Effects of Heat-Killed Probiotic Strains on Biofilm Formation, Transcription of Virulence-Associated Genes, and Prevention of UTIs in Mice.

Abstract

Urinary tract infections (UTIs) pose a substantial healthcare challenge, exacerbated by the biofilm-forming abilities and antibiotic resistance of uropathogens. This study investigated the inhibition of biofilm formation (anti-biofilm) and dispersion of pre-established biofilm properties of 18 heat-killed probiotics and their supernatants against four antibiotic-resistant uropathogens: UPEC, Klebsiella pneumoniae (KP), Methicillin-resistant Escherichia coli (MREC), and Methicillin-resistant Staphylococcus pseudintermedius (MRSP). Supernatants from 14 probiotic strains significantly (P < 0.001) inhibited UPEC biofilm formation, reducing it by 20-80%, and also showed promise in removing existing biofilms by 10-60% (P < 0.001). Eight strains significantly (P < 0.05 to < 0.001) inhibited MREC biofilm formation, with four strains achieving 50-80% dispersion. Seventeen strains of heat-killed probiotics directly inhibited UPEC biofilm formation by 10-60% (P < 0.05 to < 0.001), but were less effective against MREC and MRSP (10-50% reduction; P < 0.05 to < 0.001) and had limited impact on KP (10% reduction; P < 0.05 to < 0.001). Notably, heat-killed probiotic like LGA, LGC, LGD, TP-8, and TP-4 showed the most significant inhibitory and dispersion of biofilm activity. RT-qPCR analysis further revealed these inactivated probiotics downregulated genes associated with pili and biofilm formation (fimA, csgA) and upregulated genes linked to quorum sensing (luxS, qseBC, sdiA). Therefore, these findings suggest that paraprobiotic treatment could inhibit the formation of pili and biofilms and promote biofilm dispersion. In an animal model, mice given paraprobiotic formulations I (16 strains) and II (a specific mixture) for 2 weeks showed reduced urinary bacterial load (P < 0.05). Paraprobiotic I notably reduced morbidity from bacteriuria (> 10⁵ CFU/ml) by 5 to 30% within the first 5 days post-infection compared to placebo. These findings highlight the potential of specific heat-killed probiotics in combating biofilms and preventing UTIs.