Raw milk cheeses from Beira Baixa, Portugal-A contributive study for the microbiological hygiene and safety assessment.

Due to specific bacterial microbiota, raw milk cheeses have appreciated sensory properties. However, they may pose a threat to consumer safety due to potential pathogens presence. This study evaluated the microbiological contamination of 98 raw milk cheeses from Beira Baixa, Portugal. Presence and enumeration of Coagulase Positive Staphylococci (CPS), Listeria monocytogenes, Salmonella spp., pathogenic Escherichia coli, and indicator microorganisms (non-pathogenic E. coli and Listeria spp.) was attained. E. coli antimicrobial resistance (AMR) was also evaluated. PCR and/or Whole genome sequencing (WGS) was used to characterize E. coli, Salmonella spp. and L. monocytogenes isolates. Sixteen cheeses (16.3%) were classified as Satisfactory, 59 (60.2%) as Borderline and 23 (23.5%) as Unsatisfactory/Potential Injurious to Health. L. monocytogenes, CPS > 104 cfu g-1, Extraintestinal pathogenic E. coli (ExPEC) and Salmonella spp. were detected in 4.1%, 6.1%, 3.1% and 1.0% of the samples, respectively. Listeria innocua (4.1%) and E. coli > 104 cfu g-1 (16.3%) were also detected. AMR E. coli was detected in 23/98 (23.5%) of the cheese samples, of which two were multidrug resistant. WGS identified genotypes already associated to human disease and Listeria spp. cluster analysis indicated that cheese contamination might be related with noncompliance with Good Hygiene Practices during cheese production.

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 > 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.

Pathogenic Escherichia coli, Salmonella spp. and Campylobacter spp. in Two Natural Conservation Centers of Wildlife in Portugal: Genotypic and Phenotypic Characterization.

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.

Genotypic and Phenotypic Characterization of Pathogenic Escherichia coli, Salmonella spp., and Campylobacter spp., in Free-Living Birds in Mainland Portugal.

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 ß-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.