YOUTUBE, HARVARD MEDICAL SCHOOL
There are small petri dishes, there are large petri dishes, and then there is MEGA, an enormous, 2-foot-by-4-foot slab of black agar infused with a gradient of antibacterial drugs. Researchers built this behemoth to watch evolution in both time and space—and as they report today (September 8) in Science, MEGA has revealed that the fittest antibiotic-resistant bacteria may not necessarily grow the fastest.
On the oversized plate, “you can see evolutionary branching as it happens,” said Luke McNally, an evolutionary microbiologist at the University of Edinburgh, who co-authored an accompanying editorial. “It’s amazingly, strikingly beautiful.”
Researchers traditionally study bacterial evolution in liquid culture, which forces the bacteria to compete with the flask’s entire population for resources. By contrast, the new microbial evolution and growth arena (MEGA) plate separates bacteria both spatially and temporally, thereby reducing competition, said study coauthor Michael Baym of Harvard Medical School (HMS). The set-up shows the oft-overlooked importance of the physical space surrounding bacteria, said Viktória Lázár, a postdoc who studies evolutionary biology at the Biological Research Centre of Hungary, who was not part of the study.
Additionally, the plate can host a bacterial population much larger than a typical liquid experiment can, making it likelier to see rare mutants, said study coauthor Roy Kishony, who leads an antibiotic-resistance research group at HMS and Technion–Israel Institute of Technology. “It really allows us to see, with our own eyes, the dynamics of evolution,” he added.
Kishony and study coauthor Tami Lieberman, now a postdoc at MIT, wanted to create a vivid demonstration of evolution for their students. Together with Baym and other collaborators, they built the MEGA plate from scratch. They filled the giant acrylic dish with two layers of agar—a solid base, made up of discrete stripes laced with either trimethoprim or ciprofloxacin that increased in concentration toward the center of the plate, and a top coat of viscous “swim agar” to allow bacterial movement. The lower agar layer was mixed with India ink to provide contrast with the white bacteria seeded at the antibiotic-free ends of the dish.
For 10 days, the researchers imaged the E. coli every 10 minutes as the microbes expanded across the plate, and saw that the bacteria paused briefly at the boundaries of increasingly stringent antibiotic concentrations until a mutant struck out into the higher-drug territory. By challenging the bacteria with differing doses of antibiotic in the first step of the gradient, the team demonstrated that E. coli evolve higher resistance more quickly if they first encounter an intermediate, rather than a high, concentration of antibiotic.
Using the easy-to-see evolutionary trajectory of the bacteria as a guide, the researchers isolated and sequenced the charge-leading mutants. They found adaptive mutations in the gene for the proofreading enzyme DNA polymerase III, the target genes of the antibiotics, and in unexpected genes such as those coding for a phosphate transporter and a kinase that don’t have a known function in establishing resistance, hinting at alternative pathways that could arise.
The scientists were also intrigued to find that many bacteria behind those at the frontier—those that became resistant to antibiotics, but grew more slowly as a result—acquired mutations that further boosted both growth and antibiotic resistance later on. In fact, in a head-to-head race with the bacteria that originally outstripped them, these slow-to-grow bacteria were much more successful by the end of the experiment. Previously, it was commonly thought that regaining growth might require giving up newly acquired resistance, but these mutants suggested that wasn’t the case. “The way to overcome an evolutionary tradeoff is not always to revert back to what you were,” Baym said. “You can get growth back in more ways than just losing resistance.”
McNally said he is excited about MEGA’s potential to investigate new angles of the pressing societal problem of antibiotic resistance, such as the interactions among multiple drugs or multiple bacterial species. Yet, Julian Davies, who studies antibiotics at the University of British Columbia, is not convinced that synthetic trimethoprim and ciproflaxacin and an artificial environment are relevant to how antibiotic resistance develops in soil or the human gut. “It’s a nice paper,” he said, but “it would be really useful if you could duplicate this in the stomach system.”
Whatever the MEGA plate may yet reveal about bacterial evolution, Baym said he believes the platform will fulfill its original purpose as an educational tool. Lázár’s colleague Réka Spohn, a graduate student, agreed: “It’s a really amazing and easy way to show evolution in action to everyone,” she said, and to make abstract concepts such as evolution and mutation concrete.
M. Baym et al., “Spatiotemporal microbial evolution on antibiotic landscapes,” Science, doi:10.1126/science.aag0822, 2016.