Selection of biofilm-inhibiting ssDNA aptamers against antibiotic-resistant Edwardsiella tarda by inhibition-SELEX and interaction with their binding proteins.

Biofilms can increase bacterial resistance to antibiotic therapies. Edwardsiella tarda with biofilm is highly resistant to antibacterial treatment, especially for the antibiotic-resistant strain. In this study, we obtained biofilm-inhibiting aptamers against antibiotic-resistant E. tarda via a novel systematic evolution of ligands by exponential enrichment (SELEX) technique, called Inhibition-SELEX. After four rounds of screening and validation, we identified aptamers IB1, IB2, and IB3, which demonstrated biofilm-inhibition and biofilm-degradation rates of 69 %, 75 %, and 62 % and 51 %, 63 %, and 45 % at 2 μmol/L, respectively, against antibiotic-resistant E. tarda. Magnetic separation, SDS-PAGE, and mass spectrometry analyses revealed that all three aptamers could bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), while IB2 could also bind to formate C-acetyltransferase (FA). Through molecular docking and molecular dynamics simulations, it was found that the four complexes primarily interact through hydrogen bonding. Among them, IB1-GAPDH exhibited the strongest stability, followed by IB2-FA, then IB2-GAPDH, and IB3-GAPDH was the least stable. Our results suggest that IB1, IB2, and IB3 may inhibit and degrade E. tarda biofilm by interfering with the synthesis, secretion, and transportation of its extracellular polysaccharides and proteins by interacting with GAPDH and FA.