title: Exploring the bacterial diversity of Belgian steak tartare using metagenetics: SM2_2 Day 2
design: ""Steak tartare" is a popular meat dish in Belgium. Due to the use of raw ingredients, this dish is highly prone to bacterial spoilage. The objective of this study is to explore the bacterial flora diversity present in steak tartare in Belgium according to their origin and to determine which bacteria are able to grow during the shelf life. A total of 58 samples from butchers' shops, restaurants, sandwich shops and supermarkets were analyzed using 16S rDNA metagenetics, classical microbiological and quantitative Real-Time PCR (qPCR). Samples were analyzed at the beginning and at the end of their shelf life, except for those from restaurants and sandwich shops analyzed only at the purchase date. Metagenetic analysis identified up to 180 bacterial species and 90 genera in some samples. Depending on the sample location, the most predominant bacterial species were among Brochothrix thermosphacta, Lactobacillus algidus, Lactococcus piscium, Leuconostoc gelidum, Photobacterium kishitani, Pseudomonas spp. and Xanthomonas oryzae. Based on the metagenetic analysis, Lactobacillus genus was identified in all the samples included in this study and a specific qPCR was developed for the quantification of Lactobacillus genus. Samples with similar bacterial populations have been pooled using dissimilarity analyses giving an overview of the bacterial flora diversity in steak tartare in Belgium. In this study, different groups have been identified with different microbial quality. For some of them, a high proportion of spoiled bacteria has been identified already at the beginning of the shelf life and one group contains a high proportion of Clostridium haemolyticum. The degree of underestimation of total viable counts (TVCs) at 30oC has been outlined in comparison with the total flora estimated with classical microbiology and with qPCR combined with metagenetic results. Compared to culture-based methods, metagenetic analysis combined with qPCR targeting Lactobacillus provides valuable information, and its use should be considered as a complementary technique for characterizing the bacterial flora of raw meat products."