Evaluation of Probiotic Metabolites Mediated Antimicrobial Activity and Inhibition of Virulence Factors in Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli (APEC), a major etiological agent of colibacillosis in poultry, threatens both poultry industry and public health due to its economic impact, zoonotic potential, and rising multidrug resistance. As a safer and sustainable alternative to antibiotics, this study investigated the antagonistic effects of probiotics, specifically Lactiplantibacillus plantarum (LP) and Lacticaseibacillus rhamnosus (LR), against APEC growth and pathogenesis. Co-culture of APEC with either LP or LR at various ratios (1:1, 1:10) revealed a more rapid APEC inhibition corresponding to higher probiotics concentration with significant suppression within 24 h. The primary mechanism behind this inhibition was attributed to media acidification by LP and LR, as confirmed by pH measurements. Cell-free culture supernatants (CFCSs) collected from either LP or LR culture at both 24 and 48 h demonstrated time dependent bactericidal activity. CFCSs also significantly inhibited biofilm formation by > 3 log CFU/mL (p < 0.05) and disrupted pre-established APEC biofilms by > 2 log CFU/mL (p < 0.05). Gene expression analyses revealed marked downregulation of genes involved in adhesion, survival, membrane integrity and metabolic adaptation indicating attenuation of APEC virulence. Similarly, fluorescence microscopy analyses along with membrane permeability assays corroborated the disruption of bacterial membrane integrity due to CFCSs treatment. Together, our study demonstrates that probiotic-based strategies can be an alternative antibiotic-free intervention to prevent and control APEC infection in poultry.