New line of population health research takes guts

That’s Doug Esselman’s sales pitch for convincing people to collect a sample of their stool in the name of science — in this case, the latest addition to the Survey of the Health of Wisconsin (SHOW), a population-based study at the University of Wisconsin School of Medicine and Public Health. The school’s investigators have led SHOW since its 2008 inception.

Why stool? Because growing evidence suggests that the community of bacteria in the human gut — our microbiome — plays a critical role in keeping us healthy. Its imbalance may contribute to conditions ranging from obesity, diabetes and asthma to cancer and cardiovascular disease.

To study Wisconsin’s gut microbiome, SHOW investigators have doubled the compensation they offer to participants— now $200 — to analyze the research subjects’ stool samples in the lab and correlate that data with what they eat.

SHOW investigators have collected typical past-year diet information since Day 1, but the new microbiome study — initiated in spring 2016 — also asks participants to record everything they have consumed for the past 24 hours and the time they collected their stool samples.

Even though the sample collection involves a significant “ick” factor, the researchers were surprised to find that the 24-hour dietary recall, which takes about an hour to complete on a computer, is typically perceived as a bigger burden.

“When participants deliver their stool samples, we also collect their blood, urine and saliva, and take swabs from their skin, mouth and nose,” explains Esselman, who manages the SHOW field team and has plenty of personal experience knocking on Wisconsin’s doors to recruit participants.

“You’d be surprised by how many people don’t have an issue with any of that. But getting them to log into a website and enter everything they ate in the past 24 hours has definitely been harder.”

Kristen Malecki, PhD, MPH, and Javier Nieto, MD, PhD, MPH — then chair of the Department of Population Health Sciences — were SHOW’s co-directors until Nieto left that role in SHOW in 2015, and Paul Peppard, PhD ’99, became its co-director. Peppard admits to having had serious concerns about the success of collecting stool samples when the team first brainstormed about it.

“But apparently, Wisconsinites have few qualms about handing over their stool for research,” jokes Peppard, an associate professor of population health sciences.

And they do it for a compelling reason: to help investigators in the battle against “superbugs,” or microbes that have developed resistance to the antibiotics that have greatly reduced illness and death from infectious diseases since Alexander Fleming discovered penicillin in 1928.

According to the U.S. Centers for Disease Control and Prevention (CDC), more than 2 million Americans are infected each year with multi-drug resistant organisms (MDROs), causing at least 23,000 deaths. Since MDROs don’t know any boundaries, in 2015, the World Health Organization published the “Global Action Plan on Antimicrobial Resistance,” which states that this concern “threatens the very core of modern medicine and the sustainability of an effective, global public health response to the enduring threat from infectious diseases.”

Peppard considers antibiotic resistance “a growing, grossly underappreciated crisis” and stresses the need to direct resources to microbiome research to “help us learn a lot more about how to contain this crisis.”

From SHOW to WARRIOR

The SHOW study, which is the only statewide public health monitoring program in the nation, has enrolled more than 6,000 individuals from 62 of Wisconsin’s 72 counties since 2008. Further, it has enrolled more than 700 children since 2014.

SHOW’s methods involve having trained interviewers visit randomly selected households, within randomly selected counties, to collect survey data, biological samples and physical measurements — such as height, weight and blood pressure — from all household members who legally consent to participate.

Asked why SHOW is the only program of its kind, Peppard credits the School of Medicine and Public Health for its foresight to build important population health programs, which have been integral to the school’s transformation into a school of medicine and public health.

“It seems like an oversight by the other 49 states, especially since the importance of this type of study has been recognized for decades at the federal level,” he adds.

SHOW has been continuously funded by the School of Medicine and Public Health’s Wisconsin Partnership Program (WPP) since 2006, with intermittent additional support from federal agencies.

The beauty of the project is that its existing infrastructure is available to other investigators—who have raised independent funds—for ancillary studies of narrower research topics, such as the microbiome’s role in antibiotic resistance. SHOW interviewers refer to “the Wisconsin microbiome study” when they explain its rationale to potential participants, but the founding investigators called it the War on Antibiotic Resistance in Wisconsin, or WARRIOR.

Those leaders are Nasia Safdar, MD, PhD (PG ’00, ’09), associate professor of infectious diseases at the School of Medicine and Public Health and the William S. Middleton Memorial Veterans Hospital, and Ajay Sethi, PhD, MHS, associate professor of population health sciences. They admit that WARRIOR is not the most logical acronym, “but it seemed to give us momentum, like names sometimes do,” Safdar says.

Like SHOW, WARRIOR exists because of WPP funding. In late 2014, Safdar and Sethi pitched their idea to Peppard and Malecki, an assistant professor of population health sciences. With their support, Safdar and Sethi — along with several co-investigators — submitted their proposal to the WPP in spring 2015 and received a grant the following fall.

“WARRIOR is a perfect example of the added value that comes from the School of Medicine and Public Health’s investment in SHOW’s infrastructure,” Malecki shares. “Without that, WARRIOR would not be financially feasible. I think it also illustrates the importance of crosscampus collaborations in the pursuit of team science.”

With a shared interest in studying antibiotic resistance in the hospital setting, Safdar and Sethi recognized the tremendous opportunity SHOW provided to tackle this problem at the community level. With the WARRIOR study, which is collecting 600 stool samples and dietary information, Safdar and Sethi — in collaboration with dietary epidemiologist Julie Mares, PhD ’87, and microbiologist Garret Suen, PhD — will test whether a fiber-rich diet, consumed by less than half of Americans, is associated with changes in the microbiome and a lower prevalence of MDROs in the gut.

“To reduce the potential for outside pathogens to colonize the gut, you need to think about what causes its microbial imbalance,” Sethi explains. “We know that exposure to antibiotic medication plays a large role, but we also know that dietary fibers—from vegetables and fruits to certain kinds of grain—help maintain a healthy, diverse gut environment in which MDROs are less likely to grow.”

Despite the logical connection, Safdar and Sethi say WARRIOR explores uncharted territory because the relationship between diet and the microbiome has not been studied in a community-based setting. When stool samples arrive in Safdar’s lab, which has to happen within 48 hours from collection — or, as Sethi quips, “before the samples hit the fan” — Megan Duster and her team divide them into several portions, most of which are frozen. One fresh sample is added to a substance that promotes the growth of multiple disease-causing germs. Negative stool cultures grow no bacteria of interest for this study. On positive cultures, researchers run additional tests to identify the bugs and test their reaction to common antibiotics.

Next, investigators classify stool samples into two groups based on the presence or absence of at least one known MDRO, such as methicillin-resistant Staphylococcus aureus. Finally, statisticians compare the amount of fiber the study participants had consumed in the 24 hours before they collected their stool samples.

While this is seemingly straightforward, investigators face the big challenge of accounting for various lifestyle and long-term dietary differences between the people whose samples are in the two groups.

“More complex statistical methods are needed to attribute microbiome differences to fiber intake, rather than the fact that people with a healthier recent diet may also be healthier in general,” Sethi explains.

As a practicing physician, Safdar ultimately hopes the WARRIOR study will suggest new weapons to wield against superbugs that survive today’s antibiotic arsenal. Since probiotic yogurts and fecal transplants have had mixed success, and it’s premature to endorse them across the board, Safdar says the field is wide open to options.

“If the fiber angle becomes promising, increased dietary or supplemental fiber enrichment may be a useful measure, especially for patients who are at high risk for MDROs or have been infected by one,” says Safdar.

Sethi adds, “There is some evidence that a physician handing you a prescription that reads ‘eat more fiber’ or ‘take a fiber supplement’ may be more effective than having someone simply tell you to live a healthier lifestyle.”

Antibiotic resistance, the microbiome and lead exposure

Adding a complex ancillary study like WARRIOR to SHOW is a significant investment. With SHOW support, researchers must obtain new funding, hire and train staff, and redesign study logistics, while participants are asked to commit a substantial chunk of additional time (and overcome the “ick” factor) for the ultimate goal of improving human health.

But one way to amplify the value of these efforts is to use the newly collected data to address more research questions. The SHOW team didn’t hesitate to do just that.

Around the time Safdar and Sethi were hashing out the WARRIOR study, Malecki’s graduate student, Shannah Eggers, who has a background in microbiology, was searching for a dissertation project. Malecki’s laboratory focuses on understanding human susceptibility to environmental factors, and she encouraged Eggers to think about interactions between the environment and the microbiome. Eggers found several published studies supporting a possible relationship between superbugs and lead, but none in humans.

For instance, aquatic bacteria with a high tolerance for the heavy metal also tend to have a greater antibiotic resistance, suggesting that the two properties may be genetically linked. Other studies showed that mice exposed to lead as babies have an altered microbiome composition as adult animals, and cell assays indicate that lead compromises the body’s immune system, as does an imbalance in the gut microbiome.

“Taken together, these three lines of evidence make it highly plausible for lead exposure to increase the risk of an MDRO infection,” Eggers says. “This motivated our proposal to measure lead levels in the urine of approximately 500 WARRIOR participants to see if they are correlated with the presence of MDROs in their stools.”

When Eggers, Malecki and Safdar submitted that proposal to the Department of Medicine, the opportunity to piggyback on the existing study infrastructure — coupled with the ability to explore new scientific territory by testing the lead/MDRO hypothesis — resulted in a thumbs-up funding decision. It didn’t hurt that Malecki and Eggers’ home department has a long history of studying lead-associated health outcomes and that the Flint, Michigan, crisis of lead in its drinking water was making news headlines at the same time.

As the metric for testing the lead/MDRO hypothesis, Eggers and Malecki chose urine because it reflects 70 percent of the body’s buildup of lead. However, since blood lead is the standard most often used for monitoring human exposure to lead, they also will measure blood in a subset of samples to determine which one is more predictive of MDROs in the gut.

Reflecting upon the recent growth in the scope of SHOW’s research agenda, Malecki offers a shout-out to the thousands of Wisconsinites who make the study possible.

“I think SHOW participants are amazing people for being willing to share a large amount of time and energy with us,” she says. “As for the microbiome study’s success, many people are fascinated by the idea that our bodies are made up of all these bacteria and viruses. It’s a hot topic, and we are incredibly grateful that the research subjects want to be part of the cutting-edge research we are able to do through SHOW.”