Withaferin A inhibits biofilm formation and adhesion of neonatal meningitis Escherichia coli by downregulating ibeR expression

To investigate the effect of Withaferin A (WA) on biofilm formation and adhesion of neonatal meningitis Escherichia coli (NMEC) and the regulatory role of ibeR.

CCK-8 assay was used to determine the sub-inhibitory concentration of WA in cultured human brain microvascular endothelial cells (HBMECs). The NMEC strain RS218 (E44) and an ibeR deletion mutant (BR2) were treated with 1, 2, and 5 μmol/L WA, and the changes in biofilm formation were evaluated by crystal violet staining and SBF assay; the expression levels of ibeR, ibeA, and ibeT were detected by qPCR. HBMECs co-cultured with E44 or BR2 were examined for bacterial adhesion, and indole production in the supernatants and the effect of exogenous indole on biofilm formation were evaluated.

Based on cytotoxicity test, 2 μmol/L WA was used for subsequent experiments. WA dose-dependently reduced biofilm formation in E44 but not in the BR2 strain but did not significantly inhibit proliferation of either strain. WA treatment significantly downregulated ibeR expression in the bacteria. Deletion of ibeR did not affect bacterial growth but significantly decreased biofilm formation and adhesion to HBMECs. WA treatment also reduced cell adhesion ability of E44, which was not observed in BR2. WA significantly increased indole production and decreased biofilm content in E44 but not in BR2.

IbeR regulates NMEC biofilm formation and adhesion likely by modulating indole production. WA dose-dependently inhibits biofilm and adhesion of NMEC by downregulating ibeR without affecting bacterial growth.