Antibiotics are the cornerstone of modern medicine. Without them, anyone with open wounds or who needs to undergo surgery would be at constant risk of dangerous infections. However, we continue to face a global crisis of them, as increasingly resistant strains of bacteria are evolving, while the rate of discovery of fundamentally new antibiotics has been far slower.
But there are reasons for hope: 70% of all antibiotics currently licensed derive from soil actinobacteria and most environments on Earth have yet to be explored.
Thus, focusing research on actinobacteria in other habitats is a promising strategy — especially if this yields new molecules that do not kill the bacteria or prevent them from growing, but only reduce their “virulence” or ability to cause disease.
This is because it is difficult for the targeted pathogenic strains to develop resistance under these conditions, and it is also less likely that antivirulence compounds will cause unwanted side effects.
“Here we show how advanced screening assays can identify antivirulent and antibacterial metabolites from actinobacteria extracts,” says Päivi Tammela, professor at the University of Helsinki, Finland, and corresponding author of a new study in the Frontiers in Microbiology journal.
“We discovered a compound that inhibits the virulence of enteropathogenic E. coli (EPEC) without affecting its growth, and a growth-inhibiting compound, both in Arctic Ocean actinobacteria,” she adds.
Automated screening of candidate compounds
Tammela and her colleagues developed a new set of methods that can test antivirulence and antibacterial effects of hundreds of unknown compounds simultaneously. Their target was an EPEC strain that causes severe—and sometimes fatal—diarrhea in children under five, especially in developing countries.
EPEC causes the disease by adhering to the cells of the human intestine. After adhering to these cells, EPEC injects the so-called “virulence factors” into the host cell to hijack its molecular machinery, ultimately killing it.
The compounds tested were derived from four actinobacteria species, isolated from invertebrates collected in the Arctic Ocean, off Svalbard, during a 2020 expedition of the Norwegian research vessel Kronprins Haakon.
These bacteria were then cultured, their cells extracted and their contents separated into fractions. Each fraction was then tested in vitro, against EPEC adherent to cultured colorectal cancer cells.
The researchers found two unknown compounds with strong antivirulent or antibacterial activity: one from an unknown strain (designated T091-5) of the genus Rhodococcus and another from an unknown strain (T160-2) of Kocuria.
Potent antivirulent effects
The compounds showed two complementary types of biological activity. First, by inhibiting the formation of the so-called “actin pedestals” by the EPEC bacterium, a crucial step by which this pathogen attaches to the host’s intestinal lining.
Second, by inhibiting EPEC’s binding to the so-called Tir receptor on the surface of the host cell, a necessary step to re-engage its intracellular processes and cause disease.
Unlike the compounds from T160-2, the compound from T091-5 did not slow the growth of EPEC. This means that T091-5 is the more promising of the two, as it is less likely that EPEC will end up developing resistance to its antivirulent effects.
Using advanced analytical techniques, the authors determined that the active compound from T091-5 is most likely a phospholipid: a class of lipid molecules containing phosphorus that play important roles in cellular metabolism.
“The next steps are to optimize culture conditions for the production of the compound and to isolate sufficient quantities of each compound to elucidate their respective structures and to further investigate their bioactivities,” concludes Tammela.
