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While mammary gland tissues (MGTs) are difficult to sample without risks for cow's health or milk production, milk analysis are used in routine to assess dairy cow udder's health. This study aimed to identify, quantify, compare the milk and MGTs microbiota of macroscopically healthy dairy bovine mammary glands (MG) in order to evaluate their degree of similarity. We harvested 13 couples of milk and MGTs samples, originated from the same quarter at culling. 16S rDNA Amplicon Sequencing was performed, showing Corynebacterium as the main bacterial genus in both types of samples but generally found in the milk in higher proportions than in tissues. Species evenness was higher in MGTs while species richness was higher in milk samples. Beta diversity was significantly different between both matrices suggesting the presence of a resident microbiota in MGTs of dairy cows at time of culling partially reflected by the milk microbiota from the same quarter.

The ‘first 1000 days of life’ determine the gut microbiota composition and can have long-term health consequences. In this study, the simulator of the human intestinal microbial ecosystem (SHIME®) model, which represents the main functional sections of the digestive tract, was chosen to study the microbiota of young children. The aim of this study was to reproduce the digestive process of toddlers and their specific colonic environment. The ascending, transverse and descending colons of SHIME® model were inoculated with feces from three donors aged between 1 and 2 years-old, in three separate runs. For each run, samples from colon vessels were collected at days 14, 21 and 28 after microbiota stabilization period. Short chain fatty acid concentrations determined by HPLC showed that microbiota obtained in SHIME® model shared characteristics between adults and infants. In addition, microbial diversity and bacterial populations determined by 16S rRNA amplicon sequencing were specific to each colon vessel. In conclusion, the SHIME® model developed in this study seemed well adapted to evaluate prebiotic and probiotic impact on the specific microbiota of toddlers, or medicine and endocrine disruptor metabolism. Moreover, this study is the first to highlight some biofilm development in in vitro gastrointestinal modelling systems.

Outgrowth and toxinogenesis of Clostridium botulinum Group II (non-proteolytic) type B were studied in cooked ham prepared with different NaNO2 (ranging from 0 to 80 mg/kg) and sodium chloride (NaCl, ranging from 12 to 19 g/kg) incorporation rates. Cured ground pork batters were inoculated with a cocktail of 3 strains of C. botulinum Group II type B at 3.5 log10 CFU/g, portioned and samples of 50 g were vacuum packed then cooked and cooled based on thermal processing employed by the meat processing industry. These cooked ham model samples were stored under reasonably foreseeable conditions of use and storage i.e. for 14 days at 4 °C, followed by a cold chain break for 1 h at 20 °C then up to 33 days at 8 °C. Storage times and temperatures were used to mimic those commonly encountered along the supply chain. Enumeration of C. botulinum and detection of the botulinum neurotoxin type B (BoNT/B) were performed in triplicate at different storage times. Under these experimental conditions, incorporation rates of NaNO2 ≥ 30 mg/kg prevented the outgrowth and toxinogenesis of C. botulinum Group II type B in the cooked ham model, regardless of the NaCl concentrations tested. In contrast, total removal of nitrite allowed outgrowth and toxin production during storage of the processed meat product. Results showed that the maximum ingoing amount of nitrite (i.e. 150 mg/kg) that may be added according to the EU legislation (Regulation (EC) No 1333/2008) can be reduced in cooked ham while still ensuring control of C. botulinum Group II type B. According to the multiple factors that could affect C. botulinum behavior in processing meat products, outgrowth and toxin production of C. botulinum should be evaluated on a case by case basis, depending on the recipe, manufacturing process, food matrix and storage conditions.

The important changes in diet during the first years of life strongly modulate the intestinal microbiota of young children. Among in vitro digestive models, the simulator of human intestinal microbial ecosystem (SHIME®) model, seems particularly adapted to study the effects of prebiotics and/or probiotics on the dynamic microbiota of toddlers. The main purpose of this study was to investigate different formulations with prebiotic (3'-sialyllactose: 3'SL) and probiotic (Bifidobacterium crudilactis FR/62/b/3) effects on young child microbiota using the SHIME® model. The ascending (AC), transverse (TC) and descending (DC) colons of the SHIME® model were inoculated with feces from 3 donors aged between 1 and 2 years, in three separate vessels. After two weeks of microbiota stabilization, four treatments of one week (prebiotic, probiotic, synbiotic and cell-free spent media from the synbiotic) were administered. In all the colon vessels, the short chain fatty acid analyses, determined using high-performance liquid chromatography highlighted a ratio acetate/propionate/butyrate proportion of 5:19:6, situated between infant and adult normal values. As already observed in other validated studies focusing on the SHIME® model, the 16S rDNA sequencing highlighted a low richness and diversity in the AC, while the microbial communities in the TC and the DC remained similar to each other. Although some bacteria involved in biofilm development have been identified (Stenotrophomonas, Megasphera and Enterobacter), specific bacterial populations, proper to each colon were developed. Some bacteria associated to the upper intestinal tract, such as Lactobacillus and Veillonella genera, seemed to grow easily in the AC. The quantitative polymerase chain reaction (qPCR) targeting the hsp60 gene confirmed the ability of bifidobacteria to survive in this toddler model. In addition, the synbiotic treatment tended to a bifidogenic effect (P 0.1). On the other hand, the feces of the donors and the content of the three colon vessels were filtered and placed in contact with Escherichia coli O157:H7 ATCC 43890 to evaluate the modulation of virulence gene expression using reverse transcription PCR. Finally, filtered supernatants from donor feces significantly up-regulated the expression of the luxS gene of E. coli O157:H7 (P = 0.013). In conclusion, despite the presence of biofilms, the toddler SHIME® model used in his study shared characteristics found both in adults and infants. Although additional investigations should be performed, combining 3'SL and B. crudilactis FR/62/b/3 could lead to a beneficial effect on infant microbiota by favoring bifidobacterial presence. Finally, the filtrated supernatant from young child feces could be able to modulate the quorum sensing mechanism for E. coli O157:H7.

Complex oligosaccharides from human milk (HMO) possess an antimicrobial activity and can promote the growth of bifidobacteria such as Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis. In addition, fermentation of carbohydrates by bifidobacteria can result in the production of metabolites presenting an antivirulence effect on several pathogenic bacteria. Whey is rich in complex bovine milk oligosaccharides (BMO) structurally similar to HMO and B. crudilactis, a species of bovine origin, is able to metabolize some of those complex carbohydrates. This study focused on the ability of B. bifidum and B. crudilactis to grow in a culture medium supplemented in 3′-sialyllactose (3′SL) as the main source of carbon, a major BMO encountered in cow milk. Next, the effects of cell-free spent media (CFSM) were tested against virulence expression of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Both strains were able to grow in presence of 3′SL, but B. crudilactis showed the best growth (7.92 ± 0.3 log cfu/ml) compared to B. bifidum (6.84 ± 0.9 log cfu/ml). Then, CFSM were tested for their effects on virulence gene expression by ler and hilA promoter activity of luminescent mutants of E. coli and S. Typhimurium, respectively, and on wild type strains of E. coli O157:H7 and S. Typhimurium using RT-qPCR. All CFSM resulted in significant under expression of the ler and hilA genes for the luminescent mutants and ler (ratios of −15.4 and −8.1 respectively) and qseA (ratios of −2.1 and −3.1) for the wild type strain of E. coli O157:H7. The 3′SL, a major BMO, combined with some bifidobacteria strains of bovine or human origin could therefore be an interesting synbiotic to maintain or restore the intestinal health of young children. These effects observed in vitro will be further investigated regarding the overall phenotype of pathogenic agents and the exact nature of the active molecules.

The aims of this study were to examine the effect of mare's milk on virulence gene expression of Salmonella Typhimurium and observe its potential activity on proliferation of adenocarcinoma Caco-2 cells . Different supernatants of mare's milk, raw or heat-treated at 65°C for 15 s or 30 min, were studied. The changes in hilA gene expression of Salmonella Typhimurium in presence of mare's milk supernatants were assessed using a reporter luminescent strain. A significant decrease in hilA gene expression was observed with all tested supernatants. Virulence gene expression was then assessed using qPCR on a wild-type strain of Salmonella Typhimurium. A significant decrease of hilA and ssrB2 gene expression was observed with raw milk supernatants but not with heat-treated supernatants. The same supernatants were administered to Caco-2 cells to measure their proliferation rate. A significant reduction of proliferative effect was observed only with raw milk supernatants. This study reports that raw mare's milk was able to modulate virulence gene expression of Salmonella Typhimurium and exerts antiproliferative effects on Caco-2 cells. These results may offer new approaches for promoting gastrointestinal health.

Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis in sample D. In relation to 26S pyrosequencing, our study revealed the presence of 3 main yeast species: Naumovozyma spp., Kluyveromyces marxianus, and Kazachastania khefir. For Naumovozyma, further studies are needed to assess the isolation of new species. In conclusion, this study has proved that it is possible to establish the patterns of bacterial and yeast composition of kefir and kefir grain. This was only achieved with the use of high-throughput sequencing techniques.

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.