Antibiotic Compound Candidates Identified in Deep Sea Bacteria

Scientists at the University of Helsinki have used a screening technology that can test for the antivirulence and antibacterial effect of hundreds of unknown compounds simultaneously, to identify promising antimicrobial compounds in actinobacteria species isolated from invertebrates in the Arctic sea. The researchers discovered two compounds from marine Kocuria and Rhodococcus species that inhibited enteropathogenic Escherichia coli (EPEC) virulence or growth. “… we show how advanced screening assays can identify antivirulence and antibacterial metabolites from actinobacteria extracts,” said Päivi Tammela, PhD, a professor at the University of Helsinki, who is corresponding author of the team’s newly reported study in Frontiers in Microbiology.“We discovered a compound that inhibits enteropathogenic E. coli (EPEC) virulence without affecting its growth, and a growth-inhibiting compound, both in actinobacteria from the Arctic Ocean.” The team’s published study is titled, “Bioprospecting of Inhibitors of EPEC Virulence from Metabolites of Marine Actinobacteria from the Arctic Sea.”

While antibiotics are the linchpin of modern medicine we continue to face a global “antimicrobial crisis,” the authors wrote, as more and more resistant strains of bacteria are evolving, while the rate of discovery of fundamentally new antibiotics has been much slower.

Historically, researchers have looked for antibacterial compounds in natural products, particularly in other microbes, the team continued. “A considerable number of antibacterial agents are derived from bacterial metabolites. Similarly, numerous known compounds that impede bacterial virulence stem from bacterial metabolites. In fact, the investigators stated, soil actinobacteria have produced 80% of all currently licensed antibiotics. However, they noted, “… marine actinobacteria found in the sea, on the seafloor or within the microbiome of marine organisms have received far less attention as possible sources of antibiotics, even more so with respect to virulence-modifying compounds.”

Focusing the search on actinobacteria in other habitats could thus represent a promising strategy, the authors reason, especially if this search could yield novel molecules that neither kill bacteria outright nor stop them from growing, but only reduce their “virulence” or capacity for causing disease. This is because it is hard for targeted pathogenic strains to evolve resistance under these conditions.

“Inhibiting bacterial virulence is a well-studied alternate method to the more traditional killing of microorganisms or inhibiting their growth,” the scientists explained. “In essence, the idea is to inhibit the action of virulence-causing molecules using pharmaceutical interventions. In the best-case scenario, the treated pathogens would then remain incapable of causing symptoms, but nevertheless alive, and thus selection pressure for resistance would not form so easily.” And it’s likely that such drugs, due to their specificity, would have fewer adverse effects on normal flora, which are affected adversely by drugs that inhibit bacterial growth or viability in general, the team noted.

For their reported study, Tammela and colleagues developed a suite of methods that can test for the antivirulence and antibacterial effect of hundreds of unknown compounds simultaneously. “Our aim was to design and validate an isolation and automated screening workflow for use with fractions from microbial cultures and explore the presence of virulence-inhibitory compounds within marine bacterial fractions and their potential application for drug development as the complex nature of extracts and extract fractions may interfere with screens that have been developed and validated using pure chemical compounds only.”

They targeted an EPEC strain that causes severe, and sometimes deadly, diarrhea in children under the age of five years. Especially in developing countries, EPEC isolates have also been shown to display many different forms of antimicrobial resistance, the investigators pointed out.

EPEC causes disease by adhering to cells in the human gut. Once it adheres to these cells, EPEC injects so-called “virulence factors” into the host cell to hijack its molecular machinery, ultimately killing it. “EPEC virulence is caused by it adhering to enterocytes and causing lesions in the intestinal epithelium characterized by the destruction of microvilli, a phenomenon called attaching and effacing (A/E) lesions,” the team wrote. “Among the secreted factors is the translocated intimin receptor (Tir) which is critical for A/E lesion formation,” they wrote.

The team tested compounds derived from four species of actinobacteria, isolated from invertebrates sampled in the Arctic Sea off Svalbard during an expedition of the Norwegian research vessel Kronprins Haakon in August 2020. The bacteria were then cultured, their cells extracted, and their contents separated into fractions. Each fraction was tested in vitro, against EPEC adhering to cultured colorectal cancer cells, and further tests were carried out on the bioactive fractions to identify the relevant compounds.

“Three bioactivity screening methodologies were used for each extract,” the team further explained. “These included 1) testing for their capacity to inhibit the translocation of Tir, 2) their capacity to prevent actin pedestals, and 3) their capacity to inhibit the growth of EPEC in liquid culture … recognized active fractions were then studied further to narrow down their possible mechanism of action and to elucidate the chemical structure of the active compounds.”

The researchers found two unknown compounds with strong antivirulence or antibacterial activity: one from an unknown strain (called T091-5) of the genus Rhodococcus, and another from an unknown strain (T160-2) of Kocuria.

The compounds showed two complementary types of biological activity. One by inhibiting the formation of “actin pedestals” by EPEC bacteria, a key step by which the pathogen attaches to the host’s gut lining. The other by inhibiting EPEC from binding to the Tir receptor on the host cell’s surface, a step necessary to rewire its intracellular processes and cause disease.

Unlike the compounds from T160-2, the compound from T091-5 didn’t slow down the growth of EPEC bacteria. This means that T091-5 is the most promising strain of the two, as EPEC is less likely to ultimately evolve resistance against its antivirulence effects. “ … the specific inhibition of enteropathogenic Escherichia coli (EPEC) virulence could offer an alternative to conventional antibiotic-based approaches, helping to mitigate the issue of antimicrobial resistance over the long term,” the investigators stated.

With advanced analytical techniques, the authors determined that the active compound from T091-5 was most likely a phospholipid: a class of fatty phosphorus-containing molecules that play important roles in cell metabolism. “Our findings include the bioassay-guided identification, HPLC-MS-based dereplication, and isolation of a large phospholipid and a likely antimicrobial peptide, demonstrating the usefulness of this approach in screening for compounds capable of inhibiting EPEC virulence,” the investigators wrote. “We show that this workflow can indeed recognize bioactive compounds in these microbial fractions,” they stated.

Tammela added, “The next steps are the optimization of the culture conditions for compound production and the isolation of sufficient amounts of each compound to elucidate their respective structures and further investigate their respective bioactivities.”

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