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.
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…
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.
Courtesy of a colleague
Baym M, Lieberman TD, Kelsic ED, Chait R, Gross R, Yelin I, Kishony R.
Spatiotemporal microbial evolution on antibiotic landscapes. Science. 2016 Sep
A key aspect of bacterial survival is the ability to evolve while migrating across spatially varying environmental challenges. Laboratory experiments, however, often study evolution in well-mixed systems. Here, we introduce an experimental device, the microbial evolution and growth arena (MEGA)-plate, in which bacteria spread and evolved on a large antibiotic landscape (120 × 60 centimeters) that allowed visual observation of mutation and selection in a migrating bacterial front. While resistance increased consistently, multiple coexisting lineages diversified both phenotypically and genotypically. Analyzing mutants at and behind the propagating front, we found that evolution is not always led by the most resistant mutants; highly resistant mutants may be trapped behind more sensitive lineages. The MEGA-plate provides a versatile platform for studying microbial adaption and directly visualizing evolutionary dynamics.
For those who can access the article on line, there are videos of this process.