Our bodies are inhabited by trillions of microorganisms, with specific microbes unique to each individual. Through experiments, scientists have pinpointed certain factors responsible for variation in the intestines: diet, living conditions, exercise and maternal lineage. Now scientists at the University of California San Diego have discovered another factor that influences the composition of the gut microbiome: the time of day. In fact, the scientists have found that the time of day is such an important factor that they are calling on the National Institutes of Health (NIH) to require researchers to include it in their papers.
In new work published in Nature metabolismthe scientists report that daily fluctuations in the intestines change the microbiome so significantly that different insects populate it in the morning and evening. That means that a researcher analyzing a stool sample collected at breakfast will draw radically different conclusions than a researcher analyzing a stool sample collected just before dinner. The UC San Diego scientists say this variability prevents gut microbiome researchers from replicating each other’s experiments.
“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,” says Amir Zarrinpar, M.D., Ph.D., gastroenterologist and associate professor of medicine at UC San Diego School of Medicine and senior author of the study. “We found that when a sample is taken can dramatically affect which microbes were present and the conclusions the scientists drew 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. When researching the gut microbiome, scientists collect stool samples to discover which microorganisms are present and in what quantities. They then link those changes to disease processes.
For this study, the team compared computer analyzes of previously published studies, including their own. They found that the changes in the microbiome over time were so pronounced that they affected the results as much as the diet. “We found that within just four hours after a mouse eats breakfast, almost 80 percent of its microbiome is different,” says Zarrinpar. When analyzing the conclusions from the studies, Zarrinpar and his team found that the results and conclusions were highly dependent on when the researchers collected the samples.
Zarrinpar was inspired to do this research by a conversation he had with a colleague. “He told me that a postdoc in his lab had taken over an experiment started by someone else. The postdoc could not replicate the previous intern’s findings. That made him doubt his predecessor’s research,” Zarrinpar said. “Then the postdoc realized that a bacterium that was incredibly widespread in his findings was one that appeared late in the day. He went back to his lab and saw that the previous intern liked to collect samples in the morning, while he was collecting samples himself before going home. Therefore, he could not replicate the first intern’s findings.”
Being able 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 experiencing 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 latest findings on the importance of timing could help solve the replicability crisis in microbiome research. He explains: “If we’re ever going to be able to effectively communicate with each other about our science and what we think is going on, then we need to understand that if you get different results than I do, that maybe it could have something to do with with the time we collect samples, or not. At this point you don’t even know.”
According to Zarrinpar, scientists in other fields, such as circadian biologists, have also lobbied the NIH to be stricter about the need to report the timing of sample collections. Zarrinpar hopes that the publication of this article will convince more scientists – and the people who fund and publish their research – of the importance of timing and its potential impact on other areas, such as metabolism research.
Zarrinpar’s next steps include advocating for standardized guidelines that ensure consistency in microbiome sample collection times and methodology. “This will likely involve collaboration with other researchers, funding agencies and journal editors to promote the adoption of such standards,” he said. His next article focuses on understanding the impact of timing on people – 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 from University of California San Diego, and Vanessa A. Leone, University of Wisconsin-Madison.
