Microbiota characterization of a Belgian protected designation of origin cheese, Herve cheese, using metagenomic analysis.

Herve cheese is a Belgian soft cheese with a washed rind, and is made from raw or pasteurized milk. The specific microbiota of this cheese has never previously been fully explored and the use of raw or pasteurized milk in addition to starters is assumed to affect the microbiota of the rind and the heart. The aim of the study was to analyze the bacterial microbiota of Herve cheese using classical microbiology and a metagenomic approach based on 16S ribosomal DNA pyrosequencing. Using classical microbiology, the total counts of bacteria were comparable for the 11 samples of tested raw and pasteurized milk cheeses, reaching almost 8 log cfu/g. Using the metagenomic approach, 207 different phylotypes were identified. The rind of both the raw and pasteurized milk cheeses was found to be highly diversified. However, 96.3 and 97.9% of the total microbiota of the raw milk and pasteurized cheese rind, respectively, were composed of species present in both types of cheese, such as Corynebacterium casei, Psychrobacter spp., Lactococcus lactis ssp. cremoris, Staphylococcus equorum, Vagococcus salmoninarum, and other species present at levels below 5%. Brevibacterium linens were present at low levels (0.5 and 1.6%, respectively) on the rind of both the raw and the pasteurized milk cheeses, even though this bacterium had been inoculated during the manufacturing process. Interestingly, Psychroflexus casei, also described as giving a red smear to Raclette-type cheese, was identified in small proportions in the composition of the rind of both the raw and pasteurized milk cheeses (0.17 and 0.5%, respectively). In the heart of the cheeses, the common species of bacteria reached more than 99%. The main species identified were Lactococcus lactis ssp. cremoris, Psychrobacter spp., and Staphylococcus equorum ssp. equorum. Interestingly, 93 phylotypes were present only in the raw milk cheeses and 29 only in the pasteurized milk cheeses, showing the high diversity of the microbiota. Corynebacterium casei and Enterococcus faecalis were more prevalent in the raw milk cheeses, whereas Psychrobacter celer was present in the pasteurized milk cheeses. However, this specific microbiota represented a low proportion of the cheese microbiota. This study demonstrated that Herve cheese microbiota is rich and that pasteurized milk cheeses are microbiologically very close to raw milk cheeses, probably due to the similar manufacturing process. The characterization of the microbiota of this particular protected designation of origin cheese was useful in enabling us to gain a better knowledge of the bacteria responsible for the character of this cheese.

Assessing interventions by quantitative risk assessment tools to reduce the risk of human salmonellosis from fresh minced pork meat in Belgium.

The risk of human salmonellosis through the consumption of minced pork meat in Belgium was assessed via a modular risk model covering pork meat production from lairage to human consumption. The main goal of the model was to give concrete options to reduce effectively the risk of human salmonellosis through the consumption of minced pork meat. These options (scenarios) were elaborated with reference to the international situation and the literature to give concrete and realistic possibilities for improving the microbiological quality of pork meat and to reduce the number of human salmonellosis cases per year in Belgium. The model estimates 15,376 cases of human salmonellosis per year in Belgium due to the consumption of minced pork meat. The results of the scenarios showed that the risk of human salmonellosis could be significantly reduced by efforts all along the pork meat production chain but also by efforts made by consumers. The responsibility of food business operators for the pork meat production chain is high in relation to the microbiological quality of meat delivery, especially at the slaughterhouse. Consumers also need to be aware of good hygiene practices during preparation of the meat at home. Cross-contamination with raw food can be avoided by changing the habits and the behavior of the household cook. The results of these scenarios would be useful for the food business operators involved in the pork meat chain and for public health authorities.

Salmonella surveillance and control at post-harvest in the Belgian pork meat chain.

Salmonella remains the primary cause of reported bacterial food borne disease outbreaks in Belgium. Pork and pork products are recognized as one of the major sources of human salmonellosis. In contrast with the primary production and slaughterhouse phases of the pork meat production chain, only a few studies have focussed on the post-harvest stages. The goal of this study was to evaluate Salmonella and Escherichia coli contamination at the Belgian post-harvest stages. E. coli counts were estimated in order to evaluate the levels of faecal contamination. The results of bacteriological analysis from seven cutting plants, four meat-mincing plants and the four largest Belgian retailers were collected from official and self-monitoring controls. The prevalence of Salmonella in the cutting plants and meat-mincing plants ranged from 0% to 50%. The most frequently isolated serotype was Salmonella typhimurium. The prevalence in minced meat at retail level ranged from 0.3% to 4.3%. The levels of Salmonella contamination estimated from semi-quantitative analysis of data relating to carcasses, cuts of meat and minced meat were equal to -3.40+/-2.04 log CFU/cm(2), -2.64+/-1.76 log CFU/g and -2.35+/-1.09 log CFU/g, respectively. The E. coli results in meat cuts and minced meat ranged from 0.21+/-0.50 to 1.23+/-0.89 log CFU/g and from 1.33+/-0.58 to 2.78+/-0.43 log CFU/g, respectively. The results showed that faecal contamination still needs to be reduced, especially in specific individual plants.

Risk factors for Salmonella and hygiene indicators in the 10 largest Belgian pig slaughterhouses.

A survey was conducted to collect data on Salmonella prevalence, Escherichia coli counts (ECCs), and aerobic bacteria colony counts (ACCs) on pig carcasses after chilling at the 10 largest Belgian pig slaughterhouses during 2000 through 2004. Potential risk factors of contamination associated with production parameters, technical descriptions of the installations, and cleaning and disinfection methods were assessed during investigations in the slaughterhouses. These variables were used first in a univariate analysis and then were extended to a multivariate analysis with a logistic mixed regression model for Salmonella and a linear mixed model for ECCs and ACCs with slaughterhouses as the random effect. The results indicated high variability concerning Salmonella contamination among the 10 slaughterhouses, with prevalence ranging from 2.6 to 34.3% according to the area of origin. The median ECC and median ACC ranged from -0.43 to 1.11 log CFU/cm2 and from 2.37 to 3.65 log CFU/cm2, respectively. The results of the logistic and linear regressions revealed that some working practices such as scalding with steam, second flaming after polishing, and complete cleaning and disinfection of the splitting machine several times a day were beneficial for reducing Salmonella prevalence, ECCs, and ACCs. Changing the carcass hooks just before chilling, using water as the cleaning method, and a higher frequency of disinfection of the lairage seemed to be protective against E. coli in the multivariate mixed linear model. The monitoring of critical points, slaughterhouse equipment, good slaughtering practices, and effective washing and disinfection are the keys to obtaining good microbiological results.

Assessment of bacterial superficial contamination in classical or ritually slaughtered cattle using metagenetics and microbiological analysis.

The aim of this study was to investigate the influence of the slaughter technique (Halal vs Classical slaughter) on the superficial contamination of cattle carcasses, by using traditional microbiological procedures and 16S rDNA metagenetics. The purpose was also to investigate the neck area to identify bacteria originating from the digestive or the respiratory tract. Twenty bovine carcasses (10 from each group) were swabbed at the slaughterhouse, where both slaughtering methods are practiced. Two swabbing areas were chosen: one "legal" zone of 1600cm2 (composed of zones from rump, flank, brisket and forelimb) and locally on the neck area (200cm2). Samples were submitted to classical microbiology for aerobic Total Viable Counts (TVC) at 30°C and Enterobacteriaceae counts, while metagenetic analysis was performed on the same samples. The classical microbiological results revealed no significant differences between both slaughtering practices; with values between 3.95 and 4.87log CFU/100cm2 and 0.49 and 1.94log CFU/100cm2, for TVC and Enterobacteriaceae respectively. Analysis of pyrosequencing data showed that differences in the bacterial population abundance between slaughtering methods were mainly observed in the "legal" swabbing zone compared to the neck area. Bacterial genera belonging to the Actinobacteria phylum were more abundant in the "legal" swabbing zone in "Halal" samples, while Brevibacterium and Corynebacterium were encountered more in "Halal" samples, in all swabbing areas. This was also the case for Firmicutes bacterial populations (families of Aerococcaceae, Planococcaceae). Except for Planococcoceae, the analysis of Operational Taxonomic Unit (OTU) abundances of bacteria from the digestive or respiratory tract revealed no differences between groups. In conclusion, the slaughtering method does not influence the superficial microbiological pattern in terms of specific microbiological markers of the digestive or respiratory tract. However, precise analysis of taxonomy at the genus level taxonomy highlights differences between swabbing areas. Although not clearly proven in this study, differences in hygiene practices used during both slaughtering protocols could explain the differences in contamination between carcasses from both slaughtering groups.

Exploring the Bacterial Diversity of Belgian Steak Tartare Using Metagenetics and Quantitative Real-Time PCR Analysis.

Steak tartare is a popular meat dish in Belgium. It is prepared with raw minced beef and is eaten with sauce, vegetables, and spices. Because it contains raw meat, steak tartare is highly prone to bacterial spoilage. The objective of this study was to explore the diversity of bacterial flora in steak tartare in Belgium according to the source and to determine which bacteria are able to grow during shelf life. A total of 58 samples from butchers' shops, restaurants, sandwich shops, and supermarkets were collected. These samples were analyzed using 16S rDNA metagenetics, a classical microbiological technique, and quantitative real-time PCR (qPCR) targeting the Lactobacillus genus. Samples were analyzed at the beginning and at the end of their shelf life, except for those from restaurants and sandwich shops, which were analyzed only on the purchase date. Metagenetic analysis identified up to 180 bacterial species and 90 genera in some samples. But only seven bacterial species were predominant in the samples, depending on the source: Brochothrix thermosphacta, Lactobacillus algidus, Lactococcus piscium, Leuconostoc gelidum, Photobacterium kishitani, Pseudomonas spp., and Xanthomonas oryzae. With this work, an alternative method is proposed to evaluate the total flora in food samples based on the number of reads from metagenetic analysis and the results of qPCR. The degree of underestimation of aerobic plate counts at 30°C estimated with the classical microbiology method was demonstrated in comparison with the proposed culture-independent method. Compared with culture-based methods, metagenetic analysis combined with qPCR targeting Lactobacillus provides valuable information for characterizing the bacterial flora of raw meat.

Thermophilic and cellulolytic consortium isolated from composting plants improves anaerobic digestion of cellulosic biomass: Toward a microbial resource management approach.

A cellulolytic consortium was isolated from a composting plant in order to boost the initial hydrolysis step encountered in anaerobic digestion. Improvement of the cellulose degradation, as well as biogas production, was observed for the cultures inoculated with the exogenous consortium. Metagenomics analyses pointed out a weak richness (related to the number of OTUs) of the exogenous consortium induced by the selective pressure (cellulose as sole carbon source) met during the initial isolation steps. Main microbial strains determined were strictly anaerobic and belong to the Clostridia class. During cellulose anaerobic degradation, pH drop induced a strong modification of the microbial population. Despite the fact that richness and evenness were very weak, the exogenous consortium was able to adapt and to maintain the cellulolytic degradation potential. This important result point out the fact that simplified microbial communities could be used in order to increase the robustness of mixed cultures involved in environmental biotechnology.

Cecal drop reflects the chickens' cecal microbiome, fecal drop does not.

Microbiota in the gastro-intestinal tract are closely related to both the intestinal and overall health of the host. Experimental chickens have always been euthanized in order to identify and quantify the bacteria in cecal content. In this study, quantification and identification of the microbial populations in cecal drop, cecal content and fecal drop samples from chickens showed that cecal drop contains a bacterial community that is very similar (concerning bacterial diversity, richness and species composition) to cecal content, as opposed to the bacterial community found in fecal drop. Cecal drop analysis thus allows for longitudinal experiments on chickens' cecal bacteria. The varying results in the analysis of fecal samples question the method's reliability in reflecting the true cecal microbiota in chickens.

Retrospective analysis of a listeria monocytogenes contamination episode in raw milk goat cheese using quantitative microbial risk assessment tools.

In 2005, the Belgian authorities reported a Listeria monocytogenes contamination episode in cheese made from raw goat's milk. The presence of an asymptomatic shedder goat in the herd caused this contamination. On the basis of data collected at the time of the episode, a retrospective study was performed using an exposure assessment model covering the production chain from the milking of goats up to delivery of cheese to the market. Predictive microbiology models were used to simulate the growth of L. monocytogenes during the cheese process in relation with temperature, pH, and water activity. The model showed significant growth of L. monocytogenes during chilling and storage of the milk collected the day before the cheese production (median increase of 2.2 log CFU/ml) and during the addition of starter and rennet to milk (median increase of 1.2 log CFU/ml). The L. monocytogenes concentration in the fresh unripened cheese was estimated to be 3.8 log CFU/g (median). This result is consistent with the number of L. monocytogenes in the fresh cheese (3.6 log CFU/g) reported during the cheese contamination episode. A variance-based method sensitivity analysis identified the most important factors impacting the cheese contamination, and a scenario analysis then evaluated several options for risk mitigation. Thus, by using quantitative microbial risk assessment tools, this study provides reliable information to identify and control critical steps in a local production chain of cheese made from raw goat's milk.