Our bodies are inhabited by trillions of microorganisms, with specific microbes unique to each individual. Through experimentation, scientists have identified certain factors that account for variation in the gut: diet, living conditions, exercise, and maternal line. Now, scientists at the University of California, San Diego have discovered another factor that affects the composition of the gut microbiome: time of day. In fact, scientists have found that time of day is such an important factor that they are asking the National Institutes of Health (NIH) to require researchers to report it in their papers.
In a new paper published in Nature’s metabolismScientists report that daily fluctuations in the gut alter the microbiome so significantly that different microbes populate it in the morning and at night. That means a researcher analyzing a stool sample collected at breakfast will come to radically different conclusions than a researcher analyzing a stool sample collected just before dinner. The UC San Diego scientists propose that this variability prevents gut microbiome researchers from being able to replicate each other’s experiments.
“The unexplained variability and lack of replicability may be due to the fact that the microbiome oscillates throughout the day, with different populations of microbes dominating at different times,” said Amir Zarrinpar, M.D., Ph.D., a gastroenterologist and associate professor of medicine at UC San Diego School of Medicine and senior author of the study. “We found that the timing of a sample can dramatically impact which microbes were present and the conclusions scientists reached about the disease they were studying.”
Scientists conduct experiments for many reasons. The traditional reason is to answer a specific question, but another reason is to make a discovery or arrive at a scientific truth that others can replicate with their own experiments. In gut microbiome research, scientists collect stool samples to find out which microorganisms are present and in what amounts. They then link those changes to disease processes.
For this study, the team compared computer analyses of previously published studies, including their own. They found that changes in the microbiome were so pronounced over time that they affected the results as much as diet did. “We found that in just four hours after a mouse eats breakfast, almost 80 percent of its microbiome is different,” Zarrinpar said. When analyzing the conclusions drawn in the studies, Zarrinpar and his team found that the results and conclusions depended heavily on when the researchers collected the samples.
Zarrinpar was inspired to do this study from a conversation he had with a colleague. “He told me that a postdoc in his lab took over an experiment that someone else had started. The postdoc couldn’t replicate any of the previous researcher’s findings. That made him question his predecessor’s research,” Zarrinpar said. “Then the postdoc realized that one bacteria that was incredibly ubiquitous in his findings was one that showed up late in the day. He went back to his lab and saw that the previous researcher liked to collect samples in the morning, while he himself collected samples before he went home. That’s why he couldn’t replicate the first researcher’s findings.”
The ability to reproduce the results of a previous experiment (replicability) is a key element in knowing whether a finding reliably represents new knowledge about reality or is simply an artifact of the experiment. Microbiome research is currently undergoing a replicability crisis, in part due to the interdisciplinary nature of the field, the complicated relationship between microorganisms and their hosts, and the difficulty of controlling so many variables.
Zarrinpar believes his team’s new findings on the importance of timing may help solve the replicability crisis in microbiome research. He explains: “If we’re going to be able to communicate with each other about our science and what we think is going on in an effective way, then we have to understand that if we got different results than I did, maybe that could be because of the timing of when we collected the samples or not. Right now, you can’t even tell.”
According to Zarrinpar, scientists in other fields, such as circadian biologists, have also been lobbying the NIH to be stricter about the need to report the timing of samples. Zarrinpar hopes that the publication of this paper will convince more scientists (and the people who fund and publish their research) of the importance of timing and its potential impact on other fields as well, such as metabolism research.
Zarrinpar’s next steps include promoting standardized guidelines that ensure consistency in timing and methodology of microbiome sample collection. “This will likely involve collaboration with other researchers, funding agencies, and journal editors to promote adoption of such standards,” she said. Her next paper focuses on understanding the impact of time on humans, a variable that is much harder to control.
This research was funded in part by the National Institutes of Health, the Soros Foundation, and the American Hospital Association.
Co-authors of the study include Celeste Allaband, Amulya Lingaraju, Stephany Flores Ramos, Tanya Kumar, Haniyeh Javaheri, Maria D. Tiu, Ana Carolina Dantas Machado, Roland A. Richter, Emmanuel Elijah, Gabriel G. Haddad, Pieter C. Dorrestein, Rob Knight of the University of California, San Diego, and Vanessa A. Leone of the University of Wisconsin-Madison.