Listeria monocytogenes, a foodborne pathogen, exhibits high adaptability to adverse environmental conditions and is common in the food industry, especially in ready-to-eat foods. L. monocytogenes strains pose food safety challenges due to their ability to form biofilms, increased resistance to disinfectants, and long-term persistence in the environment. The aim of this study was to evaluate the presence and genetic diversity of L. monocytogenes in food and related environmental products collected from 2014 to 2022 and assess antibiotic susceptibility and biofilm formation abilities. L. monocytogenes was identified in 13 out of the 227 (6%) of samples, 7 from food products (meat preparation, cheeses, and raw milk) and 6 from food-processing environments (slaughterhouse-floor and catering establishments). All isolates exhibited high biofilm-forming capacity and antibiotic susceptibility testing showed resistance to several classes of antibiotics, especially trimethoprim-sulfamethoxazole and erythromycin. Genotyping and core-genome clustering identified eight sequence types and a cluster of three very closely related ST3 isolates (all from food), suggesting a common contamination source. Whole-genome sequencing (WGS) analysis revealed resistance genes conferring resistance to fosfomycin (fosX), lincosamides (lin), fluoroquinolones (norB), and tetracycline (tetM). In addition, the qacJ gene was also detected, conferring resistance to disinfecting agents and antiseptics. Virulence gene profiling revealed the presence of 92 associated genes associated with pathogenicity, adherence, and persistence. These findings underscore the presence of L. monocytogenes strains in food products and food-associated environments, demonstrating a high virulence of these strains associated with resistance genes to antibiotics, but also to disinfectants and antiseptics. Moreover, they emphasize the need for continuous surveillance, effective risk assessment, and rigorous control measures to minimize the public health risks associated to severe infections, particularly listeriosis outbreaks. A better understanding of the complex dynamics of pathogens in food products and their associated environments can help improve overall food safety and develop more effective strategies to prevent severe health consequences and economic losses.

Traditional cheeses are part of the Portuguese gastronomic identity, and raw milk of autochthonous species is a common primary ingredient. Here, we investigated the presence of Listeria monocytogenes, Coagulase Positive Staphylococci (CPS) and pathogenic Escherichia coli, as well as of indicator microorganisms (E. coli and other Listeria spp.) in 96 cured raw milk cheeses from the Alentejo region. Whole genome sequencing (WGS) of pathogenic E. coli and Listeria spp. as well as antimicrobial resistance (AMR) screening of E. coli isolates was also performed. L. monocytogenes, CPS > 10⁴ cfu/g and Extraintestinal E. coli were detected in 15.6%, 16.9% and 10.1% of the samples, respectively. Moreover, L. monocytogenes > 10² cfu/g and Staphylococcal enterotoxins were detected in 4.2% and 2.2% of the samples, respectively. AMR was observed in 27.3% of the E. coli isolates, six of which were multidrug resistant. WGS analysis unveiled clusters of high closely related isolates for both L. monocytogenes and L. innocua (often correlating with the cheese producer). This study can indicate poor hygiene practices during milk collection/preservation or during cheese-making procedures and handling, and highlights the need of more effective prevention and control measures and of multi-sectoral WGS data integration, in order to prevent and detect foodborne bacterial outbreaks.

Low-density polyethylene-based packaging with 4% lemon extract (LDPE/4LE) and two polylactic-based (PLA) packaging materials with 4% and 6% lemon extract (PLA/PEG/4LE and PLA/6LE) were produced. O₂ and water permeability tests were performed, the total and individual phenolic compounds content were measured, and the films’ antioxidant activities were determined. The films’ ability to delay lipid oxidation was tested in two model foods: almonds, packaged with LDPE/4LE, PLA/4LE and PLA/6LE for a maximum period of 60 days at 40 °C (accelerated assay); and beef meat, packaged with the PLA/6LE for a maximum period of 11 days at 4 °C. The LE improved the WVP in all of the active films by 33%, 20% and 60% for the LDPE/4LE, PLA/4LE and PLA/6LE films, respectively. At the end of 10 days, the migration of phenolic compounds through the PLA films was measured to be 142.27 and 114.9 μg/dm² for the PLA/4LE and PLA/6LE films, respectively, and was significantly higher than phenolic compounds migration measured for the LDPE/4LE (15.97 μg/dm²). Naringenin, apigenin, ferulic acid, eriocitrin, hesperidin and 4-hydroxybenzoic acid were the main identified compounds in the PLA, but only 4-hydroxybenzoic acid, naringenin and p-coumaric acid were identified in the LDPE films. Regarding the films’ ability to delay lipid oxidation, LDPE/4LE presented the best results, showing a capacity to delay lipid oxidation in almonds for 30 days. When applied to raw beef meat, the PLA/6LE packaging was able to significantly inhibit lipid oxidation for 6 days, and successfully inhibited total microorganisms’ growth until the 8th day of storage.

Birds are potential carriers of pathogens affecting humans and agriculture. Aiming to evaluate the occurrence of the top three most important foodborne pathogens in free-living birds in Portugal, we investigated 108 individual fecal samples from free-living birds and one pooled sample of gull feces (n = 50) for the presence of Escherichia coli (pathogenic and non-pathogenic), Salmonella spp. and Campylobacter spp. Virulence- and antimicrobial resistance- (AMR) associated genes were detected by PCR and Whole-Genome Sequencing (WGS), and phenotypic (serotyping and AMR profiles) characterization was performed. Overall, 8.9% of samples tested positive for pathogenic E. coli, 2.8% for Salmonella spp., and 9.9% for Campylobacter spp. AMR was performed on all pathogenic isolates and in a fraction of non-pathogenic E. coli, being detected in 25.9% of them. Ten of the tested E. coli isolates were multidrug-resistant (MDR), and seven of them were Extended-spectrum β-lactamase (ESBL) producers. Among Salmonella (n = 3) and Campylobacter (n = 9), only one strain of C. jejuni was identified as MDR. Most of the identified serotypes/sequence types had already been found to be associated with human disease. These results show that free-living birds in Portugal may act as carriers of foodborne pathogens linked to human disease, some of them resistant to critically important antimicrobials.

The search for new forms to extend foodstuffs’ shelf-life through the use of natural compounds and extracts continues to grow among researchers and the industry. In this line of thought, the main objective of this work was to develop, characterize and evaluate the effectiveness of an active food packaging based on polylactic acid (PLA) and incorporated with natural extracts obtained from grape and/or pomegranate by-products. In vitro, antioxidant methods were applied to evaluate the antioxidant capacity of three extracts: wort extract, freeze-dried pomegranate peels extract (PPE-FD), and natural pomegranate peels (PPE-N). Punicalagin (A + B) and ellagic acid were determined in PPE-FD, PPE-N, and active films by UHPLC-DAD. New PLA-based active packaging with PPE-FD (PLA/3PPE) and pomegranate peels (PLA/3PP) were developed, and their chemical, mechanical, and barrier properties were evaluated. Moreover, the effectiveness of the new film was carried out through the study of lipid oxidation state and microbial contamination of two high-fat content foodstuffs, almonds and beef meat. PPE-FD presented high antioxidant capacity and high content in total phenolics and flavonoid compounds. The PLA/3PPE and PLA/3PP prevented the lipid oxidation of meat and presented antimicrobial activity against Staphylococcus aureus. The addition of the PPE-FD and the pomegranate peels to the PLA did not affect the morphology of the polymer. Although both PPE-FD and pomegranate peels presented punicalagin (A + B) and ellagic acid, only ellagic acid was identified and quantified in the active PLA. Active PLA films were not effective in delaying the lipid oxidation of almonds, but they showed to be significantly effective in delaying the lipid oxidation of beef meat and reducing the microbial growth in this food matrix over time.

Human–wildlife coexistence may increase the potential risk of direct transmission of emergent or re-emergent zoonotic pathogens to humans. Intending to assess the occurrence of three important foodborne pathogens in wild animals of two wildlife conservation centers in Portugal, we investigated 132 fecal samples for the presence of Escherichia coli (Shiga toxin-producing E. coli (STEC) and non-STEC), Salmonella spp. and Campylobacter spp. A genotypic search for genes having virulence and antimicrobial resistance (AMR) was performed by means of PCR and Whole-Genome Sequencing (WGS) and phenotypic (serotyping and AMR profiles) characterization. Overall, 62 samples tested positive for at least one of these species: 27.3% for STEC, 11.4% for non-STEC, 3.0% for Salmonella spp. and 6.8% for Campylobacter spp. AMR was detected in four E. coli isolates and the only Campylobacter coli isolated in this study. WGS analysis revealed that 57.7% (30/52) of pathogenic E. coli integrated genetic clusters of highly closely related isolates (often involving different animal species), supporting the circulation and transmission of different pathogenic E. coli strains in the studied areas. These results support the idea that the health of humans, animals and ecosystems are interconnected, reinforcing the importance of a One Health approach to better monitor and control public health threats.