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Background: To develop a probiotic formulation for poultry feed, a few poultry gastrointestinal derived lactic acid bacteria (pGIT-d-LAB) were isolated from chicken intestinal specimens and in vitro experiment was performed to evaluate their efficacy as potential probiotic candidate. Methods: A total of 6 strains of LAB: Lactobacillus brevis (L. brevis), Lactobacillus acidophilus (L. acidophilus), Lactobacillus casei (L. casei), Pediococci spp, Lactobacillus fermentum (L. fermentum) and Lactobacillus plantarum (L. plantarum) were isolated and cultured for collection of Cell Free Supernatant (CFS). CFS collected was tested against pathogenic bacterial isolated from chicken feces as well as prevalent fungal pathogens, utilizing agar-well diffusion techniques. A preliminary investigation into the susceptibility of the pathogens to diverse antibiotics and antifungal drugs was conducted. Bacterial pathogens exhibiting resistance to a minimum of three classes of antibiotics were subsequently identified for pGIT-d-LAB CFS screening. Results: The observed results revealed that the CFS derived from the isolates exhibited varying degrees of growth inhibition against different pathogens. Among the tested pGIT-d-LAB isolates, L. acidophilus demonstrated the most prominent zone of inhibition, measuring 18 mm against Klebsiella pneumoniae ZTAC 1233. Notably, Citrobac-ter diversus ZTAC 1255 showed resistance to all tested pGIT-d-LAB. Quantification of the metabolites produced was performed, and peak production levels was determined. L. acidophilus produced the highest amount of lactic acid (1.789g/l), Pediococci spp. produced the highest amount of diacetyl and H2O2 (1.918g/l) (0.0025g/l) at 48 hr peak values respectively. Conclusion: The test isolates are potential probiotic candidates for controlling pathogens in poultry. [ABSTRACT FROM AUTHOR]