• Research

    In a first, researchers sequence single bacterial cells, paving path for rapid sepsis test

Mayo Clinic senior research fellow Yuguang Liu, Ph.D., looks into the microfluidic platform she developed for sequencing genomic contents of single bacterial cells.

For the first time, Mayo Clinic researchers are sequencing the genomic contents of single bacterial cells. The technique may pave the way for a potential lifesaving test for sepsis, a serious and sometimes deadly condition caused by the body's response to an infection. Rather than waiting for days to identify the source of a patient’s infection, the new test could provide an answer in hours and help pinpoint an effective therapy.

“When you’re dealing with bacteria, it only takes a few resistant cells to give a patient a bad outcome,” says Marina Walther-Antonio, Ph.D., associate consultant in the department of Surgery, and assistant professor in the Mayo Clinic Center for Individualized Medicine Microbiome Program, with a joint appointment in the department of Obstetrics and Gynecology.

"In principle, the research will enable the identification of pathogens within a few hours, buying precious time in what is often a life threatening battle," Dr. Walther-Antonio says.

Mayo Clinic researchers, Yuguang Liu, Ph.D. (left) and Marina Walther-Antonio Ph.D. (right), look at bacterial cells.

Rocketed to space

Mayo Clinic’s vast achievement of extracting DNA and RNA from single bacterial cells started as a study on the International Space Station as part of a large multidisciplinary team effort designated as BIOMEX (Biology and Mars Experiment), where three types of tiny microorganisms spent almost two years in orbit. Once back on Earth, Dr. Walther-Antonio, an astrobiologist who worked with NASA Astrobiology Institute during her training, set out to investigate whether the cells had mutated in order to survive in space, away from Earth’s protection. She believed the study would play a key role in understanding how to treat diseases in humans. 

The only thing missing was the tool needed to retrieve the genomic details that were locked tightly inside the cells. 

Dr. Walther-Antonio turned to Mayo Clinic senior research fellow Yuguang Liu, Ph.D., an electrical engineer.

“Dr. Liu is one of the only engineers in the world with this kind of expertise,” Dr. Walther-Antonio says. 

Dr. Liu, an expert in microfluidic platforms, recalls when she eagerly accepted the challenge.

“I knew it had never been done before, but I came here to identify problems that needed to be solved,” she says.

Yuguang Liu, Ph.D. connects a microfluidic platform to a machine to isolate and sequence single bacterial cells.

Bacterial cells, found in every habitat on Earth, are generally smaller than a pinhead, with a thick protective outer wall to enable survival in harsh environments, such as the human gut, bloodstream, soil and waters in extreme temperatures or under high radiation. Some bacterial cells help plants absorb nitrogen, others assist with human digestion. Many cause diseases. All can divide and multiply exponentially, with mutations occurring throughout the process. 

A unique tool

“Genomic sequencing has been done in human cells, but there is tremendous difficulty to do it in bacterial cells because they are very hard to break down without damaging the minute amount of DNA inside with methods compatible with downstream processing,” Dr. Liu explains.

Against great odds and in just months, Dr. Liu accomplished the unprecedented task by formulating a chemical-based “cocktail” to help break down the strong cell wall while keeping its fragile ingredients intact. She also made a special microfluidic platform — a credit card-sized piece of plastic with short, pin-like plastic spikes and raised lines that form a grid design for controlling and manipulating fluids. The chip contains nano-sized chambers for compartmentalizing single bacterial cells. 

“This tool can take the bacterial single cells and extract the DNA and RNA and amplify them and sequence them to see exactly what they are and what they are doing,” Dr. Liu explains, as she connects the chip to a machine with dozens of clear thin tubes that distribute gas pressure to operate the chip for isolating the cells and DNA/RNA amplification.

“We are now able to look at the genome to understand what drugs they are resistant to,” Dr. Liu explains.

Yuguang Liu, Ph.D., looks at single bacterial cells through a microscope.

Dr. Walther-Antonio says she was amazed with how quickly Dr. Liu accomplished the task.

“She came to me with the results and said, ‘I think it kind of worked,’” Dr. Walther-Antonio recalls. “And I said, 'Did you try it again?’ And she said, ‘Yes, 10 times.'”

Rapid sepsis diagnosis

Dr. Walther-Antonio says her team is now able to expand the technique to develop a real-time test for sepsis, which is often hard to diagnose and difficult to determine the most effective antibiotics to use on a patient. Without rapid treatment, sepsis can lead to septic shock, organ failure and death. In 2018, nearly 270,000 people in the U.S. died as a result of sepsis, according to the Centers for Disease Control. 

“The standard of care for sepsis currently involves culturing a patient’s blood sample and that always takes at least a couple of days,” she says. “In the meantime, you’re given a cocktail of antibiotics to try to save your life, and those who survive suffer lifelong side effects.”

Dr. Walther-Antonio envisions an automated process for identifying bacterial pathogens in sepsis within a few hours for time sensitive intervention, with an overall goal of saving lives.

Yuguang Liu holds up a microfluidic platform she designed for separating bacterial cells from human cells .

At the heart of the project, called “Answers in Hours,” is another microfluidic platform made by Dr. Liu — this one will separate human cells from bacterial cells.

“In a blood sample, there are very low amounts of bacteria,” Dr. Liu says. “Most are human cells, which overwhelmingly hide the bacterial cells. So in this platform, we have a measure to remove the human component so we are only detecting the bacteria.”

Dr. Walther-Antonio says knowing the genomic makeup of a tiny single bacterial cell opens the door to a world of discoveries, such as detecting the recurrence of pathogens early, and for basic science to understand what promotes the emergence of resistant strains.

She says research of patient sample testing is estimated to start in 2020, with plans to eventually incorporate the test into a clinical setting if success is reached.

The project was originally conceptualized by Heidi Nelson, M.D., and is also led by Nicholas Chia, Ph.D., Bernard and Edith Waterman co-director for the Mayo Clinic Center for Individualized Medicine Microbiome Program, and Robin Patel, M.D., chair of the Division of Clinical Microbiology and director of the Infectious Diseases Research Laboratory.

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