Turning penicillin into a lethal force against bacteria again

When many disease-causing bacteria encounter penicillin, they are not always destroyed right away, shifting into a temporary survival state called antibiotic tolerance. This state allows them to withstand drug levels that would normally eradicate them. Tolerance is not the same as full antibiotic resistance, but researchers now see it as a risky precursor towards it.

A new study led by postdoctoral researcher Megan Keller and senior author Tobias Dörr, associate professor of microbiology in the College of Agriculture and Life Sciences and faculty in the Weill Institute for Cell and Molecular Biology, reveals for the first time the metabolic changes that allow bacteria to survive high doses of penicillin, a classic β-lactam antibiotic. The study also uncovered a weakness in how the bacteria survive, which may help scientists find better ways to fight antibiotic tolerance in the future.

Published in npj Antimicrobials & Resistance, the research identifies a surprising frailty of the bacteria, Dörr said: under penicillin stress they become starved for nucleotides. These are the basic molecular building blocks essential for many key cell processes that enable the cell to survive.

The study focuses on Vibrio cholerae, the bacterium that causes cholera, and is often used to study antibiotic tolerance. "When exposed to penicillin the bacteria stop dividing while the drug is present, "but they stay metabolically active and survive," Dörr said. "Once the antibiotic fades, they can return to normal growth and continue infection."

To understand how the bacteria survive such tough conditions, Keller and the team used two main methods. They studied which genes turned on or off during penicillin treatment and measured the thousands of molecules the cell made at the same time. This gave them the most detailed map so far of what happens inside bacteria during strong β-lactam (penicillin-type) antibiotic stress.

Many expected shifts appeared, Dörr said. Genes involved in cell wall repair increased their activity as the bacteria attempted to rebuild the damage caused by the drug. Both purine and pyrimidine pathways, which help create nucleotides, also increased their activity.

"The most striking change our research revealed, though, was a sharp drop in nucleotides, the critical precursors for DNA and other important cell components," Dörr said.

"Bacteria disassemble sugars and use their molecular subcomponents to make cell wall materials, amino acids, and nucleotides," Dörr said. "Penicillin causes runaway cell wall synthesis, which essentially makes all the glucose go into the cell wall synthesis pathway. This means there is not enough material left to make, for example, nucleotides."

This led to a new question: if the bacteria are already low on nucleotides, could blocking nucleotide production even more make them easier to destroy? To test this, the team treated cholera bacteria with penicillin plus trimethoprim, which is known to interfere with nucleotide production.

The results were striking, Dörr said. Either drug alone eradicated only modest numbers of bacteria, but together they reduced V. cholerae survival by more than 100,000-fold. The same strong effect appeared in other medically important species, including Klebsiella pneumoniae, the cause of antibiotic-resistant pneumonia and urinary tract infections, as well as the food-borne pathogen E. coli.

By finding a metabolic bottleneck that bacteria must overcome to survive penicillin, the study points toward new treatment strategies. Instead of relying on higher antibiotic doses, which can fuel resistance and harm patients, future therapies might include compounds that exploit the nucleotide shortage caused by penicillin, Dörr said. It may be realistic to repurpose older drugs like trimethoprim as adjuvants to β-lactams in clinical settings, where such approaches could make existing antibiotics more effective. Combination drug "cocktails" as therapeutics are already common, Dörr said, but have not been used specifically to target nucleotide synthesis alongside β-lactam antibiotics.

This research was funded by the National Institutes of Health and partially supported by a seed grant from the Cornell University Biotechnology Resource Center (BRC) and by the Cornell Center for Antimicrobial Resistance Research and Education (CCARRE).

Henry C. Smith is communications specialist for Biological Systems at Cornell Research and Innovation.