In a creative stroke inspired by Hollywood wizardry, scientists have designed a simple way to observe how bacteria move as they become impervious to drugs. The experiments are thought to provide the first large-scale glimpse of the maneuvers of bacteria as they encounter increasingly higher doses of antibiotics and adapt to survive—and thrive—in them.
“Alright Mr. Scientists, I’m ready for my close-up,” the microbe exclaimed! While not quite the film noir of Sunset Boulevard, or acting of Norma Desmond, investigators at Harvard University Medical School (HMS) and Technion-Israel Institute of Technology have managed to design a simple way for observing how bacteria move as they become impervious to drugs.
The new study—the findings of which were published today in Science in an article entitled “Spatiotemporal Microbial Evolution on Antibiotic Landscapes”—provides the first large-scale preview of bacterial maneuvers as they encounter increasingly higher doses of antibiotics and adapt to survive, and thrive, in them. To achieve this microbial cinéma vérité, the researchers first constructed a 2-by-4-foot petri dish and filled it with 14 liters of agar.
To observe how bacteria—Escherichia coli in this instance—adapt to increasingly higher doses of antibiotics, the research team divided the dish into sections and saturated them with various doses of antibiotics. The outermost rims of the dish were free of any drug. The next section contained a small amount of antibiotic—just above the minimum needed to kill the bacteria—and each subsequent section represented a 10-fold increase in dose, with the center of the dish containing 1000 times as much antibiotic as the area with the lowest dose.
Over 2 weeks, a camera mounted on the ceiling above the dish took periodic snapshots that the researchers spliced into a time-lapse montage. The investigators were able to witness a powerful, unvarnished visualization of bacterial movement, death, and survival—evolution at work, visible to the naked eye.
The device, which the team dubbed the Microbial Evolution and Growth Arena (MEGA) plate, represents a simple, and more realistic, platform to explore the interplay between space and evolutionary challenges that force organisms to change and adapt or die.
“We know quite a bit about the internal defense mechanisms bacteria use to evade antibiotics, but we don't really know much about their physical movements across space as they adapt to survive in different environments,” explained lead study author Michael Baym, Ph.D., a research fellow in systems biology at HMS.
The inspiration for this study was a digital billboard advertisement for the 2011 film “Contagion,” a grim narrative about a deadly viral pandemic.
“This project was fun and joyful throughout,” noted senior study investigator Roy Kishony, Ph.D., visiting professor of systems biology at HMS and professor of biology at Technion. “Seeing the bacteria spread for the first time was a thrill. Our MEGA plate takes complex, often obscure, concepts in evolution, such as mutation selection, lineages, parallel evolution, and clonal interference, and provides a visual seeing-is-believing demonstration of these otherwise vague ideas. It's also a powerful illustration of how easy it is for bacteria to become resistant to antibiotics.”
Co-author Tami Lieberman, Ph.D., a postdoctoral associate at MIT and former graduate student in Dr. Kishony’s lab remarked that the images the team generated sparked the curiosity of lay and professional viewers alike. She added that “this is a stunning demonstration of how quickly microbes evolve. When shown the video, evolutionary biologists immediately recognize concepts they've thought about in the abstract, while nonspecialists immediately begin to ask really good questions.”
Beyond providing a telegenic way to show evolution, the device yielded some key insights about the behavior of bacteria exposed to increasing doses of a drug. For example, the investigators noted that at each concentration level, a small group of bacteria adapted and survived. Resistance occurred through the successive accumulation of genetic changes. As drug-resistant mutants arose, their descendants migrated to areas of higher antibiotic concentration. Multiple lineages of mutants competed for the same space. The winning strains progressed to the area with the higher drug dose, until they reached a drug concentration at which they could not survive.
Additionally, the scientists observed a dramatic demonstration of acquired drug resistance where bacteria spread to the highest drug concentration. In the span of 10 days, bacteria produced mutant strains capable of surviving a dose of the antibiotic trimethoprim 1000 times greater than the one that killed their progenitors. When researchers used another antibiotic—ciprofloxacin—bacteria developed 100,000-fold resistance to the initial dose.
Interestingly, the fittest, most resistant mutants were not always the fastest. They sometimes stayed behind weaker strains that braved the frontlines of higher antibiotic doses. The classic assumption has been that mutants that survive the highest concentration are the most resistant, but the team's observations suggest otherwise.
“What we saw suggests that evolution is not always led by the most resistant mutants,” Dr. Baym remarked. “Sometimes it favors the first to get there. The strongest mutants are, in fact, often moving behind more vulnerable strains. Who gets there first may be predicated on proximity rather than mutation strength.”