A retrospective multivariate case-control approach to investigate factors influencing the sanitary quality of raw milk cheeses

• Control farms associated with Corynebacterium spp. and higher bacterial diversity. • Key critical control points: raw milk, milking machine, wooden shelves. • Temperature and pH during cheesemaking influence cheese quality and safety. • Udder cleaning: case farms favoured pre-soaking and paper wiping. • A promising mbPLS method for analysing complex datasets including genomic data. A retrospective case control study conducted in France between 2022 and 2023, involving 20 farms (10 case and 10 control farms), investigated the factors influencing the sanitary quality of raw milk cheeses, focusing on the agricultural aspect and the bacterial microbiota characterised using 16S rRNA short reads metabarcoding from cheese production and the environment during manufacturing. The microbiological quality of raw milk cheeses is defined in Europe by Regulation (EC) No. 2073/2005. In this study, a set of food safety criteria related to the presence of food-borne pathogens such as Salmonella, Listeria monocytogenes and virulence factors of STEC in cheese for a long-time period (2017 - 2022) prior to sampling was used to select the case farms. Using multivariate analysis, this study identified multiple bacterial species and critical sampling points such as raw milk, milking machines and wooden shelves that contribute to food-borne contamination of cheeses and highlights the importance of farm management practices such as additional vaccination or transhumance, hygiene and manufacturing practices to ensure the safety of raw milk cheeses. The bacterial species from raw milk cheeses or contact materials along the production chain contributing to the case or control farms were diversified in winter and summer. This study confirms the value of multi-block analysis for managing complex datasets and suggests that 16S rRNA markers from metabarcoding can help to identify critical control points and risk factors.