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International audience; Brettanomyces bruxellensis is a unicellular fungus of increasing industrial and scientific interest over the past 15 years. Previous studies revealed high genotypic diversity amongst B. bruxellensis strains as well as strain-dependent phenotypic characteristics. Genomic assemblies revealed that some strains harbour triploid genomes and based upon prior genotyping it was inferred that a triploid population was widely dispersed across Australian wine regions. We performed an intraspecific diversity genotypic survey of 1488 B. bruxellensis isolates from 29 countries, 5 continents and 9 different fermentation niches. Using microsatellite analysis in combination with different statistical approaches, we demonstrate that the studied population is structured according to ploidy level, substrate of isolation and geographical origin of the strains, underlying the relative importance of each factor. We found that geographical origin has a different contribution to the population structure according to the substrate of origin, suggesting an anthropic influence on the spatial biodiversity of this microorganism of industrial interest. The observed clustering was correlated to variable stress response, as strains from different groups displayed variation in tolerance to the wine preservative sulfur dioxide (SO2). The potential contribution of the triploid state for adaptation to industrial fermentations and dissemination of the species B. bruxellensis is discussed.

Precedenti indagini, rivolte alla valorizzazione ed alla diffusione di pratiche enologiche basate su un ridotto uso di coadiuvanti tecnologici attraverso l’impiego di fermentazione alcoliche spontanee gestite in modo da eliminare e/o ridurre l’uso delle colture microbiche commerciali su Aglianico di Taurasi (AV), hanno permesso di isolare da mosto in fermentazione colonie di lieviti che sono state successivamente purificate e conservate in ceppoteca. Analisi microscopiche condotte sugli isolati di lievito hanno messo in evidenza la presenza di batteri endogeni presenti all’interno delle cellule del lievito. L’obiettivo di questo studio è stato rivolto all’identificazione delle specie coinvolte nell’interazione intracellulare lievito-batterio e la loro visualizzazione utilizzando appropriate sonde molecolari fluorescenti in grado di permettere la localizzazione dei batteri endogeni. Attraverso l’applicazione della tecnica FISH (Fluorescence In Situ Hybridization) in combinazione con la microscopia confocale a scansione laser (CLSM) sono state visualizzate le cellule batteriche all’interno del lievito-ospite. I risultati ottenuti, hanno permesso di identificare la specie coinvolte e di visualizzare l’interazione intracellulare tra Issatchenkia terricola e Staphylococcus sp. che rappresenta un caso particolare di endosimbiosi tra lieviti e batteri. La natura dell’ interazione rimane sconosciuta, così come i meccanismi di invasione delle cellule di I. terricola e le modalità di trasmissione durante la riproduzione asessuata del lievito. Attraverso il protocollo della tecnica FISH, opportunamente modificato, è stato possibile visualizzare con successo Staphylococcus sp. cellule all’interno di I. terricola mediante ibridazione con sonda generica per eubatteri EUB338. Ulteriori osservazioni, con sonde specifiche, sono necessarie per confermare il meccanismo di trasmissione dei batteri endogeni dai lieviti, dalle cellule madri alla figlia. Il rapporto intracellulare tra I. terricola e Staphylococcus sp. rappresenta un singolare fenomeno di relazione simbiotica tra eucarioti e procarioti, questa associazione potrebbe avere un enorme impatto sullo studio e la comprensione di questo ruolo dei microrganismi nella fermentazione spontanea del vino.

International audience; The use of sulfur dioxide (SO2) as wine additive is able to ensure both antioxidant protection and microbiological stability. In spite of these undeniable advantages, in the last two decades the presence of SO2 in wine has raised concerns about potential adverse clinical effects in sensitive individuals. The winemaking industry has followed the general trend towards the reduction of SO2 concentrations in food, by expressing at the same time the need for alternative control methods allowing reduction or even elimination of SO2. In the light of this, research has been strongly oriented toward the study of alternatives to the use of SO2 in wine. Most of the studies have focused on methods able to replace the antimicrobial activity of SO2. This review article gives a comprehensive overview of the current state-of-the-art about the chemical additives and the innovative physical techniques that have been proposed for this purpose. After a focus on the chemistry and properties of SO2 in wine(,) as well as on wine spoilage and on the conventional methods used for the microbiological stabilization of wine, recent advances on alternative methods proposed to replace the antimicrobial activity of SO2 in winemaking are presented and discussed. Even though many of the alternatives to SO2 showed good efficacy, nowadays no other physical technique or additive can deliver the efficacy and broad spectrum of action as SO2 (both antioxidant and antimicrobial), therefore the alternative methods should be considered a complement to SO2 in low-sulfite winemaking, rather than being seen as its substitutes.

Enterotoxigenic Staphylococcus (S.) aureus continue to be a great concern for raw milk and raw milk cheeses safety. Apart from the classical staphylococcal enterotoxins (SEs) (SEA-SEE), new SEs and staphylococcal enterotoxin-like toxins (SEls) have been detected and for most of them the emetic activity has been demonstrated or their genes have been found in S. aureus strains involved in staphylococcal food poisoning (SFP) cases. These findings suggest that the new SE/SEls are a potential cause of SFP outbreaks and call for studies aimed at investigating the actual and up-to-date enterotoxigenic potential of S. aureus harboring these toxins. Whole genome sequencing is being widely used to characterize S. aureus strains, but incorrect annotations and the use of pipelines targeting only a limited number of se/sel genes provide only a partial and often misleading description of the actual and overall enterotoxigenic potential of these pathogens. Confusion has further been created by the incorrect nomenclature given to the latest SEls discovered. In this study 53 S. aureus isolated from raw milk were typed by enterotoxin gene- (Seg) and 16S-23S rDNA intergenic spacer region- (ISR) typing. Six strains, one per each Seg- type and ISR-type obtained, were genome sequenced and the ability to produce classical and new SEs in milk was investigated. Seg- and ISR-typing showed the same discriminatory power, which was higher than that of multi locus sequence typing. Manual genomic analyses allowed to confirm, correct, or widen the results of the common available pipelines. In particular, novel allelic variants of se and sel genes and pseudogenes were detected. The actual location of se and sel genes was found, even for genes such as selY, whose location (in the core genome) was so far unknown. A novel type of enterotoxin gene cluster (egc), i.e. the egc-type 5 in S. aureus 356P and 364P, was detected while S. argenteus MSHR1132 harbored the novel egc-type 6. The correct annotation of se and sel genes and pseudogenes will contribute to improve and standardize the comparative genomics of S. aureus and clarify the confused scenario of the se and sel nomenclature. Four of the six S. aureus strains produced in milk sufficient amounts of SEA, SEC, SED and SEH to cause SFP, with S. aureus 372P as the highest producer of SED known to date. By contrast, S. aureus 372P released a low amount of SER in milk, most likely because the seR gene was present as a pseudogene encoding 51 amino acids. These findings confirm that not only the classical SEs but also the new ones can represent a hazard for the consumers' health and that the detection of SEs in raw milk and other matrices, especially if involved in SFP cases, should focus not only on classical, but also on all the new SEs and SEls known to date. Where reference methods are unavailable for the detection of these toxins, the presence of the relevant genes should be investigated using the (real time) PCR protocols refined by our group.

BACKGROUND: Since processing technology and storage may influence the sensory and nutritional value as well as the shelf life of pomegranate juice (PJ), mild technologies based on microfiltration may be a promising alternative to heat treatments for fruit juice preservation. In this study, physicochemical and microbiological properties of raw (RPJ), microfiltered (MPJ) and cloudy pasteurized (PPJ) PJ were compared over a period of 4weeks. RESULTS: Data demonstrated that microfiltration was comparable to pasteurization in guaranteeing microbiological stability of the juice, avoiding spoilage of the final product. After treatment, PPJ showed the highest amounts of gallic acid (GA) and ellagic acid derivatives (EAs). During storage, the amount of ellagitannins, EAs and GA similarly decreased in all types of juice. Trends towards variations of monomeric anthocyanins in MPJ and variations of polymeric and copigmented anthocyanins in both MPJ and PPJ were found over storage. CONCLUSION: The optimization of pretreatments and filtration parameters can lead to the industrial scale-up of microfiltration technology for the development of high-quality non-heat-treated PJ. © 2017 Society of Chemical Industry.