Trypticase soy broth (tsb)

During 2018, samples from intestinal content of cattle and swine (n = 300, each) at four abattoirs located in the Región Metropolitana were obtained. From these samples, 54 STEC strains were isolated from cattle (n = 51) and swine (n = 3) (25 (link)). Strains were stored in trypticase soy broth (Oxoid, Basingstoke, UK) mixed with glycerol (1:1, v/v) at −80°C. Sampling, processing, bacterial identification and characterization were detailed in a previous study (25 (link)).

Twelve H. pylori clinical isolates and one reference strain of H. pylori (NCTC 11916). The clinical strains were isolated from gastric biopsy samples obtained by a gastroenterologist of the Jordan University Hospital during a routine endoscopy. The gastric biopsy material was processed according to the standard methodology [37 ]. Briefly, each biopsy for culture was homogenized using a tissue homogenizer (IKA, Staufen, Germany). Aliquots of 100 μL of the homogenate were primarily cultured on Columbia blood agar (Oxoid, Hampshire, UK) supplemented with 7% (v/v) horse blood and Dent selective supplement (Oxoid, Hampshire, UK). Subcultures of the bacteria were performed using the same plates but without the Dent supplement. All of the plates were incubated at 37 °C under microaerophilic conditions using CampyGen atmosphere generating system (Oxoid, Hampshire, UK) in anaerobic jars for 5–7 days. Growth of H. pylori was confirmed according to colony morphology, Gram staining, microaerophilic growth (at 37 °C), biochemical tests (positive for oxidase, catalase and urease), and subsequently by standard PCR of 16S ribosomal DNA test [38 (link)]. H. pylori cultures were stored at −70 °C in Trypticase soy broth (Oxoid, Hampshire, UK) containing 10% (v/v) fetal calf serum (PAA, Pasching, Austria) and 15% glycerol.

All Shigella strains were previously cultured in trypticase soy broth (Oxoid, England) for 6 to 8 hours; Subsequently, each strain was plotted onto Salmonella–Shigella agar (Oxoid, England) and incubated at 37°C from 18 to 24 h. Presumptive identification was carried out using biochemical tests consisting of triple sugar iron agar, lysine iron agar, mobility indole ornithine agar and Simmons citrate agar (Oxoid, England). The species and serotype were determined using commercially available polyvalent and monovalent antisera (Denka Seiken Co., Ltd, Japan).

The challenge trial was conducted according to the protocol of our earlier study (37 (link)). Briefly, at the end of the 6-week feeding trial and after collecting all samples, 10 fish from each dietary treatment were retuned back to their respective tanks and injected intraperitoneally by 1-ml syringe fitted with a 27-gauge needle with 0.1 ml of pathogenic V. harveyi suspension (LD₅₀ = 1.1 × 10⁸ cfu/ml), which was supplied by Diagnostic and Laboratory Services, Department of Primary Industries and Regional Development (DPIRD), 3 Baron-Hay Court, South Perth WA 6151. V. harveyi was grown in trypticase soy broth (Oxoid, Basingstoke, UK) for 24 h at 24°C, and the culture was centrifuged (5,000 × g, 15 min) at 4°C before suspending the pellets in phosphate-buffered saline (PBS, pH 7.2) for the challenge trial. Clinical signs of vibriosis in terms of a thick layer of mucus on the body surface, congestion of the fins, and hemorrhages and ulceration of the skin and muscle tissue were observed three times a day (7:00 am, 2:00 pm, and 9:00 pm) for 14 days, and fish with such symptoms of vibriosis were subjected to euthanasia with AQUI-S at 175 mg/L for 20 min according to the protocol of the CARL SOP for euthanizing of fish.

B. coagulans was grown in Trypticase Soy broth (Oxoid) supplemented with 0.6% Yeast Extract (Oxoid), 500 mg/l of Manganese Sulfate Monohydrate and 3 mg/l of Dextrose (Sigma-Aldrich, Milan, Italy) at 37 °C for 2 days; then, it was plated on Nutrient Agar at 50 °C for 7 days. When sporulation reached 90%, spores were harvested by adding 5 mL of sterile distilled water, detaching with sterile glass beads, collecting with a pipette and re-suspending in sterile distilled water. The suspension was centrifuged five times at 4000× g at 4 °C for 10 min. Heat treatment at 80 °C for 10 min was applied to destroy any remaining vegetative cells; spore suspension was stored at 4 °C. Spore number was assessed through the spread plate count on Nutrient Agar, incubated at 40 °C for 2 days.
Spores of B. clausii were produced on Alkaline Nutrient Agar with 0.5% of NaCl, supplemented with 10.0 mg/L of Manganese Sulfate Monohydrate (Sigma-Aldrich), incubated at 30 °C for 11 days until approximately 80–90% of cells sporulated. Spores were removed and heat-treated at 80 °C for 10 min, to eliminate vegetative cells and stored at 4 °C. Spore number was assessed through the spread plate count on Alkaline Nutrient Agar with 0.5% of NaCl, incubated at 30 °C for 2 days.

TAR assembled BGCs and an empty pTARa vector control were separately integrated into the chromosome of Streptomyces albus J1074. Spore suspensions of these recombinant strains were used to seed starter cultures in 5 mL trypticase soy broth (Oxoid). These cultures were grown for 48 h (30 °C/200 rpm) and 0.4 mL of the resulting confluent culture was used to inoculate 50 mL of R5a production medium containing: 100 g/L sucrose, 10 g/L d-glucose, 5 g/L yeast extract, 10.12 g/L MgCl₂·6H₂O, 0.25 g/L K₂SO₄, 0.1 g/L casamino acids, 21 g/L MOPS, 2 g/L NaOH, 5.88 mg/L CaCl₂, 80 μg/L ZnCl₂, 400 μg/L FeCl₃·6H₂O, 20 μg/L MnCl₂, 20 μg/L CuCl₂, 20 μg/L Na₂B₄O₇·10H₂O, 20 μg/L (NH₄)₆Mo₇O₂₄·4H₂O, pH = 6.85. Cultures were fermented in 125 mL baffled flasks (30 °C, 220 rpm) for 14 days.

P. aeruginosa ATCC 9027 was used in this study. The isolates from fresh agar plates were inoculated in 5 mL of trypticase soy broth (Oxoid, Basingstoke, UK) and were kept for incubation at 37 °C for 24 h. The inoculum, measured at 10⁸ CFU/mL by spectrophotometry, was used for the biofilm formation assay on the endotracheal tube.