In vitro characterization of probiotic strains Bacillus subtilis and Enterococcus durans and their effect on broiler chicken performance and immune response during Salmonella Enteritidis infection

Authors: Shanmugasundaram, Revathi; KHOCHAMIT, NALISA - Khon Kaen University; SELVARAJ, RAMESH - University Of Georgia; MORTADA, MOHAMMAD - University Of Georgia; SIRIPORNADULSIL, SURASAK - Khon Kaen University; SIRIPORNADULSIL, WILAILAK - Khon Kaen University

Submitted to: Microorganisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2025
Publication Date: 1/21/2025
Citation: Shanmugasundaram, R., Khochamit, N., Selvaraj, R., Mortada, M., Siripornadulsil, S., Siripornadulsil, W. 2025. In vitro characterization of probiotic strains Bacillus subtilis and Enterococcus durans and their effect on broiler chicken performance and immune response during Salmonella Enteritidis infection. Microorganisms. Microorganisms 2025, 13(2), 217. https:// doi.org/10.3390/microorganisms 13020217.
DOI: https://doi.org/10.3390/microorganisms 13020217

Interpretive Summary: Salmonella Enteritidis is a major health and economic problem in poultry farming. It can infect chickens, harm their growth and health, and increase the risk of food contamination. Antibiotics are commonly used to control Salmonella, but their overuse has led to resistance and other issues. This has created a demand for safer and more sustainable solutions.We studied two probiotic strains, Bacillus subtilis CE330 and Enterococcus durans CH33, taken from the digestive tracts of broiler chickens. These probiotics showed that they could survive harsh gut conditions, like low pH and high bile salt levels. Both strains produced antimicrobial substances, such as bacteriocins, which inhibited Salmonella Enteritidis growth in vitro. In live animal trials, supplementing chicken feed with these probiotics lowered Salmonella levels in the gut by 2.8 logs within 10 days after infection.This research showed that B. subtilis CE330 and E. durans CH33 are safe and effective probiotics for controlling Salmonella in chickens. Although they did not immediately improve growth performance, their ability to reduce Salmonella levels makes them valuable for preventing infections. These probiotics could reduce the need for antibiotics, supporting healthier chickens and safer food production. Future studies will work to improve probiotic formulations to maximize their benefits for both chicken health and growth.

Technical Abstract: In vitro experiments were conducted to characterize the effect of bile salt supplementation and pH on the proliferation of Bacillus subtilis CE330 and Enterococcus durans CH33 probiotics and in vivo experiments on production performance, cecal Salmonella enterica serovar Enteritidis (S. Enteritidis) load, and the immune response of broilers. B. subtilis CE330 was more tolerant of high bile concentrations and pH levels compared to E. durans CH33. A one-way ANOVA was used to examine the effect of bile and pH on probiotic species proliferation. Bile concentrations between 3.0 - 4.0 % and a pH range between 2 - 4 decreased (P < 0.05) the proliferation of E. durans CH33. In vitro, cell-free supernatants (CFS) of B. subtilis CE330 and E. durans CH33 at a ratio of 1:1 significantly (P < 0.05) reduced S. Enteritidis proliferation, with a 5:1 ratio of E. durans CH33 CFS having maximal inhibition of S. Enteritidis proliferation. The cultures of B. subtilis CE330 and E. durans CH33 with 4% bile salt for 72 h had a higher proline concentration of 56.95 (13.1-fold) and 20.09 (2.5-fold) µmol/g of fresh weight, respectively. A total of 144 Cobb male broiler chicks were randomly allocated to four treatment groups: basal diet, basal diet + challenge, probiotics (B. subtilis CE330 and E. durans CH33, 0.5 g/kg feed), and probiotics + challenge in six replications. On day 14, all challenged birds were orally challenged with 100 µL of 1 X 108 CFU of S. Enteritidis. A two-way ANOVA was used to examine the effects of probiotic supplementation and Salmonella challenge on dependent variables after Salmonella infection. Probiotic supplementation did not alter the body weight gain, the feed conversion ratio, the intestinal histomorphology (P > 0.05), or IL-1' and IL-10 gene expression (P > 0.05) at 10 dpi. However, it decreased the salmonella load by 38% compared to the control group. In conclusion, when developing multistrain probiotic formulations, it is necessary to give the importance to the biocompatibility of various strains and understand their complex interactions within the host system between the strains. Key words: Salmonella Enteritidis, Bacillus subtilis, Enterococcus durans, Probiotics, broilers