Laked horse blood

After choosing the medium and temperature which allowed better growth of L. monocytogenes separately, the use of several supplements, such as 50 mL/L Laked Horse Blood (Oxoid, United Kingdom), 4 mL/L Campylobacter Growth Supplement (Oxoid, UK), 225 µL/225 mL Half Fraser Supplement (Biokar Diagnostics S.A., Allonne, France) and as well the combinations of these compounds together were tested in mixed cultures with the three bacteria simultaneously. The mixed cultures were prepared in 50 mL of mTA10-MOPS supplemented with the different compounds, and were inoculate with 10–10²CFU of each target species and incubated at 35 °C, 24 h. After the incubation, ten-fold serial dilutions of the culture were plated in different selective media, such as COMPASS (Biokar diagnostics S.A., Allonne, France), CHROMagar™ Salmonella Plus (CHROMagar, Paris, France) and Tryptone Bile X-Glucuronide Agar (TBX) for the quantification of L. monocytogenes, Salmonella spp. and E.coli O157, respectively. Plates of CHROMagar™ Salmonella Plus and COMPASS were incubate at 37 °C and TBX plate at 44 °C, ON.

Bacterial strains used in this study are listed in Table 1. Campylobacter jejuni strains were cultured on Mueller-Hinton (MH) agar under microaerobic conditions (85% N₂, 10% CO₂, 5% O₂) at 42°C. Incubation at 37°C was performed for assays that included comparison between temperatures. Oxygen-limited/anaerobic conditions were achieved using the BD GasPak Sachets system (BD diagnostics, Franklin Lakes, NJ) as described previously (Kassem et al. 2012 (link)). Different oxygen conditions and/or temperatures were used in some assays to be inclusive of varying conditions encountered by C. jejuni in disparate hosts and niches (Kassem et al. 2012 (link)). Laked horse blood (5%, Oxoid, Lenexa, KS), antibiotics (chloramphenicol: 20 μg mL⁻¹, kanamycin: 50 μg mL⁻¹), and the Campylobacter selective supplement (SR155E, Oxoid) were added to the MH medium when necessary.

H. pylori Sydney Strain 1 (SS1) was provided by the Helicobacter pylori Research Laboratory, University of Western Australia. H. pylori J99 strain (700824), Neisseria gonorrhoeae (BAA-1737), Staphylococcus aureus (BAA-811), and S. epidermidis (BAA-35984) were purchased from the American Type Culture Collection (ATCC). H. pylori and N. gonorrhoeae strains were grown on chocolate agar plate supplemented with 7% laked horse blood (Oxoid, Basingstoke, UK) under microaerophilic conditions at 10% CO₂, 37°C in a humidified incubator and were sub-cultured every 3 days. S. aureus and S. epidermidis were cultured on nutrient agar at 37°C in a humidified incubator.
RAW264.7 (ATCC^® TIB-71™) and human THP1 (ATCC^® TIB-202™) macrophages were purchased from the ATCC. RAW264.7 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS at 37°C, 5% CO₂. THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 mM HEPES (pH 7.4), 1 mM sodium pyruvate, 1× non-essential amino acids, and 50 μM 2-mercaptoethanol. RAW264.7 cells were seeded 1 day prior to infection, while THP-1 cells were seeded immediately before infection, at 5 × 10⁵ cells/ml. Freshly cultured bacteria were harvested in brain heart infusion (BHI) broth, measured by a spectrophotometer (OD_650nm of 1 = 1 × 10⁸ cells/ml) and infected at MOI of 1, 5 or 10.