High intraspecific variation of the cell surface physico-chemical and bioadhesion properties in Brettanomyces bruxellensis.

Brettanomyces bruxellensis is the most damaging spoilage yeast in the wine industry because of its negative impact on the wine organoleptic qualities. The strain persistence in cellars over several years associated with recurrent wine contamination suggest specific properties to persist and survive in the environment through bioadhesion phenomena. In this work, the physico-chemical surface properties, morphology and ability to adhere to stainless steel were studied both on synthetic medium and on wine. More than 50 strains representative of the genetic diversity of the species were considered. Microscopy techniques made it possible to highlight a high morphological diversity of the cells with the presence of pseudohyphae forms for some genetic groups. Analysis of the physico-chemical properties of the cell surface reveals contrasting behaviors: most of the strains display a negative surface charge and hydrophilic behavior while the Beer 1 genetic group has a hydrophobic behavior. All strains showed bioadhesion abilities on stainless steel after only 3 h with differences in the concentration of bioadhered cells ranging from 2.2 × 102 cell/cm2 to 7.6 × 106 cell/cm2. Finally, our results show high variability of the bioadhesion properties, the first step in the biofilm formation, according to the genetic group with the most marked bioadhesion capacity for the beer group.

[Use of health professional cards by general practice locums: frequency and difficulties]. / Fréquence et difficultés d'utilisation des cartes de professionnel de santé par les médecins généralistes remplaçants.

BACKGROUND: Health professional cards are used for electronic production and transmission of medical reimbursement requests. These cards are personal and cannot be lent, including to locums. However, general practice locums often report using the cards of the practitioners they replace. The aim of this study was to assess the use of general practice locum's own professional cards for patient reimbursements in 2013. METHODS: A retrospective survey by computerized questionnaire was conducted in 2014 in Lower-Normandy among general practice locums. Data were collected on their possession, knowledge,frequency and difficulties using their own cards in 2013. RESULTS: All locums were familiar with health professional cards, but 71.9% did not know about health professional trainee cards. 71.9% of respondents had a card in 2013 and only 26.1% of them had already used the card. 78.1% of respondents thought they could use the card of the practitioner that they replaced' The main difficulties encountered when using health professional cards were the failure of general practitioners to ask locums to use their own cards, problems with software configuration and card installation, and a poor understanding of the purpose of the card. CONCLUSION: Locums in Lower Normandy in 2013 rarely used their own cards when they have one. Better information concerning use of the cars is required for locums.

Genetic and phenotypic evidence for two groups of Oenococcus oeni strains and their prevalence during winemaking.

Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis was the most relevant method to follow the diversity of lactic acid bacteria during winemaking. By targeting the rpoB gene, two types of Oenococcus oeni strains were distinguished resulting from a single mutation in the rpoB region targeted in PCR and generating two different electrophoresis profiles. The first one prevailed during fermentation and the second during ageing. Some strains of each type were isolated during winemaking and were studied using several genetic methods (real-time PCR, PCR-random amplified polymorphic DNA, multiple locus sequence typing and the presence of gene markers). Physiological characters related to environmental conditions were examined. The results confirmed the relevance of the rpoB mutation for characterising the two O. oeni subgroups. The relationship between the physiological response to stress and the rpoB genetic groups raised the question of O. oeni intraspecies grouping. A possible division within this species, of great technological interest to the wine industry, was also raised.

Correlation between indigenous Oenococcus oeni strain resistance and the presence of genetic markers.

This study reports on monitoring Oenococcus oeni intraspecific diversity evolution during winemaking. Three different wines were monitored. The proportion of O. oeni species was determined by species-specific PCR and O. oeni strains were distinguished by multiplex PCR-RAPD. Each strain was tested by PCR for 16 significant markers revealed by a previous genetic comparison between a strong oenological potential strain and one with poor oenological potential. Population levels and diversity changed according to winemaking stages, oenological practices and the chemical properties of the wine. In all situations, O. oeni was the best-adapted species. Within the O. oeni group, intraspecific strain diversity decreased and the malolactic fermentation was the result of the most resistant strains with the highest number of markers.

Differentiation of wine lactic acid bacteria species based on RFLP analysis of a partial sequence of rpoB gene.

PCR-RFLP analysis of rpoB sequences were used to identify lactic acid bacteria (LAB) species commonly isolated from wine. Strains of seven cocci and 12 lactobacilli species could be identified after single digestion with AciI for the cocci and two or three digestions (AciI, HinfI and MseI) for the rods and preceded by colonies isolation on solid selective medium and microscope observation to distinguish cocci and rods cells.

Inventory and monitoring of wine microbial consortia.

The evolution of the wine microbial ecosystem is generally restricted to Saccharomyces cerevisiae and Oenococcus oeni, which are the two main agents in the transformation of grape must into wine by acting during alcoholic and malolactic fermentation, respectively. But others species like the yeast Brettanomyces bruxellensis and certain ropy strains of Pediococcus parvulus can spoil the wine. The aim of this study was to address the composition of the system more precisely, identifying other components. The advantages of the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach to wine microbial ecology studies are illustrated by bacteria and yeast species identification and their monitoring at each stage of wine production. After direct DNA extraction, PCR-DGGE was used to make the most exhaustive possible inventory of bacteria and yeast species found in a wine environment. Phylogenetic neighbor-joining trees were built to illustrate microbial diversity. PCR-DGGE was also combined with population enumeration in selective media to monitor microbial changes at all stages of production. Moreover, enrichment media helped to detect the appearance of spoilage species. The genetic diversity of the wine microbial community and its dynamics during winemaking were also described. Most importantly, our study provides a better understanding of the complexity and diversity of the wine microbial consortium at all stages of the winemaking process: on grape berries, in must during fermentation, and in wine during aging. On grapes, 52 different yeast species and 40 bacteria could be identified. The diversity was dramatically reduced during winemaking then during aging. Yeast and lactic acid bacteria were also isolated from very old vintages. B. bruxellensis and O. oeni were the most frequent.

Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries.

Brettanomyces bruxellensis spoilage is a serious problem for the wine industry. Mainly, by producing 4-ethylphenol and 4-ethylguaiacol, it confers off-odors to the wine and changes its aromatic quality. The presence of B. bruxellensis cells on the berry was speculated but it had never been clearly demonstrated. On grape berries, the microbial ecosystem is highly diverse and the population of B. bruxellensis can be very small. The aim of our study was to reveal and confirm the presence of B. bruxellensis on the surface of grape berries. We developed an enrichment medium for B. bruxellensis in order to overcome the detection limit of the molecular methods (species-specific PCR, ITS-RFLP PCR, PCR-DGGE). This medium, named EBB medium, made it possible to detect B. bruxellensis after 10 days of culture. For the first time, the presence of B. bruxellensis has been clearly established in several vineyards and at different stages of the grape development after the veraison. This work led to the conclusion that the grape berry is the primary source of B. bruxellensis. Grape growers and winemakers should take these results into account when deciding on the treatment to apply in the vineyards and the must. With the information provided here, B. bruxellensis prevention could start in the vineyard.

Evaluation of the roles of apoptosis, autophagy, and mitophagy in the loss of plating efficiency induced by Bax expression in yeast.

We found recently that, in yeast cells, the heterologous expression of Bax induces a loss of plating efficiency different from that induced by acute stress because it is associated with the maintenance of plasma membrane integrity (Camougrand, N., Grelaud-Coq, A., Marza, E., Priault, M., Bessoule, J. J., and Manon, S. (2003) Mol. Microbiol. 47, 495-506). Bax effects were neither dependent on the presence of the yeast metacaspase Yca1p and the apoptosis-inducing factor homolog nor associated with the appearance of typical apoptotic markers such as metacaspase activation, annexin V binding, and DNA cleavage. Yeast cells expressing Bax instead displayed autophagic features, including increased accumulation of Atg8p, activation of vacuolar alkaline phosphatase, and the presence of autophagosomes and autophagic bodies. However, the inactivation of autophagy did not prevent and actually slightly accelerated Bax-induced loss of plating efficiency. On the other hand, Bax expression induced a fragmentation of the mitochondrial network, which retained, however, some level of organization in wild-type cells. However, when expressed in cells inactivated for the gene UTH1, previously shown to be involved in mitophagy, Bax induced a complete disorganization of the mitochondrial network. Interestingly, although mitochondrially targeted green fluorescent protein was slowly degraded in the wild-type strain, it remained unaffected in the mutant. Furthermore, the slow loss of plating efficiency in the mutant strain correlated with a loss of plasma membrane integrity. These data suggest that Bax-induced loss of growth capacity is associated with maintenance of plasma membrane integrity dependent on UTH1, suggesting that selective degradation of altered mitochondria is required for a regulated loss of growth capacity.

Lactic acid bacteria evolution during winemaking: use of rpoB gene as a target for PCR-DGGE analysis.

Evolution of the microbial population during winemaking is crucial. Winemakers are more and more attentive to microbial aspects during fermentation. During aging, microbial stabilization is preponderant to avoid development of spoilage yeast and bacteria. Therefore, it is necessary to improve methods to study the evolution of micro-organisms and for early detection of undesirable strain. The aim of this study was to develop a culture-independent method for identifying lactic acid bacteria (LAB) and to monitoring predominant species. The benefits of PCR-DGGE for the analysis of microbial changes during winemaking were clearly demonstrated. Targeting rpoB gene allowed a reliable discrimination of each species. The primers were able to avoid the interspecies heterogeneity problem caused by the use of the 16S rRNA gene. This method was applied to study the influence of different oenological practices on LAB population and their evolution during winemaking.

rpoB gene: a target for identification of LAB cocci by PCR-DGGE and melting curves analyses in real time PCR.

Lactic acid bacteria (LAB) are essential in the quality of many fermented beverages like beer, cider and wine. In the two later cases, they convert malic acid into lactic acid during the malolactic fermentation. After fermentation, microbial stabilization is needed to prevent the development of spoilage bacteria species. Among them, cocci lead to different alterations: Pediococcus sp., and some strains of Leuconostoc mesenteroides and Oenococcus oeni can produce exopolysaccharides which modify wine viscosity and lead to ropiness. They also can produce acetic acid, biogenic amine, ethyl carbamate and volatile phenols. Therefore detection and identification are crucial. Results of phenotypic tests and DNA-DNA probes are not accurate enough. 16S RNA gene which is currently used for bacterial species identification presents intraspecies heterogeneity. The rpoB gene is an alternative to this limitation. However previous PCR targeting partial sequence of rpoB gene could not delimit cocci species. Therefore we compared the rpoB gene sequence of the six main cocci species found in fermented beverages: P. damnosus, P. dextrinicus, P. parvulus, P. pentosaceus, L. mesenteroides and O. oeni. The most discriminating partial sequence of the rpoB gene was chosen for designing primers. By PCR-DGGE the reliability of these primers was verified. It was controlled in a mixture of several cocci and other lactic acid bacteria (Lactobacillus sp.). Then we adapted the primers and the PCR conditions in order to achieve the identification of cocci species by real time PCR program including the fluorescent dye SYBR Green I, which gives faster results. PCR melt curves were established and a specific T(m) was attributed to each species.