NEW YORK – Researchers at the Broad Institute, Rutgers New Jersey Medical School, and elsewhere have found that underappreciated noncanonical genes, such as those related to central carbon and energy metabolism, could underlie antibiotic resistance in Escherichia coli.
In a paper published on Thursday in Science, the researchers noted that antibiotic resistance is usually associated with drug target modification, enzymatic inactivation, or transport processes in bacteria, rather than metabolic processes. They sequenced and analyzed E. coli strains that were adapted to representative antibiotics at increasingly heightened metabolic states, and found that metabolic alterations in the bacteria led to lower basal respiration, preventing antibiotic-mediated induction of tricarboxylic acid cycle activity. This avoided metabolic toxicity and minimized drug lethality.
Several of the metabolism-specific mutations the researchers identified were overrepresented in the genomes of more than 3,500 clinical E. coli pathogens, indicating clinical relevance.
"The results from this study expand upon the existing landscape of antibiotic resistance mutations, many of which have been widely known for a long time. That antibiotic resistance can arise by mutations in metabolic genes demonstrates the importance of metabolism in how antibiotics fundamentally work," first author Allison Lopatkin said in an email. "This gives us some new ideas as to how we might be able to increase the effectiveness of antibiotics. Testing these ideas would be a great next step."
The researchers began by evolving the BW25113 E. coli strain to the three representative bactericidal drugs, streptomycin, ciprofloxacin, and carbenicillin, along with an untreated control. After evolving all four lineages, they ended with 12 representative clones, from which they sought to identify genetic changes associated with antibiotic resistance.
An examination of passing SNPs from the clonal isolates revealed that high-frequency mutations were consistent with known resistance mechanisms. For example, 92 percent of ciprofloxacin-treated clones had mutations in the drug's target and 100 percent had acquired mutations in a known global transcriptional regulator of multidrug resistance and stress response.
However, the researchers also found that a subset of clones acquired mutations that were noncanonical and were, in several cases, related to central metabolic processes. For example, two clones had mutations in a core metabolic enzyme involved in oxidizing isocitrate during the tricarboxylic acid cycle. Other genes that appeared in only one of 12 clones were also involved in core metabolic processes.
The researchers hypothesized that maximizing metabolic rather than growth adaptation would allow them to tease out antibiotic-specific metabolic variants, so they exposed the strains to antibiotics in short-term increments and then gradually increased their metabolic activities. Indeed, they found that their samples exhibited a rich diversity of metabolic mutations and that fewer canonical mutations occurred.
To evaluate whether these metabolic mutations conferred resistance, the researchers then chose a representative subset of both genes related to metabolism and classic resistance, on the basis of their prevalence and clinical significance. They expressed the wild-type and mutant variants of each gene from a medium-copy plasmid introduced into the corresponding chromosomal knockout strain. In all cases, they found that the metabolic mutants were resistant to at least one of the antibiotics.
The plasmid-expressed wild-type metabolic genes in many cases also increased resistance, whereas the knockout strain exhibited heightened drug sensitivity, in contrast to the classic genes, in which expression of the wild-type gene from the plasmid often increased sensitivity compared with the knockout strain.
This suggested that metabolic mutations influenced expression levels or catalytic activity rather than protein structure or function, the researchers noted. Overall, these results indicated that these clinically relevant metabolic variants conferred resistance.
In an accompanying column published in Science, ETH Zurich's Mattia Zampieri wrote that the findings of Lopatkin and her colleagues raise the questions of whether metabolic mutations are necessary to facilitate the evolution of resistance or sufficient to acquire resistance.
"Lopatkin et al. showed that on average, metabolic mutations alone can confer mild, but measurable, antibiotic resistance, suggesting that increased survival is unlikely a result of increased tolerance — that is, the ability to survive antibiotic exposure for a longer time," Zampieri wrote. "These findings reinforce that antibiotic efficacy is intimately linked to the cell's metabolic state. These analyses suggest that rewiring of central metabolism may be a general stratÂegy to acquire antibiotic resistance, but the scope of metabolic changes resulting from these mutations remains to be systematiÂcally unraveled."
Technological advances that will enable the monitoring of dynamic metabolic changes in response to genetic and environmental perturbations, combined with computational models of cellular metabolism, could help reÂsearchers to investigate metabolic mutations, he said. This study shows that predicting how those muÂtations affect the metabolic state of cells, alter drug action, and fit in with other resistance mutations will be important for optimizing current treatment regimens and discovering new drugs and combination therapies.