Hektoen enteric agar

Twenty-five grams of salami samples were transferred separately into plastic one-chamber filter stomacher bags (Neomed, Milano, Italy) and then homogenized 1:10 (w:v) in sterile peptone water (PW, Conda, Madrid, Spain) for 3 min using a Stomacher 400 blender (Seward Medical, London, UK). Decimal dilutions in sterile PW were then prepared.
Mesophilic lactic acid bacteria (LAB) were enumerated in the control salami by pour plating 1 mL of appropriate dilution in de Man–Rogosa–Sharpe agar (MRSA) (Microbiol Diagnostici, Cagliari, Italy), and the plates were incubated in accordance with ISO 15214 [30 ].
In the contaminated salami, L. monocytogenes enumeration was performed according to ISO 11290-2 [31 ]. For the enumeration of Salmonella spp., appropriate dilutions were surface-plated onto Hektoen enteric agar (HEA) (Oxoid, Milano, Italy). Typical colonies were counted after the incubation of duplicate plates at 37 °C for 24 h. To verify the absence of natural contamination of raw meat, at time zero, the enumeration of pathogens was also investigated in the control salami.
The quality of each culture medium used in this study was evaluated in accordance with ISO 11133-1 [32 ] and ISO 11133-2 [33 ].

Isolation of Salmonella spp. was firstly performed by pre-enrichment of samples in lactose broth (Merck, Darmstadt, Germany) at 37 °C for 24 h. For selective enrichment, pre-enriched cultures were transferred into Selenite Cystine (SC) broth (Merck, Darmstadt, Germany) and Tetrathionate Brilliant Green bile (TBG) broth (Merck, Darmstadt, Germany), and incubated at 35 °C for 24 h. Then, these cultures were streaked onto Bismuth Sulphite agar (BSA) (Oxoid, Basingstoke, UK), Xylose Lysine Deoxycholate Agar (XLD) (Oxoid, Basingstoke, UK), and Hektoen Enteric agar (HEA) (Oxoid, Basingstoke, UK) as selective media and incubated at 35 °C for 48 h. Typical colonies were cultured on the slants of Tryptic soy agar (TSA) (Merck, Darmstadt, Germany) and subjected to biochemical tests using Lysine Iron agar (LIA) (Merck, Darmstadt, Germany), Triple Sugar Iron (TSI) agar (Merck, Darmstadt, Germany), Sulfide-Indole-Motility (SIM) medium (Merck, Darmstadt, Germany), and Christensen’s Urea agar (Merck, Darmstadt, Germany) (Table S1) [89 ].

The samples were inoculated in 40 ml of nonselective tryptic soy broth (TSB) (Oxoid Ltd., Basingstoke Hampshire, UK) and incubated for two hours at 37°C with shaking at 100 rpm. Thereafter, 0.1 ml was inoculated into 9.9 ml of Rappaport-Vassiliadis broth (Oxoid Ltd., Basingstoke, UK), a selective enrichment media for Salmonella and incubated at 42°C for 24 hours [17 (link)]. A loopful of the sample was streaked onto Brilliance Salmonella Agar (Oxoid Ltd., Basingstoke Hampshire, England) and incubated at 37°C for 24 hours [18 (link)]. Single presumptive purple/pink colonies typical of Salmonella were picked and subsequently streaked onto Hektoen Enteric Agar (HEA) (Oxoid Ltd., Basingstoke Hampshire, England) and Salmonella Shigella agar (SSA) (Oxoid Ltd., Basingstoke Hampshire, England) plates and incubated at 37°C for 24 hours. Culture plates were examined for the presence of typical colonies based on morphological characteristics, i.e., clear colonies with a black centre on HEA and SSA. Single colonies were subcultured onto nutrient agar and subsequently stored in TSB supplemented with 10% glycerol (Merck, USA) at −60°C until further analysis. Salmonella enterica subsp. enterica serovar Choleraesuis ATCC 10708 (ATCC, Manassas, Virginia, USA) was included as a control strain.