Raw milk cheeses from Beira Baixa, Portugal: A contributive study for the microbiological quality assessment

In Portugal, traditional cheeses are part of the gastronomic culture, with the accessibility to products of high microbiological quality being valued to guarantee the safety of the consumer's population. These artisanal cheeses are normally made from raw milk, having a complex microbiota that will influence the characteristics, quality and shelf life of the final manufactured products. In this study we evaluated the presence of Escherichia coli, Coagulase Positive Staphylococci (CPS), Listeria monocytogenes and Salmonella spp. in 98 cured raw milk cheeses produced in the Beira Baixa region. Conventional methods (based on ISO standards) and alternative methods (TEMPO®, VIDAS® and VITEK®2) for the quantification, detection or identification of each bacterium were used. Subsequent phenotypic characterization (serotyping and antimicrobial resistance – AMR), as well as genotyping by Whole-Genome Sequencing (WGS) were also performed. The results obtained indicate the presence of E. coli, CPS, L. monocytogenes and Salmonella spp. in 64.3%, 51.0%, 4.1% and 1.0% of the samples, respectively. AMR was present in 41.5% of the E. coli isolates, from which 5.1% were multidrug resistant. In the 2 samples with CPS>4.9x104 cfu/g, staphylococcal enterotoxins (A, B, C, D, E) were not detected in 25 g. Salmonella (S. enterica enterica ser. Duisburg) was detected in one sample. Furthermore, WGS and bioinformatics analysis of L. monocytogenes isolates unveiled clusters of high closely related isolates and correlated links to cheese processing facilities. The findings of this study may indicate, among other factors, a lack of adherence to good hygiene and manufacturing practices along one or more phases of the food chain, e.g. in the milk collection/ storage, in the cheesemaking process and in the distribution, or during shelf-life whilst placed in the market. The results also highlight for the importance of the information provided by the WGS data integration, helping in public health surveillance while revealing possible infection contamination routes and epidemiological links, which contributes to the investigation of potential foodborne outbreaks.

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