Plate count agar

To evaluate the microbiological quality, a 10 g sample of muscle was mixed with 90 mL buffered peptone water 0.1% w/w (Microkit BCD046, Madrid, Spain) in a stomacher (IUL Instruments, GmBH, Köningswinter, Germany), inside an aseptic cabinet (Telstar, Bio–II–A. Tarrasa, Spain) and serial dilutions were prepared. Bacterial counts were determined as follows: Total Aerobic were determined by the pour plate method in Plate Count Agar (Oxoid CM0325, Basingstoke, UK) incubated at 30 °C for 42 days [18 ]; Psychrophilic bacteria were analysed in Plate Count Agar (Oxoid CM0325, Basingstoke, UK) and incubated at 4 °C for 10 days [19 ]; Lactic Acid Bacteria were counted on Man Rogosa and Sharpe Agar (Oxoid CM0361, Basingstoke, UK) (30 °C for 2 days) [20 ]^; violet red bile glucose agar (Oxoid CM485, Basingstoke, UK) was used for Enterobacteriaceae incubated at 37 °C for 24 h [21 ]; Total Coliforms were determined using a violet red bile glucose agar (Oxoid CM017, Basingstoke, UK) after incubation at 37 °C for 24 h, Pseudomonas were determined by plate seeding on agar base (Oxoid, Basingstoke, UK) (28 °C-24 h), and Moulds and Yeasts was enumerated with Rose Bengal Agar + Chloramphenicol (Oxoid CM0549, Basingstoke, UK) (25 °C for 5 days) [22 ]. Plating was performed in duplicate, and the results were expressed as log colony-forming unit (CFU) g⁻¹.

Spontaneous rifampicin resistant variants of L. monocytogenes strains 0806 (isolated from hummus), 3132 (isolated from avocado), 0352 (isolated from cream cheese), and ScottA (isolated from milk [30 (link)]) were obtained by successive culturing in Brain Heart Infusion broth (BHI; Thermofisher Scientific, Waltham, MA) with increasing concentrations of rifampicin, up to the final level of resistance at 200 μg/mL. Wild-type strains were obtained from the FDA Stock Culture Collection in Bedford Park, IL. All strains were cultured individually in BHI supplemented with 200 μg/mL of rifampicin and incubated at 37°C for 16–18 h. One hundred μL of each strain was plated onto each of 5 replicate BHI agar (BHIA; Thermofisher Scientific, Waltham, MA) plates and incubated at 37°C for 24 h. Cells were harvested by adding 1.5 mL of Butterfield’s Phosphate Broth (BPB; Thermofisher Scientific, Waltham, MA) to each plate and scraping with a sterile disposable culture spreader. Five replicate plates for each strain were harvested and combined into a 50-ml tube, resulting in approximately 40 ml of cocktail (inoculum level approximately 11 log CFU/mL). The four-strain cocktail was serially diluted and plated onto Plate Count Agar (PCA; Thermofisher Scientific, Waltham, MA) with 200 μg/mL rifampicin to verify initial inoculation levels.

In the laboratory, 90 mL of 0.9% NaCl (w/v) and 0.1% (w/v) peptone water were added to each tube containing five pooled swabs and vortexed for 30 s. Tenfold serial dilutions were used for microbiological analyses. Total viable counts (TVC) were performed on spread plates with Plate Count Agar (PCA) (Thermo Fisher Scientific) as described above.