The objective of this work was to deliver a comprehensive genetic characterization of a collection of E. coli strains isolated from raw sheep milk. To complete our purpose, the technique of whole-genome sequencing, coupled with bioinformatics and phenotypic characterization of antimicrobial resistance, was performed. These Gram-negative, facultative anaerobic bacteria belong to the family Enterobacteriaceae, together with other intestinal pathogens, such as Shigella spp. and Salmonella spp. Genetic analysis was carried out on all strains (phylogram, sequence types, VFs, AMR genes, and pangenome). The results showed the presence of various genetic traits that are related to virulence factors contributing to their pathogenic potential. In addition, genes conferring resistance to antibiotics were also detected and confirmed using phenotypic tests. Finally, the genome of the E. coli strains was characterized by the presence of several mobile genetic elements, thus facilitating the exchange of various genetic elements, associated with virulence and antimicrobial resistance, within and beyond the species, through horizontal gene transfer. Contaminated raw sheep milk with pathogenic E. coli strains is particularly alarming for cheese production in artisan dairies.

The role of Enterococcus spp. in food is debated since this group of lactic acid bacteria contains opportunistic pathogenic strains, some of which exhibit a multidrug-resistant profile. In livestock farms, the use of antibiotics is the most common practice to deal with mastitis-causing bacteria. However, the heavy usage and/or misuse of antibiotics has led to the emergence of antibiotic resistance. This study aimed to genetically and phenotypically characterize Enterococcus strains isolated from raw sheep milk. Samples were collected over one year from the bulk tank of a dairy sheep farm and cultured on selective media. Isolates were purified and analyzed by whole-genome sequencing and antimicrobial susceptibility testing. The isolates were divided into clusters and the corresponding species were identified along with their genes related to virulence and antibiotic resistance. The pan-, core- and accessory-genomes of the strains were determined. Finally, the antibiotic-resistant profile of selected strains was examined and associated with their genomic characterization. These findings contribute to a better understanding of Enterococci epidemiology, providing comprehensive profiles of their virulence and resistance genes. The presence of antibiotic-resistant bacteria in raw sheep milk destined for the production of cheese should raise awareness.

Soft spread cheese is highly perishable, and conventional packaging offers limited protection against surface spoilage. Here, we present a sustainable, multifunctional solution: edible films made from whey protein concentrate (WPC), a valuable by-product of the cheese industry, incorporated with the probiotic Lactobacillus acidophilus LA5 (LA5). The objective of this study was to evaluate these films as active coatings for soft cheese, specifically assessing their physicochemical properties, probiotic viability during storage and simulated gastric transit, and their impact on cheese microbial stability and sensory quality over 60 days. Applied as active coatings on soft cheese stored at 4 °C for 60 days, these films were evaluated for their physicochemical properties, probiotic viability, microbial stability, and sensory acceptance. The incorporation of LA5 did not significantly alter film thickness (control: 0.20 ± 0.03 mm; test: 0.18 ± 0.02 mm), moisture content (control: 33.42 ± 0.54%; test: 32.34 ± 1.28%), or water solubility (control: 21.44 ± 1.14%; test: 22.89 ± 0.75%) (p > 0.05). However, mechanical properties were markedly modified: tensile strength decreased from 35.42 ± 5.38 MPa (control) to 6.04 ± 0.55 MPa (test), while elongation at break increased from 4.87 ± 0.93% to 68.23 ± 3.46% (p < 0.05), indicating a transition from rigidity to flexibility upon probiotic incorporation. The probiotic strain exhibited exceptional resilience, retaining 100% viability during simulated gastric exposure at both day 0 and day 30 of storage. During cheese storage, LA5 counts in test film-coated samples remained above the recommended therapeutic threshold (10⁶ cfu/g), starting at 7.44 ± 0.15 log(cfu/g) on day 0 and maintaining 6.56 ± 0.20 log(cfu/g) after 60 days. Critically, yeast and mold spoilage were delayed in probiotic-coated cheese, with detectable growth appearing only at day 60 (1.64 ± 1.34 log(cfu/g)), whereas uncoated cheese showed spoilage as early as day 28 (1.33 ± 1.62 log(cfu/g)). Sensory evaluation revealed no significant differences (p > 0.05) between the coated and uncoated samples for color, appearance, texture, flavor, or overall acceptability. By valorizing a dairy by-product into an active, probiotic-loaded edible film, this approach offers a sustainable, waste-reducing strategy that enhances cheese preservation while delivering added functional value—bridging the gap between food packaging and nutrition. Sensory evaluation (n = 8, preliminary) indicated no significant differences between coated and uncoated samples, but these results require confirmation with a larger, validated panel.