Tryptic soy broth (tsb)

Forty-three LAB strains, isolated from fecal samples of healthy full-term neonates from Greece, and two reference strains (Table 1) were tested for their antimicrobial capacity against ten different L. monocytogenes strains (Table 2). The LAB isolates belong to the collection of the Laboratory of Biology, Biochemistry, Physiology and Microbiology of Harokopio University of Athens, Greece, and have been previously studied for probiotic properties [26 (link),27 (link)]. The L. monocytogenes strains were kindly provided by the Laboratory of Food Quality Control and Hygiene of Agricultural University of Athens, Greece, and have been previously characterized for virulence genes and biofilm formation capacity [18 (link),29 (link)]. Prior to the experiments, the L. monocytogenes isolates were activated twice in tryptic soy broth (TSB; LabM, Lancashire, UK) supplemented with 0.6% yeast extract (YE; LabM) at 37 °C for 24 h in aerobic conditions (Memmert, Schwabach, Germany), and the lactobacilli were activated once on de Man, Rogosa and Sharpe agar (MRS; LabM) and twice in MRS broth at 37 °C under anaerobic conditions (BACTRON™ Anaerobic Chamber, Cornelius, OR, USA).

The mouse-adapted Helicobacter pylori Sydney strain 1 (SS1), originally described by Lee et al., [27 (link)], was kindly provided by Sara Lindén (Gothenburg University, Sweden). H. pylori SS1 was routinely cultured for 48 h on Tryptic Soy Agar (TSA) plates (Lab M Limited, Lancashire, UK) medium, supplemented with 5% sheep blood (Oxoid, Cambridge, UK) at 37°C under micro-aerophilic conditions (5% O₂, 10% CO₂, 85% N₂), generated by Whitley H35 Hypoxystation (Don Whitley, West Yorkshire, UK). For liquid cultures, bacteria were resuspended in Tryptic Soy Broth (TSB) (Lab M Limited, Lancashire, UK) containing 10% of fetal calf serum (FCS) (Invitrogen, Ghent, Belgium) and grown overnight at 37°C, with shaking under microaerophilic conditions.

All the microorganisms used in this study are presented in Table 1. They consist of two strains of each species, specifically for L. monocytogenes (FMCC B-125, ScottA, serotype 4b, epidemic strain, human isolate; FMCC B-129, isolated from ready-to-eat frozen meal, minced meat based), S. Typhimurium (FMCC B-137, human isolate epidemic; FMCC B-193, isolated from calf bowel), E. coli O157:H7 (FMCC B-15 and FMCC B-16, both isolated from human feces), S. aureus [FMCC B-410, methicillin-resistant (MRSA) strain COL, isolated from hospital; FMCC B-135, isolated from human lesions], and Y. enterocolitica (FMCC B-89, CITY 650; FMCC B-90, CITY 844). Before each experiment the stock cultures (frozen at -80°C) were sub-cultured twice on 10 ml of Tryptic Soy Broth (TSB, LAB M Limited, Lancashire, United Kingdom) at 37°C for 24 and 16 h, respectively (pre-cultures). Cells from exponential phase (16 h) of cultures were collected by centrifugation (5000 × g for 10 min at 4°C), washed twice with 1/4 Ringer solution and re-suspended in 1/4 Ringer solution (working cultures) in order to be used as inoculum for biofilm assays.

The TSB and TSA for bacterial growth were purchased from Lab M Ltd. (Lancashire, UK). Both types of growth media were prepared under aseptic conditions, using standard procedures. The BSM used consisted of distilled water, 1 g/L K₂HPO₄, 1 g/L KH₂PO₄, 1 g/L KNO₃, 1 g/L (NH₄)₂SO₄, 0.1 g/L MgSO₄·7H₂O, 0.1 g/L NaCl, and 10 mL/L of trace element solution. The trace element solution contained: 2 mg/L CaCl₂, 2 mg/L CuSO4·5H2O, 2 mg/L MnSO₄·5H₂O, 2 mg/L ZnSO₄·5H₂O, 2 mg/L FeSO₄, and 2 mg/L (NH₄)₆Mo₇O₂₄·4H₂O. BSM salts were purchased from BDH Chemicals Ltd. (Poole, UK). The Ringer’s solution was purchased from Lab M Ltd. (Lancashire, UK) and a quarter strength tablet was dissolved in 500 mL of deionized water. All media were sterilized in an autoclave (Priorclave Ltd, London, UK) at standard conditions (121 °C for 15 min).

Samples were enriched in 5 ml tryptone soy broth (TSB, Lab M Limited, Lancashire, UK) containing 6.5% NaCl overnight before culture on CHROM agar MRSA medium (CHROM agar, France) and incubated at 37 °C for 48 h according to Diederen et al.^{13 (link)}. Pink to mauve colonies were purified on mannitol salt agar to obtain pure colonies. Suspected S. aureus colonies were identified by Gram’s stain, hemolytic pattern and coagulase test according to ISO 688-1¹⁴ .
The resistance of Staphylococcus aureus isolates was screened in vitro for cefoxitin by the disk diffusion method according to Clinical and Laboratory Standards Institute¹⁵ on Mueller–Hinton agar (Oxoid Ltd., Hampshire, England) for MRSA identification¹⁶ .

The TSB and TSA for bacterial growth were purchased from Lab M Ltd. (Lancashire, UK). Both of these growth media were prepared using aseptic techniques under standard conditions. The BSM used consisted of distilled water, 1 g/L K₂HPO₄, 1 g/L KH₂PO₄, 1 g/L KNO₃, 1 g/L (NH₄)₂SO₄, 0.1 g/L MgSO₄7H₂O, 0.1 g/L NaCl, and 10 mL/L of a trace element solution that contained 2 mg/L CaCl₂, 2 mg/L CuSO₄5H₂O, 2 mg/L MnSO₄5H₂O, 2 mg/L ZnSO₄5H₂O, 2 mg/L FeSO₄, and 2 mg/L (NH₄)₆Mo₇O₂₄ 4H₂O. The BSM salts used were obtained from BDH Chemicals Ltd. (Poole, UK). The Ringer’s solution used throughout the study was purchased from Lab M Ltd. (Lancashire, UK). A 1/4 strength tablet was added to 500 mL of deionized water and dissolved for regular colony counting (Section 2.4). All of the media mentioned were autoclaved (Priorclave Ltd., London, UK) at 121 °C for 15 min. From 25 mL starter flasks of TSB grown at 30 °C, 1 mL of inocula were dropped into 250 mL TSB media conical flasks to establish a narrow starting range of CFUs for comparison.