A diagnostic tool that’s about the size of a credit card has identified a highly prized gut microbe.
The microbe contains interesting genetic sequences, but it has proven challenging to culture in the lab.
Researchers used the device, called SlipChip, to isolate microbes from a patient’s gut bacteria and then genetically targeted this specific bacterial species using tiny channels etched on the device.
They then grew a pure culture of this single organism in the lab.
An early guess is that this particular microbe may be linked to obesity and fatty liver disease, and could ultimately aid in finding a potential probiotic therapy.
Grow the needle without the hay
Although a few bacterial species are easy to grow in the laboratory, needing only a warm environment and plenty of food to multiply, most species that grow in and on the human body have never been successfully grown in lab conditions. It’s difficult to recreate the complexity of the microbiome—the entire human microbial community—in one small plate (a lidded dish with nutrients used to grow microbes), says Rustem Ismagilov, a professor of chemistry and chemical engineering at the California Institute of Technology (Caltech).
There are thousands of species of microbes in one sample from the human gut, Ismagilov says, “but when you grow them all together in the lab, the faster—growing bacteria will take over the plate and the slow-growing ones don’t have a chance—leading to very little diversity in the grown sample.”
Finding slow-growing microbes of interest is like finding a needle in a haystack, he says, but his group wanted to work out a way to “just grow the needle without growing the hay.”
To do this, Liang Ma, a postdoctoral scholar in Ismagilov’s lab, developed a way to isolate and cultivate individual bacterial species of interest. He and his colleagues began by looking for bacterial species that contained a set of specific genetic sequences. The targeted gene sequences belong to organisms on the list of “Most Wanted” microbes—a list developed by the National Institutes of Health (NIH) Human Microbiome Project. The microbes carrying these genetic sequences are found abundantly in and on the human body, but have been difficult to grow in the lab.
To grow these elusive microbes, the Caltech researchers turned to SlipChip, which is made up of two glass slides 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.
The researchers then needed to determine which compartment of the SlipChip contained a colony of the target bacterium—which is not a simple task, says Ismagilov.
“It’s a Catch-22—you have to kill the organism in order to find its DNA sequence and figure out what it is, but you want a live organism at the end of the day, so that you can grow and study this new microbe,” he says. “Liang solves this in a really clever way; he grows a compartment full of his target microbe in the SlipChip, then he splits the compartment in half. One half contains the live organism and the other half is sacrificed for its DNA to confirm that the sequence is that of the target microbe.”
The method of creating two halves in each well in the SlipChip will be outlined in papers slated to be published in an upcoming issue of the journal Integrative Biology.
‘Most Wanted’ list
To validate the new methodology, the researchers isolated one specific bacterium from the Human Microbiome Project’s “Most Wanted” list. The investigators used the SlipChip to grow this bacterium in a tiny volume of the washing fluid that was used to collect the gut bacteria sample from a volunteer.
Since bacteria often depend on nutrients and signals from the extracellular environment to support growth, the substances from this fluid were used to recreate this environment within the tiny SlipChip compartment—a key to successfully growing the difficult organism in the lab.
After growing a pure culture of the previously unidentified bacterium, Ismagilov and his colleagues obtained enough genetic material to sequence a high-quality draft genome of the organism. Although a genomic sequence of the new organism is a useful tool, further studies are needed to learn how this species of microbe is involved in human health, Ismagilov says.
In the future, the new SlipChip technique may be used to isolate additional previously uncultured microbes, allowing researchers to focus their efforts on important targets, such as those that may be relevant to energy applications and the production of probiotics. The technique, says Ismagilov, allows researchers to target specific microbes in a way that was not previously possible.
Additional researchers from Caltech and the University of Chicago collaborated on the project, which was funded by NIH’s National Human Genome Research Institute.
《Individually addressable arrays of replica microbial cultures enabled by splitting SlipChips》, Published on Journal 《Integrative Biologys》in June, 2014.