On species descriptions based on a single strain: proposal to introduce the status species proponenda (sp. pr.).

A survey of the descriptions of novel bacterial species published in the period 1996–2006 revealed that a large number of taxonomic descriptions are still based on one or a few strains. This situation determines that not only species descriptions, but also proposals to create higher ranks, are actually based on very few strains, which could produce a highly biased scenario. The encouragement to include a reasonable number of strains in species descriptions has been largely disregarded after its proposal, since acceptance of such descriptions relies mainly on editors' and reviewers' opinions. This observation and other considerations lead us to propose the creation of the status species proponenda (sp. pr.), as a compromise between the need for scientific description of biodiversity and exchange of data and the good taxonomic practice of including a sufficient number of strains in descriptions of species and higher taxonomic ranks

Unravelling the Impact of Grape Washing, {SO}2, and Multi-Starter Inoculation in Lab-Scale Vinification Trials of Withered Black Grapes

Wine quality is strongly affected by chemical composition and microbial population of grape must, which, in turn, are influenced by several post-harvest treatments, including grape withering. Different strategies have been suggested to manage the fermenting must microbiota, as it plays a central role in the outcomes of both spontaneous and guided fermentations. This study aimed at evaluating the impact of grape washing, SO2 addition, and selected starter culture inoculation on population dynamics, fermentation kinetics, and main oenological parameters in lab-scale trials, focusing on withered grapes usually used for Amarone production. Although grape washing treatment was effective in removing heavy metals and undesirable microorganisms from grape berry surface, inoculation of multi-starter cultures impacted more fermentation rates. Further, both grape washing and starter inoculation procedures had a remarkable impact on wine chemical characteristics, while 30 mg/L SO2 addition did not significantly affect the fermentation process. In summary, the best strategy in terms of limiting off-flavors and potentially reducing the need for SO2 addition in wine from withered grapes was the use of yeast starters, particularly mixed cultures composed by selected strains of Metschnikowia spp. and Saccharomyces cerevisiae. Application of a washing step before winemaking showed a potential to improve organoleptic characteristics of win

International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Bifidobacterium, Lactobacillus and related organisms. Minutes of the closed meeting by videoconference, 3 September 2020

International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Bifidobacterium, Lactobacillus and related organisms. Minutes of the closed meeting by videoconference, 3 September 202

Culture-independent analysis of diversity in microbial communities: pros and cons of the partial sequence-based profiling

Among the culture-independent techniques developed so far, DGGE/TGGE analysis of amplified 16S rRNA fragments is currently used to a great extent for the characterization of microbial consortia, due to its fast response and highly descriptive power. This approach relies on the separations of partial 16S rRNA gene amplicons targeting two specific hypervariable regions, namely V3 and V6-V8. Separation is followed by sequencing and identification of different bands based on BLAST comparisons. Some case studies are presented here concerning the identification of bacterial species and genera - belonging to different phyla such as Actinobacteria (Bifidobacterium, Rhodococcus), Firmicutes (Bacillus, Enterococcus, and Lactobacillus), Alphaproteobacteria (Sinorhizobium), Betaproteobacteria (Burkholderia), and Gammaproteobacteria (Pseudomonas) \u2013 all relevant to environmental and industrial biotechnology

Application of FTIR Microspectroscopy in Oenology: Shedding Light on Cell Wall Composition of Saccharomyces cerevisiae Strains

The evaluation of cell parietal components of yeasts is an important criterium for the selection of wine starters since they play a key role in the vinification process. The aim of this study was to characterize and compare the cell wall composition of four industrial (BM45, D47, EC1118, K1) and three native Saccharomyces cerevisiae (MY8, MY11, PEDRO2000E) wine strains by means of scanning and transmission electron microscopy and ATR-FTIR microspectroscopy. A statistically significant variability in the cell wall thickness and cell diameter was observed among the yeast cells, with native strains showing higher cell diameter values. FTIR microspectroscopy applied on the intact cells without any previous invasive treatment and on the separated cell walls highlighted profound differences among the strains in terms of the overall content of parietal polysaccharides as related to the thickness of the cell walls and in terms of the relative concentration of beta-glucans and mannans in the cell walls. The strains EC1118, MY11, and PEDRO2000E showed a higher overall content of beta-glucans and mannans, whose lower relative concentration in PEDRO2000E was compensated by a thicker cell wall; BM45 and D47 were characterized by a high relative concentration of polysaccharides in a thinner wall, while K1 and MY8 displayed a low relative concentration of polysaccharides. ATR-FTIR microspectroscopy allows identifying polysaccharide-rich yeast strains and can become a smart option for the selection of starter cultures to be used in oenology and for other applications in food industry, thanks to the interesting technological properties of parietal polysaccharides

Molecular Identification and Quantification of Tetracycline and Erythromycin Resistance Genes in Spanish and Italian Retail Cheeses

Large antibiotic resistance gene pools in the microbiota of foods may ultimately pose a risk for human health. This study reports the identification and quantification of tetracycline- and erythromycin-resistant populations, resistance genes, and gene diversity in traditional Spanish and Italian cheeses, via culturing, conventional PCR, real-time quantitative PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). The numbers of resistant bacteria varied widely among the antibiotics and the different cheese varieties; in some cheeses, all the bacterial populations seemed to be resistant. Up to eight antibiotic resistance genes were sought by gene-specific PCR, six with respect to tetracycline, that is, tet(K), tet(L), tet(M), tet(O), tet(S), and tet(W), and two with respect to erythromycin, that is, erm(B) and erm(F). The most common resistance genes in the analysed cheeses were tet(S), tet(W), tet(M), and erm(B). The copy numbers of these genes, as quantified by qPCR, ranged widely between cheeses (from 4.94 to 10.18 log10/g). DGGE analysis revealed distinct banding profiles and two polymorphic nucleotide positions for tet(W)-carrying cheeses, though the similarity of the sequences suggests this tet(W) to have a monophyletic origin. Traditional cheeses would therefore appear to act as reservoirs for large numbers of many types of antibiotic resistance determinants.The study was partially supported by a Spain-Italy bilateral collaboration program (Ref. IT2009-0080 and IT105MD12L). Financial support was further provided by projects from CICYT (Ref. AGL2011-24300-ALI) and INIA (Ref. RM2011-00005-00-00). A. B. Flórez and S. Delgado were supported by research contracts under Juan de la Cierva Program (Ref. JCI-2010-07457 and JCI-2008-02391, resp.). A. Alegría was awarded a scholarship of the Severo Ochoa program from FICYT (Ref. BP08-053).Peer Reviewe

Comparative genomics of the genus Lactobacillus reveals robust phylogroups that provide the basis for reclassification

The genus Lactobacillus includes over 200 species that are widely used in fermented food preservation and biotechnology or that are explored for beneficial effects on health. Naming, classifying, and comparing lactobacilli have been challenging due to the high level of phenotypic and genotypic diversity that they display and because of the uncertain degree of relatedness between them and associated genera. The aim of this study was to investigate the feasibility of dividing the genus Lactobacillus into more homogeneous genera/clusters, exploiting genome-based data. The relatedness of 269 species belonging primarily to the families Lactobacillaceae and Leuconostocaceae was investigated through phylogenetic analysis (by the use of ribosomal proteins and housekeeping genes) and the assessment of the average amino acid identity (AAI) and the percentage of conserved proteins (POCP). For each subgeneric group that emerged, conserved signature genes were identified. Both distance-based and sequence-based metrics showed that the Lactobacillus genus was paraphyletic and revealed the presence of 10 methodologically consistent subclades, which were also characterized by a distinct distribution of conserved signature orthologues. We present two ways to reclassify lactobacilli: a conservative division into two subgeneric groups based on the presence/absence of a key carbohydrate utilization gene or a more radical subdivision into 10 groups that satisfy more stringent criteria for genomic relatedness. Importance: Lactobacilli have significant scientific and economic value, but their extraordinary diversity means that they are not robustly classified. The 10 homogeneous genera/subgeneric entities that we identify here are characterized by uniform patterns of the presence/absence of specific sets of genes which offer potential as discovery tools for understanding differential biological features. Reclassification/subdivision of the genus Lactobacillus into more uniform taxonomic nuclei will also provide accurate molecular markers that will be enabling for regulatory approval applications. Reclassification will facilitate scientific communication related to lactobacilli and prevent misidentification issues, which are still the major cause of mislabeling of probiotic and food products reported worldwide

Whole genome analysis as a tool for the safety assessment of antibiotic resistance in food-processing bacteria

Trabajo presentado en la 2nd EFSA Scientific Conference, celebrado en Milán, Italia, del 14 al 16 de octubre de 2015Acquisition of antibiotic resistances (AR) by pathogens leads ultimately to a failure of antibiotic therapy. The food chain is considered a key player in the transmission of AR determinants to pathogens from reservoirs in commensal and beneficial bacteria. Therefore, the absence of transmissible AR genes in bacteria used as starter and adjunct cultures for food and feed processing is considered to be critical (EFSA, 2012; EFSA Journal, 10:2740). Genome sequencing allows the inspection of the whole genetic makeup of bacteria in the search for the basis of desirable and undesirable traits, including that of AR. Thus, in silico sequence analysis and comparison against databases can be used as a tool for the safety assessment of microorganisms intended to be used in food systems. This communication reports on the genome analysis of three Leuconostoc mesenteroides strains of dairy origin showing atypical resistances to tetracycline (LbT16), erythromycin and clindamycin (LbE15), and kanamycin, streptomycin, tetracycline and virginiamycin (LbE16). Genes encoding for erythromycin [erm(B)] and tetracycline [tet(S)] resistance had already been detected by PCR. Genome analysis confirmed the presence of these genes and identified others which encode uncommon AR in lactic acid bacteria. Analysis of the genes and their flanking regions revealed a potential of some to be horizontally transferred to other bacteria. This study demonstrates the effectiveness of combining genome sequencing and bioinformatics analysis as an affordable tool for the safety assessment of food bacteria. This innovative approach could become a novel paradigm in the selection programs of starters for the food industry.Peer Reviewe

Bark and Grape Microbiome of Vitis vinifera: Influence of Geographic Patterns and Agronomic Management on Bacterial Diversity

In recent years, the concept of “microbial terroir” has been introduced in the frame of the more renowned notion of “vitivinicultural terroir,’ since several studies demonstrated that wine characteristics are related to regional microbial community compositions. Most of the existing research focused on grape berries microbiota, since it can directly impact wine quality. In this work we studied, for the first time through next-generation sequencing, the epiphytic bacterial community of vine bark and its relationships with grape microbiota. The study was carried out in two Italian wine appellations (situated in different regions) to explore the impact of biogeography, and the influence of two agronomical practices (biodynamic and conventional) was evaluated as well. Overall, our results show that grapevine bark harbors a rich epiphytic microbiota and displays a higher microbial biodiversity than grape berry. Moreover, this study suggests that geographic and anthropogenic factors impact both bark and grape bacteriomes, but to a different extent. The evidence of a “microbial terroir” seems to be even more marked in bark than in berries, possibly due to its permanence over time and to its physical proximity with soil. The importance of vine trunk bark, as potential source of inoculum for grapes and as interesting bacterial diversity habitat, is evidenced. This opens new fields of investigation, not only for researchers that aim at describing this little-known habitat within the vineyard, but also for stakeholders from the wine industry that want to understand the roles of microorganisms on the entire winemaking process, from vineyard to cellar

Whole-metagenome-sequencing-based community profiles of Vitis vinifera L. cv. Corvina berries withered in two post-harvest conditions

Vitis vinifera L. cv. Corvina grape forms the basis for the production of unique wines, such as Amarone, whose distinctive sensory features are strongly linked to the post-harvest grape withering process. Indeed, this process increases sugar concentration and changes must characteristics. While microorganisms involved in must fermentation have been widely investigated, few data are available on the microbiota of withered grapes. Thus, in this paper, a whole metagenome sequencing (WMS) approach was used to analyse the microbial consortium associated with Corvina berries at the end of the withering process performed in two different conditions (“traditional withering,” TW or “accelerated withering,” AW), and to unveil whether changes of drying parameters could have an impact on microbial diversity. Samples of healthy undamaged berries were collected and washed, to recover microorganisms from the surface and avoid contamination with grapevine genetic material. Isolated DNA was sequenced and the data obtained were analyzed with several bioinformatics methods. The eukaryotic community was mainly composed by members of the phylum Ascomycota, including Eurotiomycetes, Sordariomycetes, and Dothideomycetes. Moreover, the distribution of the genera Aspergillus and Penicillium (class Eurotiomycetes) varied between the withered berry samples. Instead, Botryotinia, Saccharomyces, and other wine technologically useful microorganisms were relatively scarce in both samples. For prokaryotes, 25 phyla were identified, nine of which were common to both conditions. Environmental bacteria belonging to the class Gammaproteobacteria were dominant and, in particular, the TW sample was characterized by members of the family Pseudomonadaceae, while members of the family Enterobacteriaceae dominated the AW sample, in addition to Sphyngobacteria and Clostridia. Finally, the binning procedure discovered 15 putative genomes which dominated the microbial community of the two samples, and included representatives of genera Erwinia, Pantoea, Pseudomonas, Clostridium, Paenibacillus, and of orders Lactobacillales and Actinomycetales. These results provide insights into the microbial consortium of Corvina withered berries and reveal relevant variations attributable to post-harvest withering conditions, underling how WMS could open novel perspectives in the knowledge and management of the withering process of Corvina, with an impact on the winemaking of important Italian wines