Researchers have developed a large plate on which to film bacteria as they mutate in the presence of higher and higher concentrations of antibiotics, providing unprecedented insights into the phenomenon of antibiotic resistance.
"Our device allows us to systematically map the different ways by which bacteria can become resistant to a range of antibiotics and antibiotic combinations," said co-author Roy Kishony, a professor in the department of systems biology at Harvard Medical School and a principal investigator at Technion-Israel Institute of Technology.
The ultimate goal, Kishony added, is to develop tools "that can predict the evolution of pathogens under different treatments, and better guide treatment choice."
"With our plate device, the evolutionary paths that the bacteria follow to achieve antibiotic resistance appear clearly and visually," said co-author Michael Baym, a postdoctoral fellow in the Kishony Lab at Harvard Medical School, "and will hopefully let us start tailoring our approaches to treating resistance to different evolutionary modes."
The 2-by-4 foot petri dish used by the researchers to grow the bacteria contains nine bands at its base that can support varying concentrations of antibiotic. The results are reported in the 9 September issue of Science.
Antibiotics have been used to treat patients since the 1940s, greatly reducing illness and death. However, these drugs have been used so frequently that the bacteria they are designed to kill have adapted to them in many cases, making the drugs less effective. At least 2 million people become infected with bacteria resistant to antibiotics each year in the United States, according to the Centers for Disease Control and Prevention, and at least 23,000 of these die as a result.
"We know quite a bit about the internal defense mechanisms bacteria use to evade antibiotics," Baym said, "but we don't really know much about their physical movements across space as they adapt to survive in different environments."
To better understand how antibiotic resistance evolves in space and time, Baym and his colleagues developed a device called the microbial evolution growth arena plate, or MEGA-plate. The researchers used the antibiotics trimethoprim and ciprofloxacin in the MEGA plate in concentrations from zero to 10,000 times the original dose.
On the right side of the plate where antibiotic levels were zero, Baym, Kishony, and colleagues grew Escherichia coli bacteria, which appeared white on the inky black background. Over two weeks, a camera mounted on the ceiling above the plate took periodic snapshots of the bacteria mutating.
In the band with no antibiotic, the bacteria spread up until the point where they could no longer survive as they mingled with the first traces of antibiotic. Then, a small group of bacteria developed genetic mutations that allowed them to persist.
Researchers traced the branching patterns of bacterial evolution on the MEGA plate. | Katharine Sutliff/ Science
As these drug-resistant mutants arose, their descendants migrated to areas of higher and higher antibiotic concentration, developing further mutations to compete with other mutants around them. As they continued their journey to the highest antibiotic concentration level, all remaining bacterial mutants had to evolve further still.
Through this process of cumulative, successive mutations, the researchers could visualize how bacteria that are normally sensitive to antibiotics can evolve resistance to extremely high concentrations — those up to 100,000-fold higher than the one that killed their predecessors — in just over ten days.
The bacteria were unable to adapt directly from zero antibiotic to the highest concentrations, for both drugs tested, revealing that exposure to intermediate concentrations of antibiotics is essential for the bacteria to evolve resistance.
Initial mutations at each new band on the plate led to slower growth, hinting that bacteria adjusting to the antibiotic aren't able to grow at ideal speed while developing mutations. Once fully resistant, however, such bacteria regained normal growth rates.
"One of our main objectives in the lab is to reveal such evolutionary tradeoffs," said Kishony, "whereby a bacterium becoming resistant to a drug confers a cost we might be able to exploit. We might potentially use other drugs to enhance such resistance-associated weakness."
Intriguingly, the researchers also found that the location of bacterial species played a role in their success in developing resistance. For example, when the researchers moved the trapped mutants — those behind their fast-moving, fit counterparts — to the "frontlines" of the growing bacteria, they were able to grow into new regions where the frontline bacteria could not.
"What we saw suggests that evolution is not always led by the most resistant mutants," said Baym. "The strongest mutants are, in fact, often moving behind more vulnerable strains."
This overturns the assumption that mutants that survive the highest concentration of a drug drive the fitness of bacterial populations; rather, it is those mutants that are both sufficiently fit and arise sufficiently close to the advancing front that lead the evolutionary road.
The work of Baym, Kishony, and colleagues was inspired by Hollywood wizardry, the authors say. Kishony saw a digital billboard advertising the 2011 film Contagion, a grim narrative about a deadly viral pandemic. The marketing tool was built using a giant lab dish to show hordes of painted, glowing microbes creeping slowly across a dark backdrop to spell out the title of the movie.
"This project was fun and joyful throughout," Kishony said. "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."