play a role in the pathogenesis, treatment and future prevention of TB.”

Microbial communities of the oral cavity are among the most heterogeneous of the body, Genco said, with any individual housing approximately half of the more than 800 different organisms that have been identified. These communities are normally very stable, he continued, but changes in microbiota can lead to periodontal disease and dental caries. In addition, microbes normally found in the oral cavity have been detected in other anatomical sites, such as in colon cancer tumors and atheromas in heart disease.

“That is very preliminary information, but it is a provocative idea in that we may be able to prevent or moderate any one of these conditions by altering the oral flora,” Genco said.

The Developing Microbiome

Recent studies are uncovering the mechanisms behind infant microbiome assembly, transfer of the microbiota from the mother to the child, and the issues that may arise when these processes are interrupted.

“As we’re developing, so is our microbiota, and it’s clear that the microbiota helps us grow,” Martin J. Blaser, MD, the Muriel G. and George W. Singer Professor of Translational Medicine in microbiology at New York University’s School of Medicine, told Healio Gastroenterology. “It helps us develop our immunity, our metabolism and our cognition.”

According to Noel T. Mueller, PhD, assistant professor of epidemiology at the Johns Hopkins Bloomberg School of Public Health, and colleagues, changes to the composition of a woman’s vaginal and gut microbiome are thought to protect the health of the fetus and provide microbial inoculum prior to environmental exposure. This transfer was initially thought to occur through childbirth and lactation; however, recent evidence of microbes present in amniotic fluid, umbilical cord blood, fetal membranes and placenta suggests the exchange could occur earlier.

“We thought that fetal tissues were sterile or near sterile, but they’re not — the placenta has its own microbiome, and probably the key study so far of the placental microbiome, by Aagaard and colleagues, shows that it is most related to the oral microbiome,” Genco said. “We’re doing [experiments] now to see if they are the exact same organisms in the oral microbiome as one finds in the placenta.”

Blaser said interrupting the transfer of microbes could hamper an infant’s early development and, according to some experimental studies, increase the risk for obesity, asthma, juvenile diabetes or other immune illness.

“We’re disrupting it starting with antibiotics that moms get while they’re pregnant, and then by cesarean sections where the baby misses getting the mom’s microbes through the birth canal,” Blaser said. “Even then, the babies are getting washed all the time, and they’re getting antibiotics quite frequently.”

Mueller wrote that methods to restore an infant’s microbiota are being investigated, but that breast-feeding appears to be the most effective and safest route.

Probiotics’ Role

In an effort to correct dysbiosis and reduce health care’s reliance on antibiotics, many researchers and drugmakers are investigating probiotics for the treatment of several diseases.

“Clinically, I think it would be great ... if we could test and say, ‘you need to take more probiotics,’ or ‘you need to drink fermented drinks,’” Kraft said.

Genco explained that loose regulatory structures have allowed many companies to sell probiotics directly to consumers, so long as no specific disease claims are marketed.

“You can take a probiotic, and maybe it makes your stomach feel better or makes your skin look better,” Genco said. “But you can’t make a disease claim unless you go through the very rigorous clinical trials.”

“Tremendous heterogeneity” between patients’ microbiomes have complicated clinical studies of natural or engineered probiotics, which have generally produced mixed results, Mayer said. Further, a recently published systematic review of seven randomized controlled trials found no convincing evidence of the effects of probiotic supplements on the fecal microbiota composition in healthy adults.

Regardless, Mayer said these treatments remain of great interest not only to food companies, but also to many drug manufacturers.

“There’s a lot of start-up companies now in this space, using consortia of human microbes taken from the human gastrointestinal tract that have certain functions,” Mayer said. “For diseases from autism to depression to irritable bowel syndrome, there may be a particular microbe or a group of microbes that produce metabolites that play a role in symptoms.”

Experimental Data

With the possible exception of FMT for recurrent CDI, Genco and Kraft said the available data on microbiome-related care are still largely experimental and should be viewed with caution. Mayer agreed, warning that “we have to be very careful before making clinical recommendations.”

Ranallo said investigators in this field of research are interested in improving their data, with many recently attending a workshop held by the NIH and the National Institute of Standards and Technology that focused on why the evidence has been so inconclusive.

“It’s appropriate for investigators to conduct well-controlled studies that are powered correctly to look at very specific endpoints — in fact, I believe these are needed,” Ranallo said. “We still need human epidemiological studies that are adequately powered, and we need to start capturing the microbiota as people or individuals transition from a state of homeostasis to a state of dysbiosis.”

Despite these challenges, the experts agreed that microbiome-based therapies show great promise and are worth investigating.

“Unlike genetic traits, which you can’t easily change, the microbiome can be changed dramatically or subtly, so I think there’s a cause for optimism here,” Genco said. “We have learned how to tame bacteria or how to adjust the flora. We don’t yet know all the answers, but we have a lot of tricks and techniques to modify the microbiome.” – by Dave Muoio

• References:
• Alcock J, et al. Bioessays. 2014;doi:10.1002/bies.201400071.
• Crum-Cianflone NF, et al. J Clin Microbiol. 2015;doi:10.1128/JCM.00820-15.
• Grice EA. Semin Cutan Med Surg. 2014;doi:10.12788/j.sder.0087.
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• Mueller NT, et al. Trends Mol Med. 2014;doi:10.1016/j.molmed.2014.12.002.
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• For more information:
• Robert J. Genco, DDS, PhD, can be reached at rjgenco@buffalo.edu.
• Colleen S. Kraft, MD, can be reached at colleen.kraft@emory.edu.
• Emeran A. Mayer, MD, PhD, can be reached at emayer@ucla.edu.
• Ryan T. Ranallo, PhD, can be reached at ryan.ranallo@nih.gov.

Disclosures: Blaser, Cervantes, Kraft and Ranallo report no relevant financial disclosures. Genco reports probiotic research support from SunStar. Mayer reports advisory positions with Dannon and Danone, and consultation for Prolacta Bioscience and Whole Biome.