Véronique Delcenserie

Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments

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.

The impact of oregano (Origanum heracleoticum) essential oil and carvacrol on virulence gene transcription by Escherichia coli O157:H7

The aim of the current study was to determine, via reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, the effect of oregano essential oil (Origanum heracleoticum) and carvacrol, its major component, on the expression of virulence-associated genes in enterohaemorrhagic Escherichia coli (EHEC) O157:H7 ATCC strain 35150. Both oregano oil and carvacrol demonstrated their efficacy firstly, by inhibiting the transcription of the ler gene involved in upregulation of the LEE2, LEE3 and LEE4 promoters and of attaching and effacing lesions and secondly by decreasing both Shiga toxin and fliC genes expression. In addition, a decrease in luxS gene transcription involved in quorum sensing was observed. These results were dose dependent and showed a specific effect of O. heracleoticum and carvacrol in downregulating the expression of virulence genes in EHEC O157:H7. These findings suggest that oregano oil and carvacrol have the potential to mitigate the adverse health effects caused by virulence gene expression in EHEC O157:H7, through the use of these substances as natural antibacterial additives in foods or as an alternative to antibiotics.

Use of a metagenetic approach to monitor the bacterial microbiota of “Tomme d’Orchies” cheese during the ripening process

The study of microbial ecosystems in artisanal foodstuffs is important to complete in order to unveil its diversity. The number of studies performed on dairy products has increased during the last decade, particularly those performed on milk and cheese derivative products. In this work, we investigated the bacterial content of Tomme dOrchies cheese, an artisanal pressed and uncooked French cheese. To this end, a metagenetic analysis, using Illumina technology, was utilized on samples taken from the surface and core of the cheese at 0, 1, 3, 14 and 21days of ripening process. In addition to the classical microbiota found in cheese, various strains likely from environmental origin were identified. A large difference between the surface and the core content was observed within samples withdrawn during the ripening process. The main species encountered in the core of the cheese were Lactococcus spp. and Streptococcus spp., with an inversion of this ratio during the ripening process. Less than 2.5% of the whole population was composed of strains issued from environmental origin, as Lactobacillales, Corynebacterium and Brevibacterium. In the core, about 85% of the microbiota was attributed to the starters used for the cheese making. In turn, the microbiota of the surface contained less than 30% of these starters and interestingly displayed more diversity. The predominant genus was Corynebacterium sp., likely originating from the environment. The less abundant microbiota of the surface was composed of Bifidobacteria, Brevibacterium and Micrococcales. To summarize, the Tomme dOrchies cheese displayed a high diversity of bacterial species, especially on the surface, and this diversity is assumed to arise from the production environment and subsequent ripening process.

Genome of Bifidobacteria and Carbohydrate Metabolism

In recent years, the knowledge about bifidobacteria has considerably evolved thanks to recent progress in molecular biology. The analysis of the whole genome sequences of 48 taxa of bifidobacteria offers new perspectives for their classification, especially to set up limit between two species. Indeed, several species are presenting a high homology and should be reclassified. On the other hand, some subspecies are presenting a low homology and should therefore be reclassified into different species. In addition, a better knowledge of the genome of bifidobacteria allows a better understanding of the mechanisms involved in complex carbohydrate metabolism. The genome of some species of bifidobacteria from human but also from animal origin demonstrates high presence in genes involved in the metabolism of complex oligosaccharides. Those species should be further tested to confirm their potential to metabolize complex oligosaccharides in vitro and in vivo.

Cell-Free Spent Media Obtained from Bifidobacterium bifidum and Bifidobacterium crudilactis Grown in Media Supplemented with 3′-Sialyllactose Modulate Virulence Gene Expression in Escherichia coli O157:H7 and Salmonella Typhimurium

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.

In vitro screening of mare's milk antimicrobial effect and antiproliverative activity.

The aims of this study were to examine the effect of mares milk on virulence gene expression of Salmonella Typhimurium and observe its potential activity on proliferation of adenocarcinoma Caco-2 cells. Different supernatants of mares 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 mares 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 mares 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.

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.