A recent study in Science reveals a novel avenue by which genetic changes make bacteria resistant to drugs: mutations in genes involved in cellular metabolism, including some that convert food into energy. “The genes were known; their involvement in metabolism was known; but it wasn’t known that mutations in these genes can cause resistance,” says lead author Allison Lopatkin, an assistant professor of computational biology at Barnard College of Columbia University in New York. Long term, the discovery could lead to new drug candidates.
Lopatkin and coauthors set out to track how Escherichia coli adapts to new environments. First, they grew E. coli with gradually increasing concentrations of carbenicillin, streptomycin, or ciprofloxacin for 11 days. At the end of the experiment, the researchers smeared a fraction of each population onto a new agar plate, then isolated and sequenced 12 bacterial colonies from each plate. Sequencing showed that 36% of mutations occurred in genes known to cause resistance, but another 29% affected genes related to metabolism.
Growing bacteria with gradually increasing antibiotics is the classic method to study resistance—doing so selects for strains that grow the fastest in the presence of the drug. Lopatkin wondered if perhaps a different experimental design, selecting for metabolism rather than growth, would reveal more metabolic genes conferring resistance. So, in a second set of experiments, she and coauthors grew E. coli once again for 11 days. This time, instead of gradually increasing the antibiotic concentration, they gradually increased the temperature day by day, while applying a high-concentration antibiotic during a 1-hour window. The two-pronged approach of brief antibiotic exposure paired with increasingly warmer temperatures slowly increased bacterial metabolism over the 11 days, selecting for the cells that could survive under those conditions. At the end of the second experiment, the researchers once again sequenced the bacteria. This time, many of the mutations turned up in genes related to metabolism. “The next obvious question,” Lopatkin says, is “how clinically relevant are they?”
To get at the real-world relevance of metabolic mutations, the authors next compared the 109 mutations found in both experiments to a library of more than 7,000 E. coli genomes, around half of which were isolated from human patients in hospitals and clinics. Metabolic mutations were surprisingly widespread in these genomes. Further tests demonstrated that several of these mutations confer antibiotic resistance.
Finally, the researchers decided to zero in on one mutation of interest, affecting a gene called sucA, which normally encodes an enzyme involved in aerobic respiration. The mutant, they found, downregulates certain genes involved in metabolism to evade carbenicillin.
Overall, the study’s experimental design is remarkably elegant and simple, though labor-intensive, says Lingchong You, a quantitative biologist at Duke University in Durham, NC, who was not involved in the new work. Demonstrating that antibiotic-resistant E. coli from real-world clinical settings also have changes in their metabolic genes “underscores the potential relevance of these new mutations,” notes You, who was Lopatkin’s graduate advisor.
“Mutations in metabolic genes could be as relevant as classical resistance mutations,” says systems biologist Mattia Zampieri at ETH Zürich in Switzerland, who wrote an accompanying perspective for the new work. Future studies will need to clarify exactly how the different metabolic mutations confer resistance, he adds. The results will likely motivate other researchers to rethink classical experiments in favor of new and innovative selection protocols, pushing the field to appreciate the gap between “the huge complexity of evolution in the real world and the overly simplified within-tube evolution which we often employ in the lab,” notes Roy Kishony, who specializes in systematic approaches to antibiotic resistance at the Technion, Israel Institute of Technology, in Haifa.
Lopatkin views the findings as foundational work that reveals new mechanisms driving antibiotic resistance, perhaps eventually even pointing to drugs that target metabolic mutations. In the nearer term, she wants to investigate whether metabolic mutations all act to downregulate certain genes like the mutation affecting sucA. They may confer resistance in many different ways.
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