In an extensive survey, EMBL scientists tested more than 10,000 drug combinations against some of the major disease-causing bacteria that carry antimicrobial resistance and cause death. Antimicrobial resistance, in which pathogens resist antibiotic treatment, is an urgent challenge to global health. A 2022 study showed that nearly 5 million deaths in 2019 were related to bacteria that were resistant to antibiotics, and more than 1 million of these deaths were directly caused by antibiotic resistance.

A scientific illustration shows how antibacterial drugs targeting different components of bacterial cells affect each other's activity. Photo credit: Isabel Romero Calvo and Elisabetta Cacace/EMBL

In a new study, researchers from the Typas group at EMBL Laboratory in Heidelberg systematically analyzed the efficacy of more than 10,000 drug combinations against common multi-drug-resistant bacteria.

"Previously, specific drug combinations have been studied, particularly those commonly prescribed in the clinic," said Elisabetta Cacace, first author of the study and a former doctoral student in Typas' group. "However, we lacked a systematic understanding of how combinations of different classes of antibiotics, or combinations of antibiotics with non-antibiotic drugs, affect bacterial physiology, especially without considering the host."

Ezoic Cacace is an MD and currently a postdoc at ETH Zürich. During her time in the Typas group, which specialized in developing high-throughput methods to study bacterial interactions (with the environment or other species) and physiology.

Different antibiotics target different cellular structures or processes within bacteria. They can act synergistically, meaning their combined activity is greater than each drug acting alone, but they can also be mutually antagonistic, in which case the presence of one hinders the activity of the other. This antagonism could be used to mitigate the collateral damage of antibiotics to the gut microbiota.

In a previous study, researchers from Typas' group analyzed combinations of drugs that target Gram-negative bacteria, a group that includes many deadly antimicrobial-resistant pathogens such as E. coli, Salmonella Enteritidis and Pseudomonas aeruginosa. However, many deadly antibacterial bacteria are also Gram-positive, including Staphylococcus aureus, whose methicillin-resistant variant (MRSA) kills hundreds of thousands of people every year. The cell wall structure of these bacteria is different from that of Gram-negative bacteria, which affects the activity and effectiveness of the drug.

In the current study, the team used an advanced robotic device to simultaneously study the effects of hundreds of different dose combinations of antibiotic and non-antibiotic drugs on three representative Gram-positive bacteria - Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae. In addition to more than 8,000 combinations of 65 different antibiotics across all major categories, the researchers also analyzed more than 2,500 combinations of antibiotic drugs with non-antibiotic drugs.

Using this strategy, the team discovered more than a thousand interactions, including synergy and antagonism. These effects are highly species-specific and even strain-specific, distinct from interactions found previously in studies of Gram-negative bacteria. They also validated some of these results in vivo by infecting moth larvae with the pathogen and testing the ability of specific drug combinations to aid recovery.

The researchers have made their complete interaction database publicly available for other scientists to view, explore and use to find new synergies and antagonisms.

"We think the scale of this study makes it unique. It's such a rich data set that I think it will provide fodder for all kinds of hypotheses for many years to come," Kakas said. "I also find this interesting from a systems biology perspective because we are seeing interactions between drugs that target certain cellular processes that were not known before."

"We are living in an era where new strategies to combat antimicrobial resistance are urgently needed, and the development of new antibiotics is technically challenging, costly and time-consuming," said Nassos Typas, group leader at EMBL and senior author of the study. "The kind of systematic drug interaction analysis we performed in this study opens the way to alternative solutions and treatments for bacterial infections."