Busted: Most-Wanted Microbe Captured by Microfluidic Cultivation

Eugene B. Chang, MD, the Martin Boyer Professor of Medicine at the University of Chicago (Photo: Dan Dry)

Eugene B. Chang, MD, the Martin Boyer Professor of Medicine at the University of Chicago (Photo: Dan Dry)

A team led by former University of Chicago chemist Rustem Ismagilov, now at the California Institute of Technology, and three UChicago microbiologists has corralled Cecococcus microfluidicus 1, a highly sought after bacterial strain found lurking in the colon of a University of Chicago laboratory technician.

This previously unidentified organism may be of “high biomedical importance,” according to the team that brought it in alive. Microfluidicus 1 is, “to our knowledge, the first successful example of a targeted cultivation of a microorganism from the high-priority group of the Human Microbiome Project’s ‘Most Wanted’ list.” It may also be, they note in the Proceedings of the National Academy of Sciences, the “first cultured representative of a previously unidentified genus of the Ruminococcaceae family.”

ScienceLife spoke with microbiome specialist and study co-author Eugene B. Chang, MD, the Martin Boyer Professor of Medicine at the University of Chicago, about how the team isolated and cultivated this elusive microbe.

SL: Why is there a Most-Wanted list?

Chang: This was a goal of the NIH’s Human Microbiome Project—to better understand the microbes that live in the GI tract. We are most interested, of course, in those bacteria that we know least about, which often turns out to be those that are difficult to cultivate. We have a list, a very long list, of key organisms we want to study. As we identify new strains of microbes, we can build a library of their DNA that would serve as reference sources for investigators who want to explore the genetic as well as the functional basis of the gut microbiome.

How did Cecococcus microfluidicus 1 get on that list?

Microbes get on the list because they are somehow different, unknown, or unfamiliar. Being on the most-wanted list does not connote criminal or disease-causing history of bad behavior, just being poorly understood. This microbe came from a healthy human subject, part of a study that we did to characterize microbes from healthy adults, but it was not an organism we, or anyone else, had ever grown in the lab.

How do you decide which specific bacteria to go after?

There are trillions of organisms in the gut. We are trying to pick out specific strains that we believe are there, based on seeing some parts of their genome in a general survey of all microbial genes present in a sample. The research team chose a unique strain that had not been previously characterized. We used a known genetic snippet as an ‘address label’ to probe thousands of strains and identify the one that fit. We were able to identify that organism and then—and this is the hard part—grow enough of it to sequence its genome.

How many strains of bacteria are found in the human bowel?

Talking about strains could lead us to a huge number. At the species level, we believe the number of different bacterial types represented in the GI tract is on the order of several hundreds. The bacteria that we discovered was named Cecococcus microfluidicus 1.

Where did that name come from?

It tells the story of where and how we found it. So Cecocossus, the genus, comes from cecum, the part of the bowel where this bacterium lives, plus coccus, which tells you its shape. Cocci are spherical, from the Greek word for berry. The interesting part, though, is the species name, microfluidicus. This essentially describes the technique, invented by Rustem Ismagilov, now a professor of chemistry and chemical engineering at Caltech. His approach enabled the team to isolate and grow this organism, a novel new strain. Hence, the “1.”

So, what is microfluidics?

This is a new way to scale the entire process to optimize conditions for growth for a single bacterium. Ordinarily, you begin with a microbe and grow it in a large liquid-filled flask. That approach doesn’t work for many bacteria because the large liquid volume dilutes out critical growth factors and molecular signals often made by the bacteria themselves to regulate the growth and eventual community behavior. Without the proper concentration of these factors, many bacteria cannot grow and will die.

Microfluidics is a way to grow an entire culture from a single organism in a micro-droplet of fluid. That reduces the volume such that it can produce concentrations of growth factors that allow it to multiply. They eventually grow enough pure culture for our CalTech colleagues to sequence and identify the organism.

How does this work?

Here’s how the Caltech team described it: To grow these elusive microbes … “We turned to SlipChip, a microfluidic device previously developed in Ismagilov’s lab. SlipChip is made up of two glass slides, each the size of a credit card, that have tiny etched grooves which become channels when the grooved surfaces are stacked atop one another. When a sample—say, a jumbled-up assortment of bacteria species collected from a colonoscopy biopsy—is added to the interconnected channels of the SlipChip, a single “slip” of the top chip will turn the channels into individual wells, with each well ideally holding a single microbe. Once sequestered in an isolated well, each individual bacterium can divide and grow without having to compete for resources with other types of faster-growing microbes.”

How big a deal is that?

I have to say, Rustem’s approach to this problem is very clever. This technique is going to be enormously useful for most of us studying microbiomes. This was a major challenge, a bottleneck in the first phase of the project. Now we have an experimental approach that can rapidly identify and cultivate rare or novel microbes. We can build libraries of information about the creatures that live within us.

Does Cecococcus microfluidicus 1 cause a disease?

Not that we know of. We already know some of its functions based on what we learned from the sequence information. It’s not a bad player. It belongs to a family group of microbes called Ruminococcaceae.

Are there other Most Wanted bacteria in the microfluidics pipeline?

About 100 new reference strains have already been identified and characterized, using this approach. It’s a stepping stone, a tool we can use to learn more about our microbes.

So, what are the hot topics in microbiome research these days?

There’s a great deal going on. This field has exploded. But here are the three that most interest me.

  • First: We want to learn how our gut microbiome helps regulate our metabolism, impacting our notions of hunger and satiety. We believe changes in the gut microbiome, brought on by Westernization, play an important role in the development of obesity and type-2 diabetes.
  • Second: We are just beginning to learn about other types of microbes that live within us. Many bacteria harbor microbial passengers, called bacteriophages, which are a type of virus they acquire. Recent studies suggest these can impart functional properties to the bacteria. This increases the functional heterogeneity of our gut microbes.
  • Third, there is growing interest in how the gut microbiome affects tissues outside of the GI tract, for example the brain. Several studies suggest that gut microbes play a role in the development of autism, and various behavioral disorders. This is new territory. We only have descriptions and correlations right now. We need to know what these microbes are doing and how they are impacting the central nervous system.

There was a CSI episode in which they identified the bad guys by swabbing keyboards tied to the crime and then sequencing the bacteria from the suspect’s fingertips. There is a recent, real-life study, done in mice, that enables researchers to back-calculate the time of death from changes in the gut microbiome. Is there a forensic application for the flora of the human bowel?

“The Case of the Flatulent Felon?” Let’s not go there.

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Ma L., R. Hatzenpichler, M. A. Karymov, N. Hubert, I. M. Hanan, E. B. Chang & R. F. Ismagilov (2014). Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project’s Most Wanted taxa, Proceedings of the National Academy of Sciences, DOI: http://dx.doi.org/10.1073/pnas.1404753111

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