Listeria monocytogenes , Escherichia coli and Coagulase Positive Staphylococci in Cured Raw Milk Cheese from Alentejo Region, Portugal.

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 > 10 4 cfu/g and Extraintestinal E. coli were detected in 15.6%, 16.9% and 10.1% of the samples, respectively. Moreover, L. monocytogenes > 10 2 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.