Objectives Biofilm 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. Methods 5-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. Results 5-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 β-lactams drastically reduced the viability of biofilm-resident bacteria. Conclusions 5-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 β-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.

The antimycotic 5-fluorocytosine is a virulence inhibitor of uropathogenic Escherichia coli and eradicates biofilm-embedded bacteria synergizing with β-lactams / S. Ravishankar, A.L. Conte, S.J. Carrasco Aliaga, V. Baldelli, K.L. Nielsen, M.P. Conte, P. Landini, E. Rossi. - (2024 Nov 22). [10.1101/2024.11.20.624304]

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

S. Ravishankar;S.J. Carrasco Aliaga;V. Baldelli;P. Landini
Penultimo
;
E. Rossi
Ultimo
2024

Abstract

Objectives Biofilm 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. Methods 5-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. Results 5-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 β-lactams drastically reduced the viability of biofilm-resident bacteria. Conclusions 5-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 β-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.
Settore BIOS-15/A - Microbiologia
Settore MEDS-03/A - Microbiologia e microbiologia clinica
22-nov-2024
https://www.biorxiv.org/content/10.1101/2024.11.20.624304v1
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/1122765
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