Bhi medium

Attenuated B. anthracis Sterne (pXO1⁺, pXO2⁻) was cultivated in Brain-Heart Infusion (BHI) medium (Merck, Darmstadt, Germany) under aerobic conditions at 37 °C. The non-sporulating, Gram-negative control organism Francisella tularensis subsp. holarctica live vaccine strain (LVS) was grown in Mueller–Hinton (MHII) broth (Becton Dickinson, Heidelberg, Germany), supplemented with 2% IsoVitaleX (Becton Dickinson, Heidelberg, Germany) at 37 °C, under 5% CO₂ atmosphere for 48 h.

L. tarentolae T7-TR strain (Cat.-No. EGE-1410, Jena Bioscience, Germany), were cultivated on brain heart infusion (BHI) medium (Merck, Germany), with the addition of 15 μg/L of hemin (Jena Bioscience), 50 IU/mL of penicillin, and 50 μg/mL of streptomycin (Jena Bioscience). As used L. tarentolae is T7-TR, the inducible host, two more antibiotics, hygromycin (50 µg/ml) and nourseothricin (50 µg/ml; Jena Bioscience), were added. Cells were cultivated in 50-ml ventilated flasks (Orange, USA) at 26 °C in two styles: static and agitated culture. The suspension culture of L. tarentolae was propagated by dilution ratio of 1:10 to 1:100 into a fresh medium when it reached the stationary growth phase. After dilution, the number of cells was typically 10⁷/ml.

As the vaccine strain, the LC-α strain, and as the control, the LCP strain, were formerly prepared in our laboratory as described in the previous study [16 ]. These strains were grown at 37 °C in deMan, Rogosa, and Sharpe (MRS) medium (Himedia, Thane, India) under the anaerobic condition without shaking. Erythromycin (5 μg/ml) was added whenever required. C. perfringens strain CP58 was used in the challenge experiment [45 (link)]. C. perfringens was grown in brain heart infusion (BHI) medium (Merck, Germany) for colony differentiation, and 5% sheep blood agar for hemolytic activity evaluation. Also, cooked meat medium (CMM) (Merck, Darmstadt, Germany), and fluid thioglycolate (FTG) medium (Merck, Darmstadt, Germany) were used to cultivate C. perfringens in large quantities, and for animal inoculation in the challenge experiment, respectively. All C. perfringens cultures were anaerobically grown at 37 °C without shaking.

Biopsy specimens were carefully homogenized and inoculated onto Brucella agar plates (Merck, Germany) supplemented with 7% (v/v) horse blood, 10% fetal calf serum (FCS), Campylobacter-selective supplement (vancomycin 2.0 mg, polymyxin 0.05 mg, trimethoprim 1.0 mg), and amphotericin B (2.5 mg/l). The incubation was performed at 37 °C for 3–7 days under a microaerophilic atmosphere (5% O₂, 10% CO₂, and 85% N₂) in a CO₂ incubator (Innova® CO-170; New Brunswick Scientific, USA). The suspected colonies were identified as H. pylori based on colony morphology, Gram staining, positive reaction for oxidase, catalase, as well as urease tests, and also by H. pylori gene-specific PCR following the previously described protocols^{27 (link),28 (link)}. Pure cultures from confirmed isolates were kept in 0.5 ml of brain heart infusion (BHI) medium (Merck, Germany) containing 15% glycerol plus 20% FCS, and stored at − 80 °C until further analysis.

Listeria monocytogenes (ATCC19115) and Escherichia coli O157:H7 (ATCC1533), stored at the Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran, were used in the study. E. coli and Listeria monocytogenes were cultured in BHI medium (Merck, Darmstadt, Germany) at 37°C for 24 h. After incubation, the grown cells were harvested by centrifugation (4500 × g, 15 min, 4°C) and washed twice with a sterilized peptone water solution (0.1 g 100 mL⁻¹) to be added later to milk for the yogurt preparation.
Each pathogenic strain was added to the milk for the yogurt preparation at a concentration of about 1 × 10³ cfu mL⁻¹.

Corneal scrapings were collected on an applicator using a Bard–Parker 15 number surgical blade (Beckton-Dickinson, Mississauga, ON, Canada). The applicator was transferred onto a sterile cotton swab immersed in liquid blood heart infusion (BHI) medium (Merck KGaA, Darmstadt, Germany). BHI was sent to the laboratory and inoculated on blood agar, chocolate agar, and Sabouraud agar (Merck KGaA, Darmstadt, Germany). The media were incubated for 18 h at 37 °C in aerobic, anaerobic, and microaerophilic atmospheres obtained using the Genbag system (bioMérieux, Marcy l’Etoile, France). Gram and Giemsa smears were prepared from the developed colonies on agar culture media. Bacterial and fungal species were identified using the VITEK® 2 bacterial identification and antimicrobial susceptibility testing system (bioMérieux, Marcy l’Etoile, France), along with antibiotic susceptibility testing, using cards AST-GP67 and AST-P592 (for Staphylococcus spp.), AST-ST03 (for Streptococcus viridans group), and AST-P576 (for Streptococcus pneumoniae). Antibiotic susceptibility testing of Gram-negative strains was performed using the Vitek2 system with AST-N233 and AST-XN05 cards [31 (link)].