4.1. Sample Collection and Bacterial Isolation

Samples were taken from three meat processing units in Timiș County, Romania. Each was sampled during a single visit. Scrapings and swabs were used to collect samples from the surface of carcasses that had been kept in cooling chambers for at least three days. The aseptically collected samples were each packaged in a polyethylene bag (Sterile Ziploc^®, Sklar Instruments, West Chester, PA, USA), labeled with the sampling date and the area, and delivered under refrigeration conditions (4 °C) to the Food Hygiene and Microbiological Risk Assessment Laboratory, Faculty of Veterinary Medicine, Timisoara [37].

Twenty samples were collected for biofilm screening by scraping the surfaces of the carcasses using sterile surgical blades (SMI, Steinerberg, Belgium) that were subsequently placed in sterile tubes (Deltalabs, Barcelona, Spain).

For the pork carcasses, 10 sampling points were chosen, namely: the masseter muscles, neck area, chest, sternum, intercostal region, axillary region, ventral abdominal area, lumbar region, coccygeal area, and inner thigh area.

For the beef carcasses, seven sampling points were established, namely: the neck area, chest, rib cage area, thoracoabdominal region (dorsal and ventral), inner thigh area, and gluteal muscles area.

For the poultry carcasses, the samples were collected from three sampling points: the chest, axillary region, and back area. The sampling points were adapted from the protocols presented by Barros et al. [38]. The 20 resulting samples were stained with acridine orange (Sigma-Aldrich, Darmstadt, Germany) for one minute, with the dehydrated samples being treated with Hanks modified solution (without D glucose and phenol red) and fixed in ethanol 96% for two minutes before being placed on the slide. A Leica DM 2500 epifluorescent microscope (Leica Microsystems, Wetzlar, Germany) with Leica EL 6000 external UV light source was used to visualize the biofilm. The machine was calibrated at a wavelength of 488 nm, and the visualization was performed with 63× and 100× immersion objectives.

Sample collection for the microbiological testing was performed from a surface of 100 cm², using sterile swabs (Deltalabs, Barcelona, Spain) immersed in physiological peptone solution (PBS, ThermoFisher Scientific, Canada) [38]. The sampling points were identical to the points established for biofilm detection. The number of analyzed carcasses was higher, namely five pork carcasses (50 samples), five beef carcasses (35 samples), and 15 poultry carcasses (45 samples), resulting in a total of 130 swab samples. The sampling protocol was conducted according to ISO 17604/2015 [39].

In the laboratory, the swab samples were immersed in 9 mL of preheated (45 °C) peptone-buffered solution (PBS; pH = 7.5 ± 0.1). Next, serial dilutions of up to 10⁻³ (1:1000) in sterile 0.5% peptone water were prepared; subsequently, 1 mL from each dilution was transferred into a sterile Petri dish (in duplicate) with specific isolation media.

The total aerobic colony count (TACC) was assessed according to ISO 4833/2003 [40] and Commission Regulation (EC) No 1441/2007 using a PCA culture medium and an incubation period of 72 h at 30 °C [17].

The Enterobacteriaceae level was assessed according to SR ISO 21528-2/2007 [41]. The samples were cultured on VRBG agar (violet, red, bile, and glucose agar; Biokar Diagnostics, Allone, France) at 37 °C for 24 h. Specific red and violet colonies were subjected to oxidase production and glucose fermentation tests.

According to the regulatory standards established by Commission Regulation (EC) No. 1441/2007 [17] for Enterobacteriaceae and TACC, the results were categorized into the following three categories: (1) satisfactory (if the daily mean Log is m, an acceptable level of contamination); (2) acceptable (if it is between m and M, a dangerous level of contamination); or (3) unsatisfactory (if the daily mean is M). The m and M corresponded to 3.5 and 5.0 log CFU/cm² for TACC and to 1.5 and 2.5 log CFU/cm² for Enterobacteriaceae, respectively.

The identification of E. coli was carried out according to ISO 16649-2/2001 [42] on a chromogenic selective medium consisting of tryptone bile with X-glucuronide (TBX) agar (Oxoid, Basingstoke, Hampshire, UK) at 37 °C, for 4 h and then at 43.5 °C for 24 h. Pseudomonas spp. were isolated on Cetrimid agar (Biokar Diagnostics, Allone, France) at a temperature of 37 °C for 18–24 h according to ISO 13720:2010 [43].