Seeing microbes as individuals
By Patti Koning
Microbes, the most abundant life form on earth, live everywhere — in air and water, in extreme heat and freezing cold, in soil and rocks, and inside our bodies, where they outnumber human cells by a factor of 10:1. Despite this ubiquity, we know very little about microbes because the vast majority, 90 percent to 99 percent, cannot be cultured and characterized using traditional laboratory techniques.
STUDYING MICROBES ONE AT A TIME µFlowFISH, shown here with researchers Peng Liu and Robert Meagher, is an integrated microfluidics device that combines fluorescence in situ hybridization (FISH) and flow cytometry, enabling single-cell analysis. The device, currently being used to analyze samples from the Hanford Superfund site, could become a powerful tool in understanding bacteria from the human microbiome. (Photo by Randy Wong)
One solution to this problem is to take culturing in the laboratory, in the traditional sense, out of the picture.
“Our idea is to develop culture-independent techniques for the microbial communities that are hard to access using existing technologies, whether it’s because the sample is too small or the environment is too complex to replicate in a lab,” says Anup Singh (8621).
One of the harshest environments is at the Hanford Superfund Site in southeastern Washington state, where cleanup, described as one of the largest and most complex projects in the country, has been ongoing since plutonium production ceased in 1987. A multitude of cleanup strategies are being employed at Hanford, including bioremediation.
“There is evidence to suggest that naturally occurring bacteria in harsh environments like Hanford may be cleaning up some of the contamination,” says Robert Meagher (8621). “If we could somehow harness and leverage the bacteria’s capability to assist in cleanup, that would be huge. But first, you have to understand the bacteria, what they are doing, and what we can do to encourage that natural process.”
There has been a lot of research over the years using traditional microbiology techniques, including culture-independent techniques like PCR and micro arrays, to understand the dynamics of that microbial community. While that work has yielded some good information, Robert says a complementary approach is needed, one that looks at the bacteria on a single-cell basis.
“When you work with a large sample from a place like Hanford, you have radionuclides from the uranium that was processed there and many different heavy metals,” Anup says. “On a broad level, you can see what is there and what is happening, but you can’t connect any particular function back to specific bacteria. This shotgun approach can give you a lot of information quickly, but not much detail.”
Over the past year, the Sandia team adapted fluorescence in situ hybridization and flow cytometry onto an integrated microfluidics device, called µFlowFISH, that can analyze small samples, one cell at a time. “With our device, once you insert the cell sample, the whole process is contained, so the chance of introducing
contamination is very low,” says Peng Liu (8621). “It’s also very efficient because all of the operations are automated and can work with such small sample volumes.”
The team analyzed two samples of less than 100 microliters each, taken from the Hanford site at different times of the year (October and February), and looked for changes in population of Pseudomonas, which is believed to play an important role in the microbial community. As a proof-of-concept, they compared those results with analysis of the same samples performed by traditional benchtop methods and found them to be in excellent agreement. These results were published in a paper titled “Microfluidic fluorescence in situ hybridization and flow cytometry (µFlowFISH)” that appeared in Lab on a Chip in August.
While the paper focuses on the ability to analyze very small samples, the ability to study cells one at a time has other advantages. “With population-level measurements, subtle differences between bacteria can get averaged out,” says Anup. “Bacteria can react differently to the same thing, just like people. There is a lot of value in determining the common mechanisms that bacteria use to cope with a common stressor and the things that change from cell to cell. The only way to get this information is to look at each cell individually.”
In addition to environmental samples, the researchers are also analyzing microbes found in the human body.
“In recent years, there is a growing awareness that microbes within our body do much more than was previously thought,” says Robert. “For a long time we only studied the bacteria that made us sick, but the vast majority of microbes in our bodies are not pathogens. They are supposed to be there, and some of them are helpful to us, but we’re just beginning to understand how.”
Just like microbes from harsh environments, many of these microbes are difficult to culture and analyze. The Sandia team is collaborating with the New York University College of Dentistry on an oral cancer study. Evidence suggests that bacteria normally present in the mouth may have an indirect role in the onset or progression of the disease.
The link is not as simple as the presence of a particular bacterium causing oral cancer. Rather, it’s likely a very complex chain of events.
“For example, some small change in the mouth allows a certain type of bacteria to colonize, which changes the environment of the mouth, and causes the body to respond in a particular way,” explains Robert.
Conventional microbiology methods focus on the most abundant bacteria found in a sample. “If there are 15 bacteria, for example, current techniques might allow you to look at only five that make up 99 percent of the population,” says Anup. “The other 1 percent are just noise in your experiment. But if you look at those five and cannot correlate them with what is happening, it begs the question, who are those other 10 bacteria and what are they doing? This is where we come in.”
The device could be a powerful tool in fighting foodborne illnesses. It took health officials in Germany more than a month to characterize the source of the recent E. coli outbreak, which killed at least 50 people.
“They were able to culture the bacteria, which was very lucky,” Anup says. “But if you can’t culture the bacteria — which could happen — then you could be treating people in ways that cause more harm than good.”
Anup says he expects that the team will complete a device that can take a single cell all the way to sequencing within the next two years.