How do intestinal bacteria evolve antibiotic resistance? Inquiring minds want to know, and an American-Israeli team from the Technion-Israel Institute of Technology and Harvard inspired by a horror flick built a giant Petri dish to investigate that very thing. And while about it, they discovered that bacteria can evolve powerful drug resistance a lot faster than had been thought.
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The biochemistry of bacterial drug resistance is clear. But how bacteria behave had been less clear.
Normally bacteria are cultivated in normal dishes that would fit on the palm of your hand, unless your hands are tiny Trumpian mitts. The Technion and Harvard people created a vast two-by-four-foot Petri dish. A normal dish takes maybe 50 to 100 milliliters of agar. This tub contained with 14 liters of the seaweed-derived jelly. In short, it was huge.
No, it didn't house commensurately gigantic germs. The scientists set out to observe how normally sized intestinal bacteria, Escherichia coli, adapt to increasingly higher doses of antibiotic.
To achieve this, the scientists divided the dish into sections, in which the agar was saturated with increasingly higher doses of antibiotic. The agar around the rim was drug-free. The closer to the center, the more concentrated the antibiotic became. The center of the bacterial mansion had 1,000 times as much antibiotic as the area with the lowest dose.
After the drug-free rim, the next section contained a small amount of antibiotic and each subsequent section represented a 10-fold increase in dose.
Then for two weeks, a camera above the dish took shots from which the researchers created a time-lapsed montage. They discovered that multiple resistant lineages developed and coexisted, though there were states of competition. And they discovered that evolution in motion is much faster than thought, as described in their seminal Science magazine paper, "Spatiotemporal microbial evolution on antibiotic landscapes".
Evolution on steroids
The concept of the giant Petri dish, which the team dubbed MEGA (Microbial Evolution and Growth Arena), was born out of the acknowledgement that millennials get bored very easily. Their teachers thought it might help to teach evolution in a "visually captivating way," they explain. Enter Hollywood and the 2011 flick “Contagion," which moved scientist Roy Kishony in an unexpected way.
The movie, which was about a deadly viral pandemic – there are a few of those – was being marketed using a giant lab dish in which glowing germs crawl to spell out the title of the movie.
In real life, the Harvardians and Technionites were not surprised to find that the bacteria proliferated until reaching the area with enough antibiotics to kill them. Or, really, most of them. Again unsurprisingly, at any concentration of antibiotic, somebody would survive, thanks to mutation.
Fascinatingly, though, as these drug-resistant mutant lines arise – their descendants spread to areas of higher antibiotic concentration. Moreover, these descendent lines of resistant bacteria would "fight" over space in the dish – and as they progressed sequentially through increasingly higher doses of antibiotic, low-resistance mutants gave rise to moderately resistant mutants, which eventually spawned highly resistant strains able to fend off the highest doses of antibiotic.
And there you have it.
"Ultimately, in a dramatic demonstration of acquired drug resistance, bacteria spread to the highest drug concentration," the team stated.
In just 10 days, the E. coli birthed mutants that could survive a dose of the antibiotic trimethoprim 1,000 times higher than the one that killed their progenitors. When researchers wielded yet another antibiotic—ciprofloxacin—bacteria developed 100,000-fold resistance to the initial dose.
Another observation was that the fittest, most resistant mutants were not always the fastest. "The fittest mutants stayed behind weaker strains that braved the frontlines of higher antibiotic doses," the team stated.
“What we saw suggests that evolution is not always led by the most resistant mutants,” said study first author Michael Baym. “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." Co-investigators included Eric Kelsic, Remy Chait, Rotem Gross and Idan Yelin.