Draft Genome Sequence of Oenococcus kitaharae CRBO2176, Isolated from Homemade Water Kefir.

Here, we announce the draft genome sequence of an Oenococcus kitaharae strain isolated from homemade water kefir in Bordeaux, France. O. kitaharae CRBO2176 is deposited at the Biological Resources Center Oenology (CRBO) of the Institute of Vine and Wine Science (ISVV; Villenave d'Ornon, France).

Phage-host interactions as a driver of population dynamics during wine fermentation: Betting on underdogs.

Winemaking is a complex process in which numerous microorganisms, mainly yeasts and lactic acid bacteria (LAB), play important roles. After alcoholic fermentation (AF), most wines undergo malolactic fermentation (MLF) to improve their organoleptic properties and microbiological stability. Oenococcus oeni is mainly responsible for this crucial process where L-malic acid (MA) in wine converts to softer L-lactic acid. The bacterium is better adapted to the limiting conditions imposed by the wine matrix and performs MLF under regular winemaking conditions, especially in wines with a pH below 3.5. Traditionally, this process has been conducted by the natural microbiota present within the winery. However, the start, duration and qualitative impact of spontaneous MLF are unpredictable, which prompts winemakers to use pure starter cultures of selected bacteria to promote a more reliable, simple, fast and efficient fermentation. Yet, their use does not always ensure a problem-free fermentation. Spontaneous initiation of the process may prove very difficult or does not occur at all. Such difficulties arise from a combination of factors found in some wines upon the completion of AF (high ethanol concentration, low temperature and pH, low nutrient concentrations, presence of free and bound SO2). Alongside these well documented facts, research has also provided evidence that negative interactions between O. oeni and other biological entities such as yeasts may also impact MLF. Another insufficiently described, but highly significant factor inhibiting bacterial growth is connected to the presence of bacteriophages of O. oeni which are frequently associated to musts and wines. The purpose of this review is to summarize the current knowledge about the phage life cycles and possible impacts on the trajectory of the microbiota during winemaking.

Assessment of the lysogenic status in the lactic acid bacterium O. oeni during the spontaneous malolactic fermentation of red wines.

After alcoholic fermentation, most wines undergo malolactic fermentation (MLF), driven by the lactic acid bacterium Oenococcus oeni, which improves their organoleptic properties and microbiological stability. Prophages were recently shown to be notably diverse and widely disseminated in O. oeni genomes. Such in silico predictions confirmed previous cultivation-based approaches which showed frequent lysis of strains upon treatment with the inducing agent mitomycin C. Both strategies used to assess lysogeny in the species were so far applied to a number of strains collected from distinct countries, wineries, cepages and fermentation processes. Results may not therefore be representative of the lysogenic population in natural communities driving the MLF during winemaking. Here we report the prevalence of lysogeny during winemaking in three wineries in the Bordeaux area. The dominant LAB population was collected in 11 red wines upon completion of MLF. Using VNTR and prophage typing analyses, our data confirm the presence of lysogens in the population driving the spontaneous MLF in all tested wines, although lysogeny rates varied across wineries. Higher prevalence of lysogeny was associated to a reduced diversity in VNTR profiles, the dominance of a few prophage-types and presence of some bacterial genetic backgrounds that were particularly prone to lysogenization.

Phage Encounters Recorded in CRISPR Arrays in the Genus Oenococcus.

The Oenococcus genus comprises four recognized species, and members have been found in different types of beverages, including wine, kefir, cider and kombucha. In this work, we implemented two complementary strategies to assess whether oenococcal hosts of different species and habitats were connected through their bacteriophages. First, we investigated the diversity of CRISPR-Cas systems using a genome-mining approach, and CRISPR-endowed strains were identified in three species. A census of the spacers from the four identified CRISPR-Cas loci showed that each spacer space was mostly dominated by species-specific sequences. Yet, we characterized a limited records of potentially recent and also ancient infections between O. kitaharae and O. sicerae and phages of O. oeni, suggesting that some related phages have interacted in diverse ways with their Oenococcus hosts over evolutionary time. Second, phage-host interaction analyses were performed experimentally with a diversified panel of phages and strains. None of the tested phages could infect strains across the species barrier. Yet, some infections occurred between phages and hosts from distinct beverages in the O. oeni species.

Distribution of Prophages in the Oenococcus oeni Species.

Oenococcus oeni is the most exploited lactic acid bacterium in the wine industry and drives the malolactic fermentation of wines. Although prophage-like sequences have been identified in the species, many are not characterized, and a global view of their integration and distribution amongst strains is currently lacking. In this work, we analyzed the complete genomes of 231 strains for the occurrence of prophages, and analyzed their size and positions of insertion. Our data show the limited variation in the number of prophages in O. oeni genomes, and that six sites of insertion within the bacterial genome are being used for site-specific recombination. Prophage diversity patterns varied significantly for different host lineages, and environmental niches. Overall, the findings highlight the pervasive presence of prophages in the O. oeni species, their role as a major source of within-species bacterial diversity and drivers of horizontal gene transfer. Our data also have implications for enhanced understanding of the prophage recombination events which occurred during evolution of O. oeni, as well as the potential of prophages in influencing the fitness of these bacteria in their distinct niches.

Wine Phenolic Compounds Differently Affect the Host-Killing Activity of Two Lytic Bacteriophages Infecting the Lactic Acid Bacterium Oenococcus oeni.

To provide insights into phage-host interactions during winemaking, we assessed whether phenolic compounds modulate the phage predation of Oenococcus oeni. Centrifugal partition chromatography was used to fractionate the phenolic compounds of a model red wine. The ability of lytic oenophage OE33PA to kill its host was reduced in the presence of two collected fractions in which we identified five compounds. Three, namely, quercetin, myricetin and p-coumaric acid, significantly reduced the phage predation of O. oeni when provided as individual pure molecules, as also did other structurally related compounds such as cinnamic acid. Their presence was correlated with a reduced adsorption rate of phage OE33PA on its host. Strikingly, none of the identified compounds affected the killing activity of the distantly related lytic phage Vinitor162. OE33PA and Vinitor162 were shown to exhibit different entry mechanisms to penetrate into bacterial cells. We propose that ligand-receptor interactions that mediate phage adsorption to the cell surface are diverse in O. oeni and are subject to differential interference by phenolic compounds. Their presence did not induce any modifications in the cell surface as visualized by TEM. Interestingly, docking analyses suggest that quercetin and cinnamic acid may interact with the tail of OE33PA and compete with host recognition.

"French Phage Network" Annual Conference-Fifth Meeting Report.

Attracting about 100 participants, the fifth edition of our French Phages.fr annual conference was once more a success. This year's conference took place at the Institute for Structural Biology on the European Electron and Photon Campus in Grenoble, October 8 and 9, 2019. Similar to previous years, our meeting gathered scientists mainly working in France, from academic labs and hospitals as well as from industry. We also had the pleasure of welcoming attendees from different European countries and even beyond. The conference was divided into four sessions: Ecology and Evolution, Phage Therapy and Biotechnology, Structure and Assembly and Phage-Host Interaction, each opened by a keynote lecture. The talks, selected from abstracts, gave the opportunity for young scientists (especially students and post-docs) to present their project and results in a friendly atmosphere. Poster sessions also favoured interactions and discussions between young researchers and more senior scientists. Here, we provide a summary of the topics developed during the conference.

Isolation and CryoTEM of Phages Infecting Bacterial Wine Spoilers.

With the objective to isolate phages infecting wine bacterial spoilers, we designed a method for the isolation and purification of phages infecting grape-associated bacteria. The method proved successful to isolate GC1 tectivirus infecting the acetic acid bacterium Gluconobacter cerinus. The isolated phage represents a new genus within the Tectiviridae, named "Gammatectivirus". Using a traditional technique for the concentration of phage particles involving several steps of centrifugation, further insights in the ultrastructure of GC1 could be observed by cryo electron microscopy, saving time and effort. The simple workflow presented may be applied to other viruses infecting bacteria inhabiting other vegetal niches.

Characterization of the First Virulent Phage Infecting Oenococcus oeni, the Queen of the Cellars.

There has been little exploration of how phages contribute to the diversity of the bacterial community associated with winemaking and may impact fermentations and product quality. Prophages of Oenococcus oeni, the most common species of lactic acid bacteria (LAB) associated with malolactic fermentation of wine, have been described, but no data is available regarding phages of O. oeni with true virulent lifestyles. The current study reports on the incidence and characterization of the first group of virulent oenophages named Vinitor, isolated from the enological environment. Vinitor phages are morphologically very similar to siphoviruses infecting other LAB. Although widespread during winemaking, they are more abundant in musts than temperate oenophages. We obtained the complete genomic sequences of phages Vinitor162 and Vinitor27, isolated from white and red wines, respectively. The assembled genomes shared 97.6% nucleotide identity and belong to the same species. Coupled with phylogenetic analysis, our study revealed that the genomes of Vinitor phages are architecturally mosaics and represent unique combinations of modules amongst LAB infecting-phages. Our data also provide some clues to possible evolutionary connections between Vinitor and (pro)phages associated to epiphytic and insect-related bacteria.

Lysogeny in the Lactic Acid Bacterium Oenococcus oeni Is Responsible for Modified Colony Morphology on Red Grape Juice Agar.

Oenococcus oeni is the lactic acid bacterium (LAB) that most commonly drives malolactic fermentation in wine. Although oenococcal prophages are highly prevalent, their implications on bacterial fitness have remained unexplored and more research is required in this field. An important step toward achieving this goal is the ability to produce isogenic pairs of strains that differ only by the lysogenic presence of a given prophage, allowing further comparisons of different phenotypic traits. A novel protocol for the rapid isolation of lysogens is presented. Bacteria were first picked from the center of turbid plaques produced by temperate oenophages on a sensitive nonlysogenic host. When streaked onto an agar medium containing red grape juice (RGJ), cells segregated into white and red colonies. PCR amplifications with phage-specific primers demonstrated that only lysogens underwent white-red morphotypic switching. The method proved successful for various oenophages irrespective of their genomic content and attachment site used for site-specific recombination in the bacterial chromosome. The color switch was also observed when a sensitive nonlysogenic strain was infected with an exogenously provided lytic phage, suggesting that intracolonial lysis triggers the change. Last, lysogens also produced red colonies on white grape juice agar supplemented with polyphenolic compounds. We posit that spontaneous prophage excision produces cell lysis events in lysogenic colonies growing on RGJ agar, which, in turn, foster interactions between lysed materials and polyphenolic compounds to yield colonies easily distinguishable by their red color. Furthermore, the technique was used successfully with other species of LAB.IMPORTANCE The presence of white and red colonies on red grape juice (RGJ) agar during enumeration of Oenococcus oeni in wine samples is frequently observed by stakeholders in the wine industry. Our study brings an explanation for this intriguing phenomenon and establishes a link between the white-red color switch and the lysogenic state of O. oeni It also provides a simple and inexpensive method to distinguish between lysogenic and nonlysogenic derivatives in O. oeni with a minimum of expended time and effort. Noteworthy, the protocol could be adapted to two other species of LAB, namely, Leuconostoc citreum and Lactobacillus plantarum It could be an effective tool to provide genetic, ecological, and functional insights into lysogeny and aid in improving biotechnological processes involving members of the lactic acid bacterium (LAB) family.

Contributions and lessons learned from virology in contemporary biology.

Virology is a young discipline at the origin of discoveries that revolutionized our vision of biology. Often associated to pathological studies, this science raises more fundamental questions and brings molecular tools required for cellular manipulation. If viruses are considered as our enemies, they are used, sometimes as a last attempt, against multidrug resistant bacteria or to treat cancer.

Clinical trials: odds ratios and multiple regression models--why and how to assess them.

Odds ratios (ORs), unlike chi2 tests, provide direct insight into the strength of the relationship between treatment modalities and treatment effects. Multiple regression models can reduce the data spread due to certain patient characteristics and thus improve the precision of the treatment comparison. Despite these advantages, the use of these methods in clinical trials is relatively uncommon. Our objectives were (1) to emphasize the great potential of ORs and multiple regression models as a basis of modern methods; (2) to illustrate their ease of use; and (3) to familiarize nonmathematical readers with these important methods. Advantages of ORs are multiple. (1) They describe the probability that people with a certain treatment will have an event, versus those without the treatment, and are therefore a welcome alternative to the widely used chi2 tests for analyzing binary data in clinical trials. (2) statistical software of ORs is widely available. (3) Computations using risk ratios (RRs) are less sensitive than those using ORs. (4) ORs are the basis for modern methods such as meta-analyses, propensity scores, logistic regression, and Cox regression. For analysis, logarithms of the ORs have to be used; results are obtained by calculating antilogarithms. A limitation of the ORs is that they present relative benefits but not absolute benefits. ORs, despite a fairly complex mathematical background, are easy to use, even for nonmathematicians. Both linear and logistic regression models can be adequately applied for the purpose of improving precision of parameter estimates such as treatment effects. We caution that, although application of these models is very easy with computer programs widely available, the fit of the regression models should always be carefully checked, and the covariate selection should be carefully considered and sparse. We do hope that this article will stimulate clinical investigators to use ORs and multiple regression models more often.

Novel procedures for validating surrogate endpoints in clinical trials.

BACKGROUND: The International Conference of Harmonisation (ICH) Guideline E9 Statistics Principles for Clinical Trials recommends that surrogate endpoints in clinical trials be validated using either (1) the sensitivity-specificity approach or (2) regression analysis. The problem with (1) is that an overall level of validity is hard to achieve, and with (2) is that a significant correlation between the surrogate and true endpoint is not enough to indicate that the surrogate is a valid predictor. OBJECTIVE: To provide for a nonmathematical readership, procedures that avoid the above two problems. RESULTS AND CONCLUSIONS: 1. Instead of the sensitivity-specificity approach, we used an overall validity level, expressed as the percentage of patients with a true surrogate test, either positive or negative. We calculated confidence intervals of this estimate, and assessed whether they were entirely within the prespecified interval of validity. If so, the surrogate marker was validated for use in subsequent trials. 2. For validating continuous surrogate variables, regression analysis was used, accounting for both the correlation between the surrogate and true endpoints, and the associations between these two variables and the treatment modalities to be tested. If the proportion of variability in the surrogate endpoint explained the true endpoint by 70% or more, the surrogate test was validated. A wrong conclusion here would be to accept validity if the surrogate endpoint was an independent determinant of the true endpoint, but not of the treatment modality. It is to be hoped that this paper will affect the validity of future clinical trials constructed with surrogate endpoints.