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Evaluation of lactic acid bacteria for application as direct-fed microbials in food animals

Rumbaugh, Kaylee Marie
Shiga-toxin-producing Escherichia coli (STEC) and Salmonella enterica are important foodborne pathogens, commensal to food animal gastrointestinal (GI) tract, and shed in their feces. Reduction of pathogens before their introduction to the food chain is critical. Direct-fed-microbials (DFM) are living microorganisms used for the reduction or control of enteric pathogens. Lactic acid bacteria (LAB) have been studied as potential DFM due to their inhibition of STEC, and Salmonella. The purpose of this study was to evaluate and select LAB strains for application as DFMs in food-animal GI-tracts. Viability of 250 LAB strains was measured after extended frozen storage (-80℃), by determining growth under optimal conditions (24-48h at 30/37/42℃, anaerobic incubation). Those showing excellent or very-good growth were evaluated for initial inhibition against pathogens (1.0x10⁴ CFU/mL)–STEC-O157, NonO157-STEC, and Salmonella Typhimurium, using agar-spot-tests; tolerance to acid (pH 2, 4, 5, 7) and bile (0, 0.1, 0.3, 0.5%) at 0, 1, 3, and 6h; resistance to common antibiotics (ampicillin, clindamycin, erythromycin, gentamycin, streptomycin, tetracycline, vancomycin), through disk-diffusion-assay. Selected LAB were evaluated for their biofilm-forming-ability and inhibition (48h at 30/37/42℃) on polystyrene surfaces, and their capacity to prevent or correct pathogen attachment to intestinal cell lines. Of the 250 LAB strains, 65 were tested further. Initial inhibition revealed, 40%, 42%, and 92% of the LAB exhibited very-good-to-excellent (>10mm) inhibition against NonO157-STEC, STEC-O157, and Salmonella Typhimurium, respectively. Thirteen percent showed excellent, 35% very-good, and 39% good tolerance to acidic conditions, while 29% exhibited excellent, 16% very-good, and 52% good tolerance to bile. All LAB strains formed biofilms, with 33% forming strong, 50% moderate, and 17% weak biofilms. The majority (96.7%) of the LAB were sensitive (S or I) to ampicillin, erythromycin (89.8%), tetracycline (78%), clindamycin (76.3%), vancomycin (44.1%), streptomycin (15.3%), and gentamicin (8.5%). Out of the LAB strains tested, L. animalis, L. lactis and L. salivarius performed the best in both corrective and preventive treatments, reducing STEC-O157 and Salmonella Typhimurium to undetectable levels. LAB tested in cell culture studies, reduced STEC-O157 attached population by half. Selected LAB strains could be used for DFM development based on their survival under GI-tract conditions and inhibition against foodborne-pathogens and their biofilms.