The Enterotoxin Gene Profiles and Enterotoxin Production of Staphylococcus aureus Strains Isolated from Artisanal Cheeses in Belgium.

A Staphyloccoccus aureus is one of the leading causes of food poisoning outbreaks (FPOs) worldwide. Staphylococcal food poisoning (SFP) is induced by the ingestion of food containing sufficient levels of staphylococcal enterotoxins (SEs). Currently, 33 SEs and SE-like toxins (SEls) have been described in the literature, but only five named "classical" enterotoxins are commonly investigated in FPOs due to lack of specific routine analytical techniques. The aims of this study were to (i) establish the genetic profile of strains in a variety of artisanal cheeses (n = 30) in Belgium, (ii) analyze the expression of the SE(l)s by these strains and (iii) compare the output derived from the different analytical tools. Forty-nine isolates of S. aureus were isolated from ten Belgian artisanal cheeses and were analyzed via microbiological, immunological, liquid chromatography mass spectrometry, molecular typing and genetic methods. The results indicated that classical SEs were not the dominant SEs in the Belgian artisanal cheeses that were analyzed in this study, and that all S. aureus isolates harbored at least one gene encoding a new SE(l). Among the new SE(l)s genes found, some of them code for enterotoxins with demonstrated emetic activity and ecg-enterotoxins. It is worth noting that the involvement of some of these new SEs has been demonstrated in SFP outbreaks. Thus, this study highlighted the importance of the development of specific techniques for the proper investigation of SFP outbreaks.

Integration of Multiomic Data to Characterize the Influence of Milk Fat Composition on Cantal-Type Cheese Microbiota.

A previous study identified differences in rind aspects between Cantal-type cheeses manufactured from the same skimmed milk, supplemented with cream derived either from pasture-raised cows (P) or from cows fed with maize silage (M). Using an integrated analysis of multiomic data, the present study aimed at investigating potential correlations between cream origin and metagenomic, lipidomic and volatolomic profiles of these Cantal cheeses. Fungal and bacterial communities of cheese cores and rinds were characterized using DNA metabarcoding at different ripening times. Lipidome and volatolome were obtained from the previous study at the end of ripening. Rind microbial communities, especially fungal communities, were influenced by cream origin. Among bacteria, Brachybacterium were more abundant in P-derived cheeses than in M-derived cheeses after 90 and 150 days of ripening. Sporendonema casei, a yeast added as a ripening starter during Cantal manufacture, which contributes to rind typical aspect, had a lower relative abundance in P-derived cheeses after 150 days of ripening. Relative abundance of this fungus was highly negatively correlated with concentrations of C18 polyunsaturated fatty acids and to concentrations of particular volatile organic compounds, including 1-pentanol and 3-methyl-2-pentanol. Overall, these results evidenced original interactions between milk fat composition and the development of fungal communities in cheeses.

Study of the microbial diversity of a panel of Belgian artisanal cheeses associated with challenge studies for Listeria monocytogenes.

High throughput sequencing could become a powerful tool in food safety. This study was the first to investigate artisanal cheeses from Belgium (31 batches) using metagenetics, in relation to Listeria monocytogenes growth data acquired during a previous project. Five cheese types were considered, namely unripened acid-curd cheeses, smear- and mold-ripened soft cheeses, and Gouda-type and Saint-Paulin-type cheeses. Each batch was analyzed in triplicate the first and the last days of storage at 8 °C. Globally, 2697 OTUs belonging to 277 genera and to 15 phyla were identified. Lactococcus was dominant in all types, but Streptococcus was co-dominant in smear-ripened soft cheeses and Saint-Paulin-type cheeses. The dominant population was not always associated with added starter cultures. Bacterial richness and diversity were significantly higher in both types of soft cheeses than in other categories, including particular genera like Prevotella, Faecalibacterium and Hafnia-Obesumbacterium in mold-ripened cheeses and Brevibacterium, Brachybacterium, Microbacterium, Bacteroides, Corynebacterium, Marinilactibacillus, Fusobacterium, Halomonas and Psychrobacter in smear-ripened soft cheeses. A strong correlation was observed between no growth of L. monocytogenes in a smear-ripened cheese and the presence of an unknown Fusobacterium (relative abundance around 10%). This in silico correlation should be confirmed by further experiments in vitro and in situ.

Study of the bacterial profile of raw milk butter, made during a challenge test with Listeria monocytogenes, depending on cream maturation temperature.

Bacteria can play different roles and impart various flavors and characteristics to food. Few studies have described bacterial microbiota of butter. In this study, next-generation sequencing was used to determine bacterial content of raw milk butter, processed during a challenge test, depending on cream maturation temperature and on the presence or not of L. monocytogenes. Two batches were produced. pH and microbiological analyses were conducted during cream maturation and butter storage. DNA was also isolated from all samples for 16S rRNA amplicon sequencing analysis. For butter made from cream matured at 14 °C, a growth potential of L. monocytogenes of - 1.72 log cfu/g was obtained. This value corresponds to the difference between the median of counts at the end of storage and the median of counts at the beginning of storage. This butter (pH value of 4.75 ± 0.04) was characterized by a dominance of Lactococcus. The abundance of Lactococcus was significantly higher in inoculated samples than in control samples (p value < 0.05). Butter made from cream matured at 4 °C (pH value of 6.81 ± 0.01) presented a growth potential of 1.81 log cfu/g. It was characterized by the abundance of psychrotrophic bacteria mainly Pseudomonas. This study demonstrated that cream maturation temperature impacts butter microbiota, affecting thus product's characteristics and its ability to support or not the growth of pathogens like L. monocytogenes.

Determination of the growth potential of Listeria monocytogenes in various types of Belgian artisanal cheeses by challenge tests.

Cheese potentially allowing the growth of Listeria monocytogenes must be free of the pathogen in 25 g before being put on the market, while 100 cfu/g is tolerated when the pathogen is unable to grow. Challenge tests were performed in order to assess the growth potential of L. monocytogenes in at least one batch of 32 Belgian cheese varieties from 32 factories. All varieties were grouped in four categories: unripened acid-curd cheeses, mold-ripened soft cheeses, smear-ripened soft cheeses and ripened semi-hard cheeses. Associated microflora and cheese physicochemical characteristics were also studied. A cocktail of three strains was used to inoculate cheese on the first day of shelf-life, and samples were stored until the end of shelf-life at 7-9 °C. Growth potential was considered as the difference (a) between median contamination at the end and at the beginning of the test or (b) between the highest value at the end of the test and the lowest value at its beginning. L. monocytogenes always decreased in unripened acid-curd cheeses but showed extended growth in 21 out of 25 batches of ripened soft cheese. Contrasting results were obtained for semi-hard cheeses, as important intra- and inter-batch variability was observed. For the latter, the recommended method based on medians to calculate the growth potential led to erroneous food safety considerations, and it should always be advised to focus on absolute levels.

Assessment of growth and survival of Listeria monocytogenes in raw milk butter by durability tests.

Butter is a complex matrix characterized by a high fat content. Existing publications on the behavior of Listeria monocytogenes in this type of food reported contrasted results. This study was performed to provide further information and data about raw milk butter's ability to support survival or growth of L. monocytogenes. Durability tests were performed on naturally contaminated samples of raw milk butter with various physico-chemical characteristics. At the end of shelf life, no growth of L. monocytogenes was observed in the studied butters, regardless of their physico-chemical characteristics (pH, aw, water dispersion index and salt concentration) and the initial level of contamination. The number of positive samples and the colony counts of L. monocytogenes were even decreased at the end of the storage period.

Development of real-time PCR tests for the detection of Tenebrio molitor in food and feed.

Insects are rich in proteins and could be an alternative source of proteins to feed animals and humans. Numerous companies have started the production of insects for feed purposes. In Europe, these processed animal proteins are not yet authorised by legislation as many questions still need to be answered concerning this 'novel food'. Authorisations will be possible when methods of authentication of the products are available. In this study we propose real-time PCR methods for the specific detection of the mealworm (Tenebriomolitor), one of the most widely used insects for food and feed production. Two PCR assays are proposed: the first based on the wingless gene and the second based on the cadherin gene. The PCR tests amplify fragments of 87 bp. These qualitative methods were tested according to several performance criteria. The specificity was tested on 34 insect species' DNA, but also on non-insect species including crustacean, mammals, birds and plants. The limit of detection was determined and was below 20 copies for the two PCR tests. The applicability of the tests was demonstrated by the analysis of real-life processed samples containing T. molitor.