Methanol and chloroform were purchased from Fisher. Tryptone Soya Broth (TSB) was purchased from Difco Laboratories, UK, and Fluid Thioglycollate Medium (FTM) from Oxoid Microbiology Products, U.K. L929 mouse fibroblasts were acquired from Adolfo Lutz Institute, São Paulo, Brazil. All other materials, including polycaprolactone (PCL; average molecular weight 80 kDa), were purchased from Sigma Aldrich, Brazil.
Tryptone soya broth (tsb)
Manufactured by BD
Sourced in United States
Tryptone Soya Broth (TSB) is a general-purpose microbiological culture medium. It is designed to support the growth of a wide range of aerobic and facultative anaerobic bacteria. The medium contains peptones and other nutrients that provide carbon, nitrogen, and other elements necessary for bacterial growth and cultivation.
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Lab products found in correlation
Tryptone soya agar, by BD (3 mentions)
Polycaprolactone pcl, by Merck Group (1 mentions)
Microflex lt spectrometer, by Bruker (1 mentions)
Ultrospec 10 cell density meter, by Harvard Bioscience (1 mentions)
Lactobacilli mrs broth, by BD (1 mentions)
Peptone, by Merck Group (1 mentions)
Potassium phosphate, by Merck Group (1 mentions)
Magnesium sulfate heptahydrate, by Merck Group (1 mentions)
Peptone, by BD (1 mentions)
Leucine, by Merck Group (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Yeast extract, by BD (1 mentions)
L threitol, by Tokyo Chemical Industry (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Methanol, by Avantor (1 mentions)
Sucrose, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Sodium nitrate, by Samchun (1 mentions)
Sucrose, by Samchun (1 mentions)
Potassium chloride (kcl), by Samchun (1 mentions)
10 protocols using tryptone soya broth (tsb)
1
Fibroblast cell culture protocol
2
Quantifying Biofilm Formation and Growth Kinetics
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Biofilm formation was quantified using a 96-well plate and accompanying peg-lid of the MBEC (Minimum Biofilm Eradication Concentration) assay device (Innovatech, Edmonton, Canada). Wells received 150 µL bacterial suspension of B. seminalis standardized to 107 CFU.mL−1 in Tryptone Soya Broth (TSB) (Difco Laboratories). Eight wells were inoculated in at least three independent experiments. The peg-lid was placed on the plate and incubated at 28 °C or 37 °C for 24 h. The peg-lid was transferred to a fresh 96-well plate containing pre-warmed TSB and incubated for more than 24 h. The peg-lid was rinsed with PBS and then baked at 60 °C for 20 minutes, followed by staining with crystal violet 0.1% (w/v) (200 µL per well) and incubation for 30 minutes at room temperature. Three wash plates containing PBS (200 µL per well) were used to rinse the pegs following staining; subsequently, the crystal violet was solubilized with 95% ethanol prior to measurement at OD59567 . In addition, a growth curve was prepared. Flasks containing 100 mL of TSB were each inoculated with a bacterial suspension to reach OD595 equal to 0.05. Then, flasks were shaken at 150 rpm at 28 °C or 37 °C. Aliquots were removed at periods of 2 h until the stationary phase, and OD595 readings were measured.
3
Antimicrobial Potential of Moringa Extracts
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Three microorganisms were used to test the antimicrobial potential of the prepared Moringa plant extracts, they were: Staphylococcus aureus (S. aureus) and Streptococcus mutans (S. mutans) and Candida albicans (C. albicans). S. aureus was isolated and serologically identified by dairy microbiological Lab., National Research Center, Giza, Egypt. While S. mutans were isolated from the dental plaque of high caries index patients by swabbing method from the oral cavity of the patients according to Sanchez et al., 2004 [21 (link)]. Selective media Mitis salivarius-bacitracin agar (MSB; BD Difco, Paris) was used to isolate and grow S. mutans. Both pathogenic bacteria were routinely transferred into Tryptone Soya broth (TSB; Difco laboratories, Detroit, MI, USA) and incubated at 37°C for 24 hours. C. albicans (ATCC 10231) was provided by the Department of Microbiology, Cairo University and maintained on Sabouraud Liquid Medium (1% peptone, 4% glucose and adjusted to pH 5.8) to grow at a temperature of 35-37°C for 48 hours.
4
Phage Isolation and Characterization
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Bacteria strains used in this study are listed in Table S1 . Bacterial strains were grown in tryptone soya broth (Becton Dickinson, Sparks, MD, USA) at 37 °C overnight. These strains were applied for phage isolation, propagation, purification, as well as for phage lytic range and lytic activity determination in liquid cultures and in food matrix.
5
Bacterial Isolation and Identification
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Six bacterial isolates of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae were collected from the “Collection de Souches de l’Unité des Rickettsies” (CSUR, WDCM 875; Table 1 ). Bacteria were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a Microflex LT spectrometer (Bruker Daltonics, United States; Seng et al., 2009 (link)). The bacterial strains were grown overnight in tryptone soya broth (TSB; Becton Dickinson, United States) at 37°C under aerobic conditions. Then, the fresh cultures were diluted using TSB and adjusted at an optical absorbance of 0.18 (106–107 CFU/ml), at a wavelength of 600 nm measured by an Ultrospec 10 cell density meter (Biochrom, United Kingdom). Experiments were carried out on 4 ml of bacterial suspension per condition and bacterial concentrations were validated by the colony counting method.
6
Culturing Highly Pathogenic S. suis Strain
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S. suis serotype 2 strain 05ZY (also known as SC-19) was isolated from the brain of a diseased piglet during the outbreak of S. suis diseases in China in 2005. The strain expresses muramidase-released protein, extracellular protein factor, and suilysin and is highly pathogenic to mice and pigs, causing STSLS (Zhang et al., 2011 (link), 2012 (link)).
The bacteria were cultured for 12 h in Tryptone Soya Broth (BD) plus 10% bovine sera at 37°C to reach stationary phase. Then 500 μl of the bacteria suspension was added into 50 ml of fresh Tryptone Soya Broth plus 10% bovine sera and cultured for 4–6 h to reach log-phase. Then the bacteria suspension was placed on ice to stop growth and then washed with PBS for two times. Finally, the concentration of the bacteria suspension was adjusted to 8 × 108 CFU/ml for animal experiments. CFU per ml values were further confirmed by spreading the serially diluted bacteria suspension on Tryptone Soya Agar (BD) plates.
The bacteria were cultured for 12 h in Tryptone Soya Broth (BD) plus 10% bovine sera at 37°C to reach stationary phase. Then 500 μl of the bacteria suspension was added into 50 ml of fresh Tryptone Soya Broth plus 10% bovine sera and cultured for 4–6 h to reach log-phase. Then the bacteria suspension was placed on ice to stop growth and then washed with PBS for two times. Finally, the concentration of the bacteria suspension was adjusted to 8 × 108 CFU/ml for animal experiments. CFU per ml values were further confirmed by spreading the serially diluted bacteria suspension on Tryptone Soya Agar (BD) plates.
7
Olive Pomace Fermentation Protocol
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In the preparation of olive pomace, OP was obtained from a local olive-pressing factory and then anti-mycotoxin (Mycofix, Biomin) was added. In the next step, OP was dried at 70°C using hot air and sieved (1.5 mm mesh diameter) to remove part of the stones (seeds). Bacillus subtilis var. natto N21 (BS) and Lactobacillus casei were used for dried olive pomace fermentation.
For microbial activation, tryptone soya broth (BD) was used for the incubation of BS at 37°C in a 150-rpm Erlenmeyer flask. Lactobacilli MRS broth (BD) was used for the incubation of L. casei at 37°C in a 100-rpm Erlenmeyer flask. After that, the broth was centrifuged for 10 min, the supernatant was discarded, and sterile water was added to obtain 109 CFU/mL. For the preparation of fermented feed, B. subtilis was added at a concentration of 106 CFU/g of feed and with 10% water for 2 days of aerobic fermentation at 37°C in the first phase, and then LC was added (106 CFU/g feed) with 13% water for 5 days of anaerobic fermentation at 25 to 35°C in the second phase.
For microbial activation, tryptone soya broth (BD) was used for the incubation of BS at 37°C in a 150-rpm Erlenmeyer flask. Lactobacilli MRS broth (BD) was used for the incubation of L. casei at 37°C in a 100-rpm Erlenmeyer flask. After that, the broth was centrifuged for 10 min, the supernatant was discarded, and sterile water was added to obtain 109 CFU/mL. For the preparation of fermented feed, B. subtilis was added at a concentration of 106 CFU/g of feed and with 10% water for 2 days of aerobic fermentation at 37°C in the first phase, and then LC was added (106 CFU/g feed) with 13% water for 5 days of anaerobic fermentation at 25 to 35°C in the second phase.
8
Synthetic Media Composition for Microbial Growth
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Sucrose, potassium chloride, sodium nitrate, magnesium sulfate heptahydrate, and potassium phosphate were obtained from Samchun Pure Chemicals Co. Ltd. (Pyeongtaek, Gyeonggi-do, Korea). L-Threitol was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Peptone, yeast extract and tryptone soya broth (TSB) were purchased from Becton Dickinson (Sparks, MD, USA). Acetonitrile and methanol were purchased from J.T.Baker (Phillipsburg, NJ, USA). All the other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
The synthetic medium contained 1% leucine, 2%, Sucrose, 0.5% Peptone, 0.1% yeast extract, 0.1% potassium phosphate, 0.05% of potassium chloride and magnesium sulfate heptahydrate, trace element (5 g of citric acid, 1 g of ZnSO4·H2O, 5 g of Fe(NH4)2 (SO4)2·6H2O, 250 mg of CuSO4·5H2O, 50 mg of boric acid, 50 mg of MnSO4 and 50 mg of Na2MoO4·2H2O in distilled water), and vitamin solution (4 g of inositol, 200 mg of pantothenic acid, 200 mg of choline, 100 mg of thiamine, 75 mg of pyridoxine, 75 mg of nicotinamide, 30 mg of riboflavin, 5 mg of p-aminobenzoic acid, 5 mg of ascorbic acid, 5 mg of folic acid and 5 mg of biotin in 50% ethanol).
The synthetic medium contained 1% leucine, 2%, Sucrose, 0.5% Peptone, 0.1% yeast extract, 0.1% potassium phosphate, 0.05% of potassium chloride and magnesium sulfate heptahydrate, trace element (5 g of citric acid, 1 g of ZnSO4·H2O, 5 g of Fe(NH4)2 (SO4)2·6H2O, 250 mg of CuSO4·5H2O, 50 mg of boric acid, 50 mg of MnSO4 and 50 mg of Na2MoO4·2H2O in distilled water), and vitamin solution (4 g of inositol, 200 mg of pantothenic acid, 200 mg of choline, 100 mg of thiamine, 75 mg of pyridoxine, 75 mg of nicotinamide, 30 mg of riboflavin, 5 mg of p-aminobenzoic acid, 5 mg of ascorbic acid, 5 mg of folic acid and 5 mg of biotin in 50% ethanol).
9
Aeromonas Bacterial Infection Protocol
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The study was performed with 24 strains (10 clinical and 14 environmental) of six different species of which A. dhakensis, A. media, A. jandaei, Aeromonas piscicola, and A. caviae have not been studied until now, being the only exception A. veronii (Table 1
10
Salmonella Strain Characterization and Phage Isolation
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The details of all Salmonella strains used in this study are listed in Table 1 . Standard strains were purchased from the American Type Culture Collection (ATCC, Gaithersburg, MD, United States) and the National Centre for Medical Culture Collections (CMCC, Beijing, China). Other strains were isolated from poultry, swine, and environmental sewage, and were preserved in 25% (v/v) glycerol at –80°C and cultured in tryptone soya broth (TSB; Becton Dickinson, Sparks, MD, United States) or tryptone soya agar (TSA; Becton Dickinson, Sparks, MD, United States) at 37°C. A total of 88 Salmonella strains were used to determine the lytic range of phages, including the most prevalent serovars recovered from livestock and poultry clinical samples between 2015 and 2021. S. Enteritidis ATCC 13076, S. Enteritidis ATCC13311, and S. Typhimurium CMCC50115 were selected for phage isolation, propagation, and purification. The serovars of all clinical isolates were characterized using whole genome sequencing (WGS) performed by ANNOROAD Gene Technology Co., Ltd (Beijing, China). AMR identification was performed using the disk diffusion method (Bauer et al., 1966 (link)).
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