We believe what we see … so how do unseen ideas become believable?
Antibiotic resistance could have a drastic impact on all of our lives, but is invisible to the naked eye and impossible for most to comprehend. So researchers at Harvard Medical School and the Technion-Israel Institute of Technology decided to make it obvious: They designed a model to show bacteria mutating to overcome drugs meant to stop and destroy them.
Along the way, their experiments, which have been described and videotaped for the journal Science, revealed something even more profound. Not only does the model vividly illustrate how antibiotic resistance happens, it also demonstrates “survival of the fittest” and other Darwinian concepts that have been often discussed but never once seen.
“When I saw those videos it kinda hit me viscerally — I’m watching evolution, I don’t have to think about it, there it is, I can see it,” said Sam P. Brown, an evolutionary biologist at Georgia Institute of Technology, who was not involved in the experiments. He and Luke McNally of the University of Edinburgh, co-wrote a commentary published with the research.
To observe the do-or-die encounter between bacteria and an antibiotic drug, the research team constructed a two-foot by four-foot petri dish — dubbed the Microbial Evolution and Growth Arena or MEGA plate — and filled it with agar, a jellylike nourishment used in labs to feed growing organisms. Next, they searched among bacteria for the right one to work with and landed on E. coli.
“In order to grow bacteria on a petri dish of that size, it needs to be able to swim, which is something E. coli can do but many other model organisms cannot,” said Dr. Michael Baym, first author of the study and a postdoctoral fellow in microbial evolution at Harvard Medical School. E. coli also possesses fundamental mechanisms in common with infection-causing bacteria, explained Baym, and quite simply, “we knew how to work with it.”
Next, the researchers divided the MEGA plate into sections and added increasing doses of an antibiotic, trimethoprim. This antibiotic was chosen because it is well-known, explained Baym. “We have a lot of experience understanding how resistance of trimethoprim evolves,” said Baym. “We were developing a new system to study evolution and so we wanted to work with as many parts that we sort of understood as possible.”
Preparing the MEGA plate for their experiments, the research team left the outermost area clean of trimethoprim. In the area nearest this outermost section, they added a single dose of the drug and then, as the sections progressed to the center, the dosage kept increasing until it reached 1,000 times the initial dose.
Mutations ‘right in front of you’
Over two weeks, a ceiling-mounted camera snapped periodic shots of what transpired in the MEGA plate below. Later, the researchers spliced these shots into a time-lapsed videotape that showed how most of the bacteria spread until they reached the antibiotic dose that was too strong for them to continue growing or living.
However, at each dosage level, a small group of bacteria were able to survive. Bacteria, like other living beings, evolve to adapt to changes in their environment and one way they do this is through genetics. New genes can arise through mutations and these then get passed down to subsequent generations.
The MEGA plate revealed all this and more: Descendants of the drug-resistant mutants instinctively migrated to new territories, the areas of fresh agar nourishment and also higher antibiotic concentration. Once there, multiple lineages of mutants competed for dominance within the same space. And so, over a span of just days, the wily E. coli strains made their way from the good life of easy nourishment in a drug-free outer layer through sections of the MEGA plate containing increasingly higher doses of antibiotic.
The early low-resistance mutants soon gave rise to moderately resistant mutants and ultimately these intermediate bacteria spawned highly resistant strains able to overcome a dose of trimethoprim 1,000 times more intense than the one that killed their ancestors.
“You can just see mutations and selections and trade-offs happening right in front of you,” said Baym.
Along with the usual package of genes inherited from their forebears, bacteria also contain plasmids, small rings of additional DNA. Scientists have known that plasmids enable wider and faster spread of resistance among bacteria. Within the MEGA plate, the bacteria strains traded plasmids containing genes essential to survival.
Branches of a tree
The team tried another antibiotic in their giant petri dish: ciprofloxacin. As Baym explained, the team believed cipro’s path to resistance would be different from that of trimethoprim.
“Trimethoprim is bacteria static — it stops bacteria but it does not kill them, whereas cipro actually directly kills bacteria,” said Baym. Would this fundamental difference between the two drugs change the view?
In this second experiment, the E. coli mutated to develop 100,000-fold resistance to an initial dose of cipro and, as the researchers anticipated, the evolutionary process “looked quite different” from that of trimethoprim, according to Baym. Still, the branches of a tree could be traced along the ever-more-resistant bacteria.
In both cases, though, location of a particular bacterial strain determined its success — or failure — in developing resistance. When the researchers moved mutants trapped behind their parents to the so-called “frontlines” of the culture, they were able to grow into new regions where the parents could not. Survival may not be driven by the fittest mutants, suggest Baym and his colleagues; what matters most for survival is a combination of sufficient fitness and sufficient closeness to the advancing front.
Visualizing the branches of evolution is all well and good, said Professor Roy Kishony of Harvard Medical School and the Technion-Israel Institute of Technology. Still, one of the main objectives of these experiments was “to identify evolutionary tradeoffs whereby becoming resistant to one drug confers a cost to the bacteria that we might be able to exploit,” he said.
Future MEGA plate studies may help reveal some resistance-associated weakness of bacteria, which scientists can exploit for practical purposes such as treating patients, explained Kishony, the senior author of the paper.
Practical application of a unique model
“A lot of modern medicine depends on antibiotics that kill bacterial pathogens,” said Georgia Tech’s Brown, and “a failure to treat infections due to the evolution of resistance in bacteria…is a huge and growing problem.”
Without effective antibiotics, common infections, including everything from strep throat and tuberculosis to sexually transmitted diseases, could become extremely risky, while surgeries would be near impossible.
“The value of this work is not a clinical model — this doesn’t look like a patient’s leg,” said Brown. In fact, the researchers themselves do not claim their MEGA plate is a mirror of how bacteria thrive in the world. Still, Brown noted, this “technically difficult” experiment offers a more closely related view of real environments than any previous studies, which relied on flasks and beakers filled with bacteria or even more sophisticated, though very small scale, microfluidic devices.
Evolutionary biologists always think of the tree as a sort of metaphor, said Brown — a Darwinian organizational chart on which they place organisms. By creating the MEGA plate, Kishony and his team rendered this concept real, Brown said: “It’s a beautiful visualization of the evolutionary process.”