Lactobacillus helveticus

Saccharomyces cerevisiae strain PE-2 was kindly provided by professor Thiago Olitta Basso. Strains of Lactobacillus amylovorus and Lactobacillus fermentum were isolated from stored industrial samples. Strains of Pediococcus claussenii, Lactobacillus helveticus, Lactobacillus buchneri, and Zymomonas mobilis were purchased from ATCC (Manassas, VA, USA).

In this study, we used two Salmonella enterica (SE) serovars, which are pathogenic to poultry, specifically SE serovar Gallinarum (CAT 375, Presque Isle Cultures, Erie, PA, USA) and SE serovar Pullorum (ATCC 13036). Additionally, we used S. Pullorum and S. Gallinarum samples which were isolated previously from farm environments or poultry products in our lab. Three common chicken gut microbiomes, including Enterococcus faecalis (PIC 522A), Streptococcus thermophilus (ATCC 19258), and Lactobacillus helveticus (ATCC 8018), were used. As a probiotic reference, Escherichia coli Nissle 1917 (Mutaflor, Herdecke, Germany) was employed. Before each experiment, S. Gallinarum, S. Pullorum, and E. coli Nissle were cultivated and maintained on Luria-Bertani (LB) agar (Millipore, Billerica, MA, USA); L. helveticus was cultured in de Man Rogosa Sharpe (MRS) agar (Merck, Germany); and E. faecalis and S. thermophilus were cultured in Tryptic Soy (TS) agar (Hardy Diagnostic, Santa Maria, CA, USA) plates, all at 37 °C for 18–24 h from −80 °C glycerol stock. The bacterial strains were stored at −80 °C in glycerol, and prior to each experiment, they were cultured on their respective agar plates for the specified duration.

The PP mixture comprised various Lactobacillus strains supplied by the American Type Culture Collection (ATCC) (Manassas, VA, USA). The strains included Lactobacillus gasseri (ATCC 33323), Lactobacillus plantarum (ATCC BAA-793), Lactobacillus reuteri (ATCC 23272), Lactobacillus helveticus (ATCC BAA-2840), Lactobacillus fermentum (ATCC 23271), Lactobacillus rhamnosus (ATCC BAA-2836), and Lactobacillus casei (ATCC BAA-2843). Following the supplier’s protocol, we cultured the strains in MRS broth (Beckton Dickinson, Sparks, MD), and preserved them as glycerol stocks at −80°C. To initiate culture growth, we prepared 5 mL of pre-warmed MRS broth with the glycerol stocks and then incubated these starter cultures at 37°C in a 5% CO₂ environment for 2 h. The bacteria were cultured overnight in 1 L of MRS broth under identical conditions until they reached the logarithmic growth phase, confirmed by measuring optical density at 600 nm (OD600). After growth, the bacterial cells were collected through several centrifugation steps at 3000×g and 4°C, followed by a wash in cold PBS. We then resuspended the bacterial pellets in 10% glycerol in PBS and promptly froze them in 1 mL aliquots for future administration to mice. The viability and concentration of the bacteria were verified by serial dilution and colony-forming unit (CFU) enumeration on agar plates.