We isolated and characterized bacteriocin producers Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC from raw milk artisanal cheeses. Their bacteriocins were tolerant to temperatures from 4°C to 100°C and under sterilization conditions (121°C for 15 min). Additionally, the tested bacteriocins remained active after being exposed to pH 2.0 to 10.0 for 2 h. The activity of the bacteriocins was affected by proteolytic enzymes but remained stable after treatment with EDTA, sodium dodecyl sulfate, NaCl, skim milk, and Tween 80. Cell-free supernatants were capable of inhibiting Listeria innocua and several strains of Listeria monocytogenes obtained from different sources and belonging to different serotypes. When L. monocytogenes 211 and L. monocytogenes 422 were treated with bacteriocins, growth was completely inhibited over 12 h. Cocultures of bacteriocinogenic strains and L. monocytogenes 422 in skim milk showed that E. hirae ST57ACC could control the growth of the pathogen in the matrix after 48 h. None of the selected isolates presented positive results on a screening panel for 25 bacteriocin-related genes, however, indicating that both strains might express novel bacteriocins.

Pseudomonas spp. are usually associated with spoilage microflora of dairy products due to their proteolytic potential. This is of particular concern for protein-based products, such as goat milk cheeses and fermented milks. Therefore, the goal of the present study was to characterize the proteolytic activity of Pseudomonas spp. isolated from goat milk. Goat milk samples (n=61) were obtained directly from bulk tanks on dairy goat farms (n=12), and subjected to a modified International Organization for Standardization (ISO) protocol to determine the number and proteolytic activity of Pseudomonas spp. Isolates (n=82) were obtained, identified by PCR, and subjected to pulsed-field gel electrophoresis with XbaI macro-restriction. Then, the isolates were subjected to PCR to detect the alkaline protease gene (apr), and phenotypic tests were performed to check proteolytic activity at 7°C, 25°C, and 35°C. Mean Pseudomonas spp. counts ranged from 2.9 to 4.8 log cfu/mL, and proteolytic Pseudomonas spp. counts ranged from 1.9 to 4.6 log cfu/mL. All isolates were confirmed to be Pseudomonas spp., and 41 were identified as Pseudomonas fluorescens, which clustered into 5 groups sharing approximately 82% similarity. Thirty-six isolates (46.9%) were positive for the apr gene; and 57 (69.5%) isolates presented proteolytic activity at 7°C, 82 (100%) at 25°C, and 64 (78%) at 35°C. The isolates were distributed ubiquitously in the goat farms, and no relationship among isolates was observed when the goat farms, presence of apr, pulsotypes, and proteolytic activity were taken into account. We demonstrated proteolytic activity of Pseudomonas spp. present in goat milk by phenotypic and genotypic tests and indicated their spoilage potential at distinct temperatures. Based on these findings and the ubiquity of Pseudomonas spp. in goat farm environments, proper monitoring and control of Pseudomonas spp. during production are critical.

Consumption of goat milk has been increasing due to its nutritional characteristics and health benefits. Therefore, assessment of the presence of foodborne pathogens in this product is critical to ensure its safety to consumers. The present study aimed to identify common foodborne pathogens in raw goat milk. Fifty-three samples of raw goat milk from 11 farms were collected and cultured for the presence of Salmonella spp. and Listeria monocytogenes, as well as for enumeration and isolation of coagulase-positive and coagulase-negative Staphylococcus (CPS and CNS, respectively). All samples tested negative for Salmonella spp. and L. monocytogenes. The CPS counts in raw goat milk samples were predominantly less than 2 log cfu/mL (n=39), and CNS counts were predominantly higher than 3 log cfu/mL (n=42). Based on Staphylococcus counts, 51 isolates were selected (CPS=26; CNS=25) and tested by PCR for the presence of classic enterotoxin-encoding genes (sea, seb, sec, sed, and see). Only 3 isolates (CPS=2, CNS=1) were negative for all enterotoxin-encoding genes tested, and the genotype sec and see was the most frequent (n=16), followed by sea, sec, and see (n=13) and sec (n=13); sed was not detected in any isolate. The frequencies of enterotoxin-encoding genes for CPS and CNS were similar, demonstrating the equivalence of both groups in harboring these virulent markers. These results suggest that Salmonella and L. monocytogenes are not frequent contaminants of raw goat milk, but that Staphylococcus spp. that are capable of producing enterotoxins are prevalent; therefore, consumers of raw goat milk and products made from raw milk are at risk.

Utensils and equipment from meat-processing facilities are considered relevant cross-contamination points of Listeria monocytogenes to foods, demanding tracking studies to identify their specific origins, and predict proper control. The present study aimed to detect L. monocytogenes in a beef-processing facility, investigating the diversity of serotypes and pulsotypes in order to identify the possible contamination routes. Surface samples from knives ( n =26), tables ( n =78), and employees hands ( n =74) were collected before and during the procedures from a beef-processing facility, in addition to surface samples of end cuts: round ( n =32), loin ( n =30), and chuck ( n =32). All samples were subjected to L. monocytogenes screening according ISO 11.290-1, and the obtained isolates were subjected to serotyping and pulsed-field gel electrophoresis. Listeria spp. were identified in all processing steps, in 61 samples, and L. monocytogenes was detected in 17 samples, not being found only in knives. Eighty-five isolates were identified as L. monocytogenes , from serotypes 1/2c ( n =65), 4b ( n =13), and 1/2b ( n =7), being grouped in 19 pulsotypes. Considering these results, cross-contamination among hands, tables, and beef cuts could be identified. The obtained data indicated the relevance of cross-contamination in the beef-processing facility, and the occurrence of serotypes 1/2b and 4b in beef cuts distributed for retail sale is a public health concern.