Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 16 4 2024 08 14 497 508 Mahdi Asghari Ozma Student Research Committee, Baqiytallah University of Medical Sciences, Tehran, Iran Hamideh Mahmoodzadeh Hosseini Applied Microbiology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran Mohammad Hossein Ataee Applied Microbiology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran Seyed Ali Mirhosseini Applied Microbiology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran 2024 04 19 2024 07 17 Background and Objectives: The most common cause of healthcare-associated diarrhea is Clostridium difficile infection (CDI), which causes severe and recurring symptoms. The increase of antibiotic-resistant C. difficile requires alternate treatments. Postbiotics, metabolites produced by probiotics, fight CDI owing to their antibacterial capabilities. This study aims to evaluate the antibacterial, antibiofilm, and anti-toxigenic potential of postbiotics in combating CDI. Materials and Methods: GC-MS evaluated postbiotics from Bifidobacterium bifidum and Lactobacillus plantarum. Disk diffusion and broth microdilution determined C. difficile antibacterial inhibition zones and MICs. Microtiter plates assessed antibiofilm activity. MTT assay evaluated postbiotics anti-viability on HEK293. ELISA testing postbiotic detoxification of toxins A and B. Postbiotics were examined for tcdA and tcdB genes expression using real-time PCR. Results: The most identified B. bifidum and L. plantarum postbiotic compounds were glycolic acid (7.2%) and butyric acid (13.57%). B. bifidum and L. plantarum displayed 13 and 10 mm inhibition zones and 2.5 and 5 mg/ml MICs against C. difficile. B. bifidum reduced biofilm at 1.25 mg/ml by 49% and L. plantarum by 31%. MTT assay showed both postbiotics had little influence on cell viability, which was over 80%. The detoxification power of postbiotics revealed that B. bifidum decreased toxin A and B production more effectively than L. plantarum, and also their related tcdA and tcdB genes expression reduction were statistically significant (p < 0.05). Conclusion: Postbiotics' ability to inhibit bacterial growth, biofilm disruption, and toxin reduction makes them a promising adjunctive for CDI treatment and a good solution to pathogens' antibiotic resistance. https://ijm.tums.ac.ir/index.php/ijm/article/view/4733 https://ijm.tums.ac.ir/index.php/ijm/article/download/4733/1694