Making antibiotics more potent against drug-resistant bacteria

Researchers have created a pipeline for discovering unique combinations of molecules that increase the effectiveness of antibiotics against drug-resistant bacteria. The team, led by scientists at the Broad Institute and the Tufts University School of Medicine, used a microfluidic approach to screen more than 1 million combinations of antibiotics, small molecules, and bacteria. They identified a small molecule that boosts the power of the antibiotic rifampin in certain bacteria by weakening their defenses. The team also developed another molecule that was even more potent. 

The findings were recently published in PNAS and come from the Center for Innovation to Transform Antibiotic Discovery (CITADel) at the Broad Institute and MIT. CITADel aims to accelerate discovery of antibiotics against Gram-negative bacteria that can be particularly difficult to treat and often develop resistance.

Paul Blainey, core member of the Broad and a professor of biological engineering at MIT, was a co-senior author on the study along with Ralph Isberg, a professor at the Tufts University School of Medicine. Joan Mecsas and Bree Aldridge, both from Tufts, and CITADel leaders Deb Hung, Broad core institute member, and Laura Kiessling, Broad institute member, also helped drive the work.

ESKAPE route

More than 1 million people die of antibiotic-resistant infections every year and doctors need new ways to treat these patients. None of the antibiotics discovered in the last 10 years kill bacteria through new mechanisms, and it is likely that bacteria will evolve to resist these newer treatments through existing resistance mechanisms. One potential approach to addressing this is to identify other molecules, called adjuvants, that can make bacteria more susceptible to existing antibiotics.

“A novel adjuvant could really help us overcome resistance mechanisms and better address the antibiotic crisis,” said Megan Tse, who is a co-first author on the study along with Meilin Zhu. Both Tse and Zhu were graduate students in Blainey’s lab when the work began. 

Tse, Zhu, and their team wanted to use a combinatorial approach to treat so-called “ESKAPE” pathogens, a priority designation by the World Health Organization that includes Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. In their new study, the scientists focused on these three Gram-negative bacteria. 

While it might be straightforward to test a single compound against a bacterial species, testing millions of combinations of molecules quickly becomes an “absurdly large” problem requiring specialized equipment, Tse says. 

So the team turned to a technology called DropArray, developed by Blainey’s lab in 2018. This technique uses microarrays of hundreds of thousands of wells to combine bacteria and compounds in thousands of nanoliter-sized droplets. Fluorescent barcodes mark the precise combination in each droplet. The researchers then use a microscope to track the growth of bacteria in each well, looking for wells where bacteria don’t grow at all due to an effective combination of bacterial-killing molecules. 

To identify molecules that might help target ESKAPE pathogens, Tse, Zhu, and their colleagues scaled up the approach to analyze a panel of six bacteria, 22 known antibiotics at different concentrations, and 30,000 chemical compounds — testing about 1.3 million combinations in just one month. The team looked for molecules that had effects in multiple species.

One compound, a molecule called P2-56, stood out. It made multiple antibiotics more effective in A. baumannii and K. pneumoniae and at least one antibiotic more effective in all of the ESKAPE species. The team also developed a more potent version of the molecule called P2-56-3 that made the antibiotic rifampin even more effective.

Membrane disruption

In additional experiments, Tse working in collaboration with Isberg lab members found that P2-56-3 disrupts the outer membrane of A. baumannii’s cell envelope, potentially by disrupting lipooligosaccharide transport. This may increase the bacteria’s ability to take up antibiotics such as rifampin that don’t normally enter the cell.

Additional work is needed to determine whether P2-56-3 and rifampin could be a viable combination therapy in people. But Blainey says their technology makes possible a previously inaccessible goal of large-scale combination screening. He hopes other groups will use his team’s dataset to interpret their own results and train new computational models to predict antibiotic activity.

“We’ve shown that with this technology and throughput, combination screening is a doable thing,” Blainey said. “I hope this expands people’s imaginations of what can be done.”

A video about the original DropArray technique, a tool for identifying compounds that improve the efficacy of antibiotics against bacteria.