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.

Introdução: Nas últimas décadas, o número de casos de infeções de origem alimentar provocadas por Listeria monocytogenes tem vindo a aumentar. Objetivo: Deteção e quantificação de Listeria monocytogenes em queijos de pasta mole produzidos na região a Sul do Tejo. Metodologia: Foram analisadas 30 amostras de queijos de pasta mole de diferentes fabricantes, dos quais 66,7% produzidas a partir de leite de ovelha cru. Resultados: Das amostras analisadas, 10% (n = 3) estavam contaminadas com Listeria monocytogenes e 17% (n = 5) com Listeria innocua. Conclusão: O consumo de queijos de pasta mole está associado com o aumento do risco de ocorrência de listeriose.

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.

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.

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