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.

New Pathophysiological Insights from Serum Proteome Profiling in Equine Atypical Myopathy.

Equine atypical myopathy (AM) is a severe environmental intoxication linked to the ingestion of protoxins contained in seeds and seedlings of the sycamore maple (Acer pseudoplatanus) in Europe. The toxic metabolites cause a frequently fatal rhabdomyolysis syndrome in grazing horses. Since these toxic metabolites can also be present in cograzing horses, it is still unclear as to why, in a similar environmental context, some horses show signs of AM, whereas others remain clinically healthy. Label-free proteomic analyses on the serum of 26 diseased AM, 23 cograzers, and 11 control horses were performed to provide insights into biological processes and pathways. A total of 43 and 44 differentially abundant proteins between "AM vs cograzing horses" and "AM vs control horses" were found. Disease-linked changes in the proteome of different groups were found to correlate with detected amounts of toxins, and principal component analyses were performed to identify the 29 proteins representing a robust AM signature. Among the pathway-specific changes, the glycolysis/gluconeogenesis pathway, the coagulation/complement cascade, and the biosynthesis of amino acids were affected. Sycamore maple poisoning results in a combination of inflammation, oxidative stress, and impaired lipid metabolism, which is trying to be counteracted by enhanced glycolysis.

Senescence Induced by UVB in Keratinocytes Impairs Amino Acids Balance.

Skin is one of the most exposed organs to external stress. Namely, UV rays are the most harmful stress that could induce important damage leading to skin aging and cancers. At the cellular level, senescence is observed in several skin cell types and contributes to skin aging. However, the origin of skin senescent cells is still unclear but is probably related to exposure to stresses. In this work, we developed an in vitro model of UVB-induced premature senescence in normal human epidermal keratinocytes. UVB-induced senescent keratinocytes display a common senescent phenotype resulting in an irreversible cell cycle arrest, an increase in the proportion of senescence-associated ß-galactosidase‒positive cells, unrepaired DNA damage, and a long-term DNA damage response activation. Moreover, UVB-induced senescent keratinocytes secrete senescence-associated secretory phenotype factors that influence cutaneous squamous cell carcinoma cell migration. Finally, a global transcriptomic study highlighted that senescent keratinocytes present a decrease in the expression of several amino acid transporters, which is associated with reduced intracellular levels of glycine, alanine, and leucine. Interestingly, the chemical inhibition of the glycine transporter SLC6A9/Glyt1 triggers senescence features.

Evaluation of Enzymatic Cleaning on Food Processing Installations and Food Products Bacterial Microflora.

Biofilms are a permanent source of contamination in food industries and could harbor various types of microorganisms, such as spoiling bacteria. New strategies, such as enzymatic cleaning, have been proposed to eradicate them. The purpose of this study was to evaluate the impact of enzymatic cleaning on the microbial flora of installations in a processing food industry and of the final food product throughout its shelf life. A total of 189 samples were analyzed by classical microbiology and 16S rDNA metagenetics, including surface samples, cleaning-in-place (CIP) systems, and food products (at D0, Dend of the shelf life, and Dend of the shelf life +7 days). Some surfaces were highly contaminated with spoiling bacteria during conventional cleaning while the concentration of the total flora decreased during enzymatic cleaning. Although the closed circuits were cleaned with conventional cleaning before enzymatic cleaning, there was a significant release of microorganisms from some parts of the installations during enzymatic treatment. A significant difference in the total flora in the food products at the beginning of the shelf life was observed during enzymatic cleaning compared to the conventional cleaning, with a reduction of up to 2 log CFU/g. Metagenetic analysis of the food samples at the end of their shelf life showed significant differences in bacterial flora between conventional and enzymatic cleaning, with a decrease of spoiling bacteria (Leuconostoc sp.). Enzymatic cleaning has improved the hygiene of the food processing instillations and the microbial quality of the food throughout the shelf life. Although enzymatic cleaning is not yet commonly used in the food industry, it should be considered in combination with conventional sanitizing methods to improve plant hygiene.

Modeling the Growth and Interaction Between Brochothrix thermosphacta, Pseudomonas spp., and Leuconostoc gelidum in Minced Pork Samples.

The aim of this study was to obtain the growth parameters of specific spoilage micro-organisms previously isolated in minced pork (MP) samples and to develop a three-spoilage species interaction model under different storage conditions. Naturally contaminated samples were used to validate this approach by considering the effect of the food microbiota. Three groups of bacteria were inoculated on irradiated samples, in mono- and in co-culture experiments (n = 1152): Brochothrix thermosphacta, Leuconostoc gelidum, and Pseudomonas spp. (Pseudomonas fluorescens and Pseudomonas fragi). Samples were stored in two food packaging [food wrap and modified atmosphere packaging (CO2 30%/O2 70%)] at three isothermal conditions (4, 8, and 12°C). Analysis was carried out by using both 16S rRNA gene amplicon sequencing and classical microbiology in order to estimate bacterial counts during the storage period. Growth parameters were obtained by fitting primary (Baranyi) and secondary (square root) models. The food packaging shows the highest impact on bacterial growth rates, which in turn have the strongest influence on the shelf life of food products. Based on these results, a three-spoilage species interaction model was developed by using the modified Jameson-effect model and the Lotka Volterra (prey-predator) model. The modified Jameson-effect model showed slightly better performances, with 40-86% out of the observed counts falling into the Acceptable Simulation Zone (ASZ). It only concerns 14-48% for the prey-predator approach. These results can be explained by the fact that the dynamics of experimental and validation datasets seems to follow a Jameson behavior. On the other hand, the Lotka Volterra model is based on complex interaction factors, which are included in highly variable intervals. More datasets are probably needed to obtained reliable factors, and so better model fittings, especially for three- or more-spoilage species interaction models. Further studies are also needed to better understand the interaction of spoilage bacteria between them and in the presence of natural microbiota.

Assessment of Spoilage Bacterial Communities in Food Wrap and Modified Atmospheres-Packed Minced Pork Meat Samples by 16S rDNA Metagenetic Analysis.

Although several studies have focused on the dynamics of bacterial food community, little is known about the variability of batch production and microbial changes that occur during storage. The aim of the study was to characterize the microbial spoilage community of minced pork meat samples, among different food production and storage, using both 16S rRNA gene sequencing and classical microbiology. Three batches of samples were obtained from four local Belgian facilities (A-D) and stored until shelf life under food wrap (FW) and modified atmosphere packaging (MAP, CO2 30%/O2 70%), at constant and dynamic temperature. Analysis of 288 samples were performed by 16S rRNA gene sequencing in combination with counts of psychrotrophic and lactic acid bacteria at 22°C. At the first day of storage, different psychrotrophic counts were observed between the four food companies (Kruskal-Wallist test, p-value < 0.05). Results shown that lowest microbial counts were observed at the first day for industries D and A (4.2 ± 0.4 and 5.6 ± 0.1 log CFU/g, respectively), whereas industries B and C showed the highest results (7.5 ± 0.4 and 7.2 ± 0.4 log CFU/g). At the end of the shelf life, psychrotrophic counts for all food companies was over 7.0 log CFU/g. With metagenetics, 48 OTUs were assigned. At the first day, the genus Photobacterium (86.7 and 19.9% for food industries A and C, respectively) and Pseudomonas (38.7 and 25.7% for food companies B and D, respectively) were dominant. During the storage, a total of 12 dominant genera (>5% in relative abundance) were identified in MAP and 7 in FW. Pseudomonas was more present in FW and this genus was potentially replaced by Brochothrix in MAP (two-sided Welch's t-test, p-value < 0.05). Also, a high Bray-Curtis dissimilarity in genus relative abundance was observed between food companies and batches. Although the bacteria consistently dominated the microbiota in our samples are known, results indicated that bacterial diversity needs to be addressed on the level of food companies, batches variation and food storage conditions. Present data illustrate that the combined approach provides complementary results on microbial dynamics in minced pork meat samples, considering batches and packaging variations.

Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus.

The lactic acid bacterium Streptococcus thermophilus is widely used for the manufacture of yogurt and cheese. This dairy species of major economic importance is phylogenetically close to pathogenic streptococci, raising the possibility that it has a potential for virulence. Here we report the genome sequences of two yogurt strains of S. thermophilus. We found a striking level of gene decay (10% pseudogenes) in both microorganisms. Many genes involved in carbon utilization are nonfunctional, in line with the paucity of carbon sources in milk. Notably, most streptococcal virulence-related genes that are not involved in basic cellular processes are either inactivated or absent in the dairy streptococcus. Adaptation to the constant milk environment appears to have resulted in the stabilization of the genome structure. We conclude that S. thermophilus has evolved mainly through loss-of-function events that remarkably mirror the environment of the dairy niche resulting in a severely diminished pathogenic potential.