Xylose lysine deoxycholate (xld)

Four whole lettuce leaves were submerged in 100 ml of minimal medium containing 4.4 log CFU/ml of S. Enteritidis SE86 and incubated at 36 ± 1°C for 120 h.
Aliquots of 6 ml were removed every two hours for up to 60 and, after each time period, aliquots were withdrawn every 24 h for up to 120 h of culture in order to determine the emulsification index (IE24), pH, and bacterial count. Bacterial counts were performed in triplicate by seeding the samples onto plates containing xylose lysine deoxycholate agar (XLD: Merck, Darmstadt, Germany) and incubated at 36 ± 1°C for 24 h. The pH was evaluated by aliquots (10 ml) of the samples and then analyzed with a pH meter (PHTECK). The emulsification index (IE24) was assessed using the method described by Cooper and Goldenberg (1987) (link).
All experiments were repeated three times and the averages were subsequently expressed as the final result.

Previous studies have shown, Salmonella Typhimurium, Salmonella Derby and Salmonella London to be the most common serovars in pig carcasses and retailed pork in Vietnam [12 (link),13 (link),27 ]. These serovars of S. enterica, isolated from pig carcasses and retailed pork in a recent investigation by Dang-Xuan [27 ], were used to make an inoculation medium. First, each serovar was recovered and then cultured separately in a 150 mL-conical flask containing 50 mL Buffered Peptone Water (BPW) (Merck, Darmstadt, Germany) at 37 °C overnight (without agitation). The following day, Salmonella concentration was determined applying a plate count technique using Xylose Lysine Desoxycholate (XLD, Merck) agar for each cultured medium. Duplicate plates were made by spreading 0.1 mL of cultured BPW, diluted 10-fold with Maximum Recovery Diluent (MRD, Merck), on XLD plates and incubated at 37 °C for 20–24 h. After determining the Salmonella concentration, the culture was diluted to a concentration of 10⁵ CFU/mL. Following this 5 mL of medium containing each incubated serovar of Salmonella with 10⁵ CFU/mL were mixed, and 15 mL of the medium prepared. Then, MRD (90 mL) was added to 10 mL of the inoculated medium to achieve a concentration of 10⁴ CFU/mL medium (100 mL), which was then used to inoculate the pork.

Each raw beef patty sample was collected under aseptic conditions and mixed with buffered peptone water (Panreac, Darmstadt, Germany). To determine the TVC, Plate Count Agar (PCA; Panreac) was employed and incubated at 37 °C for 48 h. Enumeration of the LAB was performed using De Man, Rogosa, and Sharpe agar (MRS; Merck, Darmstadt, Germany), with an incubation period of 48 h at 37 °C. For Enterobacteriaceae enumeration, Violet Red Bile Glucose Agar (VRBG; Panreac) with a double layer was used and incubated at 37 °C for 24 h. Microbiological analyses were conducted in duplicate, and microbial counts were expressed as logarithms of colony-forming units per gram (log CFU/g). Additionally, the presence of Salmonella sp. was assessed in 25 g of the beef patties. For this purpose, a 25 g sample was mixed with 225 mL of sterile buffered peptone water solution (0.1%, Panreac) for bacterial prepropagation. Selective proliferation of bacteria was carried out using Rappaport-Vassiliadis Soy Broth medium (Merck) and bacterial isolation was performed using Hektoen (Merck KGaA, Darmstadt, Germany) and Xylose Lysine Deoxycholate (XLD; Merck) agars. Previously, the microbiological characterization of CF was conducted, and the results are as follows: TVC: 6.83 log CFU/g; Enterobacteriaceae: <1.6 log CFU/g; LAB: 2.71 log CFU; Salmonella sp.: absence; and Listeria monocytogenes: absence.

The detection of Salmonella spp. was performed in accordance with DIN EN ISO 6579-1. For pre-enrichment, the swabs were immersed in pre-warmed buffered peptone water (BPW) (Merck, Darmstadt, Germany) and incubated at 37 ± 1 °C for 18 h. The swab samples were fully covered with peptone water during this process.
For cultivation, three droplets of the incubated BPW (≥0.1 mL in total) were pipetted separately onto a modified semi-solid Rappaport–Vassiliadis (MSRV) medium (Oxoid, Waltham, MA, USA). The plates were then incubated at 41.5 °C for 24 ± 3 h.
After the initial 24 ± 3 h of incubation, the plates were examined for any signs of growth. If no growth was observed, the plates were further incubated for an additional 24 ± 3 h and rechecked.
In the event that a swarming zone was detected, colonies were subcultured from the outer swarming zone onto agar modified with xylose lysine deoxycholate (XLD; Merck, Darmstadt, Germany) and Rambach agar (Merck, Darmstadt, Germany). The subcultures were then incubated at 34–38 °C for 24 ± 3 h. Presumptive Salmonella isolates were subsequently transferred onto Columbia agar (Merck, Darmstadt, Germany) with sheep blood (Thermo Scientific, Waltham, MA, USA) and incubated for 24 ± 3 h. The confirmation of Salmonella spp. was achieved using MALDI-TOF mass spectrometry (Bruker, Billerica, MA, USA) after this step.

The bacterial strains that were selected for the host range screen and other experiments are listed in Table 1. In addition, S. enterica subsp. enterica serotype Typhi NCTC 160 was used as a host strain for the isolation and characterization of the phage. The bacterial culture was grown at 37 °C in tryptone soy broth (TSB) (Oxoid, Basingstoke, UK) or xylose lysine deoxycholate (XLD) (Merck, Darmstadt, Germany).