Scientists at the 91̽�� and the University of Idaho have discovered just how readily MDR bacteria can emerge. In a published April 6 in Nature Ecology & Evolution, the researchers report that, for a bacterial pathogen already resistant to an antibiotic, prolonged exposure to that antibiotic not only boosted its ability to retain its resistance gene, but also made the pathogen more readily pick up and maintain resistance to a second antibiotic and become a MDR strain.

The team’s experiments indicate that prolonged exposure to one type of antibiotic essentially “primed” the bacteria. This priming effect made it more likely that the bacteria would acquire resistance to additional antibiotics, even in the absence of further antibiotic exposure, and helped the strain hold on to those antibiotic-resistance traits for generations.

“Exposure to antibiotics appears to select indirectly for more stable antibiotic resistance systems,” said , a 91̽��professor of biology and co-senior author on the paper. “A more stable system in a strain will increase the chances that it will acquire resistance to multiple antibiotics.”

Their findings also show how antibiotic exposure affects the evolutionary dynamics within bacteria.

“This could help explain not only the rise of multidrug resistance in bacteria, but also how antibiotic resistance persists and spreads in the environment — in health care settings, in soil from agricultural runoff — even long after the antibiotic exposure has ended,” said co-senior author , a professor of biology at the University of Idaho.

The researchers tested a common mechanism for the spread of antibiotic resistance: plasmids. These are circular strands of DNA that can contain many types of genes, including genes for antibiotic resistance. Bacteria easily share plasmids, even across species.

Yet plasmids have their downsides, and past research has shown that bacteria readily shed them.

“Even though they can carry beneficial genes, plasmids can also interfere with many types of processes inside a bacterial cell, such as metabolism or DNA replication,” said lead author , a 91̽��research scientist in biology. “So, scientists have generally thought of plasmids as costly and burdensome to the host cell.”

The UW-University of Idaho team worked with cells containing a -resistance plasmid and cells containing a -resistance plasmid. Both hosts, which had not been grown in the presence of antibiotics before, showed no great loyalty to their plasmids. After nine days in an antibiotic-free environment, the fraction of Klebsiella still harboring a plasmid dropped to less than 50%. For E. coli, less than 20% kept their plasmid.

When the researchers exposed the strains to antibiotics, growing each for 400 generations in their respective antibiotic, the strains showed greater affinity for their plasmids even after the antibiotic threat was lifted. After nine days in an antibiotic-free growth medium, more than half of E. coli and Klebsiella cells held on to their respective plasmid.

“Of course, the cells needed their plasmids to help them survive the antibiotic exposure. But even after we took away that selective pressure, both strains retained their plasmids at significantly higher levels than they had before the antibiotic exposure,” said Jordt.

In addition, other experiments showed that antibiotic exposure increased the occurrence of MDR Klebsiella. Even without exposure to antibiotics, Klebsiella pneumoniae can acquire multiple plasmids. For example, when the researchers grew antibiotic-naive Klebsiella and E. coli plasmid-bearing strains together, a small fraction of Klebsiella became MDR by retaining their chloramphenicol-resistance plasmid and acquiring tetracycline-resistance plasmids from E. coli. But when the researchers repeated the experiment using antibiotic-exposed bacteria, they found roughly 1,000 times more MDR Klebsiella.

Prior prolonged exposure to just one antibiotic — chloramphenicol — had increased the likelihood that chloramphenicol-resistant Klebsiella would acquire the tetracycline-resistance plasmid from E. coli in an antibiotic-free environment. In addition, the team’s experiments also showed that, when MDR cells were grown later in an antibiotic-free environment, chloramphenicol-exposed Klebsiella more readily held on to both resistance plasmids.

Evolution can explain both the persistence of the antibiotic-resistance plasmids and the increase of MDR in Klebsiella, the researchers say: Exposing the strains to their respective antibiotic selected for mutations in their genomes to minimize the clash between plasmid and host, making it less costly to keep that plasmid as well as others.

“We believe that by stabilizing one plasmid, these mutations make them more likely to stabilize additionally acquired plasmids,” said Kerr.

Additional experiments may identify the specific mutations that helped E. coli and Klebsiella keep their plasmids, and why Klebsiella was more able to develop into a MDR strain than E. coli. Even though antibiotic exposure creates selective pressure to keep plasmids and acquire new ones, there is still widespread variation in how different species keep, share and receive plasmids.

“There are many, many details to be worked out in the future,” said Top. “But what we see here is that even short-term exposure to just one antibiotic accelerates the development of multidrug resistance, which should give us pause as we use these drugs in health care, agriculture and other settings.”

Co-authors on the paper are 91̽��doctoral student ; Thibault Stalder, a research scientist at the University of Idaho; and , an associate professor of biology at the University of Florida. The research was funded by the National Institutes of Health and the National Science Foundation.

For more information, contact Kerr at kerrb@uw.edu and Top at evatop@uidaho.edu.

“BEACON was founded on the premise that the study of evolutionary processes not only enriches our understanding of the natural world, but also has applications in other fields, such as computer science and engineering,” said 91̽��biology professor , who manages the BEACON project for the university. “We want to understand evolution in biological systems better, but we also want to use evolution to improve computer software, to solve engineering problems and other projects of practical value.”

The BEACON center was founded in 2010, and the 91̽��received $2.5 million in funding for the first five years. This year’s renewal — $22.5 million split among the five universities — will support the center for a final five years. The majority will go to , where BEACON is headquartered. The other partner institutions — the , and the — will receive amounts comparable to the UW.

BEACON was founded in part to pursue interdisciplinary and innovative approaches to evolutionary studies. Participating faculty receive funds to begin new types of projects that fall under BEACON’s overall mission.

“These are often seed grants to professors — starter funds for getting new projects and collaborations off the ground,” said Kerr.

The 91̽��researchers involved in BEACON projects include faculty from traditional fields such as biology to unexpected ones such as electrical engineering. The experiments they have pursued include new approaches to biofuel production, flower pollination by insects, chemical communication among bacteria and the evolution of multicellular organisms. At other institutions, BEACON projects include robotics, evolution of computer programs, algorithms for facial recognition software and cancer detection, the origin of new species and computational techniques to understand the genetics of complex diseases.

BEACON researchers also benefit from annual meetings at Michigan State University to share research updates and hold courses and tutorial sessions on new techniques and experimental approaches. Kerr and several 91̽��colleagues attended this year’s meeting in August shortly before the NSF announced that the center would receive five more years of funding.

“We’re thrilled that this has happened,” said Kerr. “We’ve another five years, so let’s see what more we can do in that time.”

91̽��faculty and staff also use BEACON funds for public outreach and education. Using BEACON seed funds in conjunction with a grant from the Howard Hughes Medical Institute, Kerr and biology department lecturer are developing a laboratory module for introductory biology courses where students can design their own evolution experiments with bacteria, such as seeing how quickly the microbes evolve resistance to antibiotics. BEACON funds have also been used to develop new courses at the UW’s in the San Juan Islands and hold outreach events at the Seattle Aquarium, the Pacific Science Center, Seattle Town Hall and local schools.

In addition to Kerr, 91̽��faculty in BEACON leadership roles include biology professor , who is BEACON education coordinator for the UW, and affiliate professor of biology at the , who coordinates efforts to enhance participant diversity with the center.

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For more information, contact Kerr at 206-221-3996 or kerrb@uw.edu.

Previous studies have shown that the more gradual the change, the better the chances for “evolutionary rescue” – the process of mutations occurring fast enough to allow a population to avoid extinction in changing environments. One obvious reason is that more individuals remain alive when change is gradual or moderate, meaning there are more opportunities for a winning mutation to emerge.

Now 91̽�� biologists using populations of microorganisms have shed light for the first time on a second reason. They found that the mutation that wins the race in the harshest environment is often dependent on a “relay team” of other mutations that came before, mutations that emerge only as conditions worsen at gradual and moderate rates.

Without the winners from those first “legs” of the survival race, it’s unlikely there will even be a runner in the anchor position when conditions become extreme.

“That’s a problem given the number of factors on the planet being changed with unprecedented rapidity under the banner of climate change and other human-caused changes,” said Benjamin Kerr, 91̽��assistant professor of biology.

Kerr is corresponding author of a paper in the advance online edition of Nature the week of Feb. 9.

Unless a species can relocate or its members already have a bit of flexibility to alter their behavior or physiology, the only option is to evolve or die in the face of challenging environmental conditions, said lead author Haley Lindsey of Seattle, a former lab member. Other co-authors are Jenna Gallie, now with ETH Zurich, the Swiss Federal Institute of Technology, and Susan Taylor of Seattle.

The species studied was Escherichia coli, or E. coli, a bacterium commonly found in the lower intestine and harmless except for certain strains that cause food-poisoning sickness and death in humans. The 91̽��researchers evolved hundreds of populations of E.coli under environments made ever more stressful by the addition of an antibiotic that cripples and kills the bacterium. The antibiotic was ramped up at gradual, moderate and rapid rates.

Mutations at known genes confer protection to the drug. Researchers examined these genes in surviving populations from gradual- and moderate-rate environments, and found multiple mutations.

Using genetic engineering, the scientists pulled out each mutation to see what protectiveness it provided on its own. They found some were only advantageous at the lower concentration of the drug and unable to save the population at the highest concentrations. But those mutations “predispose the lineage to gain other mutations that allow it to escape extinction at high stress,” the authors wrote.

“That two-step path leading to the double mutant is not available if a population is immersed abruptly into the high-concentration environment,” Kerr said. For populations in that situation, there were only single mutations that gave protection against the antibiotic.

“The rate of environmental deterioration can qualitatively affect evolutionary trajectories,” the authors wrote. “In our system, we find that rapid environmental change closes off paths that are accessible under gradual change.”

The work was funded by the National Science Foundation, including money through the consortium known as the Beacon Center for the Study of Evolution in Action, and 91̽��Royalty Research Funds.

The findings have implications for those concerned about antibiotic-resistant organisms as well as those considering the effects of climate and global change, Kerr said. For instance, antibiotics found at very low concentrations in industrial and agricultural waste run-off might be evolutionarily priming bacterial populations to become drug resistant even at high doses.

As for populations threatened by human-caused climate change, “our study does suggest that there is genuine reason to worry about unusually high rates of environmental change,” the authors wrote. “As the rate of environmental deterioration increases, there can be pronounced increases in the rate of extinction.”

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For more information:
Kerr, 206-221-3996, 206 221-7026, kerrb@uw.edu