The antimycotic 5-fluorocytosine is a virulence inhibitor of uropathogenic Escherichia coli and eradicates biofilm-embedded bacteria synergizing with β-lactams

Abstract

ObjectivesBiofilm increases bacterial antibiotic tolerance, posing a challenge for treating biofilm-associated infections in clinical settings. 5-fluorocytosine (5-FC), an FDA-approved antifungal and antitumor drug, has been shown to inhibit virulence factor production and biofilm formation in Gram-negative bacteria. This work aims to determine whether 5-FC antivirulence and antibiofilm activity are preserved in clinical E. coli isolates and to test possible synergies with antibiotics in treating preformed biofilm. Methods5-FC ability to inhibit biofilm formation, disrupt mature biofilm, and modulate virulence determinants was tested on uropathogenic E. coli (UPEC) clinical strains using the Crystal Violet-based biofilm adhesion and minimum biofilm eradication concentration assays. Gene expression was measured by RT-qPCR. The effect on bacterial viability within preformed biofilm was monitored using fluorescein diacetate and by determining colony-forming units in biofilm treated with or without 5-FC and specific antibiotics. Bladder epithelial cell cultures were used to assess the effect of 5-FC on UPEC adherence and cytotoxicity. Results5-FC inhibited biofilm formation in all tested UPEC strains. Gene expression analysis suggested that 5-FC concurrently inhibits the expression of genes encoding curli fibers, a major adhesion factor in E. coli, as well as other virulence determinants such as secreted toxins and type I and P fimbriae. Accordingly, 5-FC reduced UPEC adherence to epithelial cells and promoted host cell survival. Although 5-FC could not disrupt preformed biofilm, combining 5-FC with {beta}-lactams drastically reduced the viability of biofilm-resident bacteria. Conclusions5-FC shows antibiofilm and antivirulence activity against uropathogenic E. coli strains by reducing the expression of several virulence factors and the overall UPEC pathogenic potential. In combination with {beta}-lactams, 5-FC eradicates bacteria within mature biofilms, which are known to be highly refractory to antibiotic treatment. Our data suggest that 5-FC is an excellent candidate for preventing and treating bacterial infections associated with recalcitrant biofilms.