Finding a correct species assignment for a Metschnikowia strain: insights from the genome sequencing of strain DBT012.

Metschnikowia pulcherrima is an important yeast species that is attracting increased interest thanks to its biotechnological potential, especially in agri-food applications. Phylogenetically related species of the so-called 'pulcherrima clade' were first described and then reclassified in one single species, which makes the identification an intriguing issue. Starting from the whole-genome sequencing of the protechnological strain Metschnikowia sp. DBT012, this study applied comparative genomics to calculate similarity with the M. pulcherrima clade publicly available genomes with the aim to verify if novel single-copy putative phylogenetic markers could be selected, in comparison with the commonly used primary and secondary barcodes. The genome-based bioinformatic analysis allowed the identification of 85 consensus single-copy orthologs, which were reduced to three after split decomposition analysis. However, wet-lab amplification of these three genes in nonsequenced type strains revealed the presence of multiple copies, which made them unsuitable as phylogenetic markers. Finally, average nucleotide identity (ANI) was calculated between strain DBT012 and available genome sequences of the M. pulcherrima clade, although the genome dataset is still rather limited. Presence of multiple copies of phylogenetic markers as well as ANI values were compatible with the recent reclassification of the clade, allowing the identification of strain DBT012 as M. pulcherrima.

Non-conventional yeasts for food and additives production in a circular economy perspective.

Yeast species have been spontaneously participating in food production for millennia, but the scope of applications was greatly expanded since their key role in beer and wine fermentations was clearly acknowledged. The workhorse for industry and scientific research has always been Saccharomyces cerevisiae. It occupies the largest share of the dynamic yeast market, that could further increase thanks to the better exploitation of other yeast species. Food-related 'non-conventional' yeasts (NCY) represent a treasure trove for bioprospecting, with their huge untapped potential related to a great diversity of metabolic capabilities linked to niche adaptations. They are at the crossroad of bioprocesses and biorefineries, characterized by low biosafety risk and produce food and additives, being also able to contribute to production of building blocks and energy recovered from the generated waste and by-products. Considering that the usual pattern for bioprocess development focuses on single strains or species, in this review we suggest that bioprospecting at the genus level could be very promising. Candida, Starmerella, Kluyveromyces and Lachancea were briefly reviewed as case studies, showing that a taxonomy- and genome-based rationale could open multiple possibilities to unlock the biotechnological potential of NCY bioresources.

A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae.

The genus Lactobacillus comprises 261 species (at March 2020) that are extremely diverse at phenotypic, ecological and genotypic levels. This study evaluated the taxonomy of Lactobacillaceae and Leuconostocaceae on the basis of whole genome sequences. Parameters that were evaluated included core genome phylogeny, (conserved) pairwise average amino acid identity, clade-specific signature genes, physiological criteria and the ecology of the organisms. Based on this polyphasic approach, we propose reclassification of the genus Lactobacillus into 25 genera including the emended genus Lactobacillus, which includes host-adapted organisms that have been referred to as the Lactobacillus delbrueckii group, Paralactobacillus and 23 novel genera for which the names Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus are proposed. We also propose to emend the description of the family Lactobacillaceae to include all genera that were previously included in families Lactobacillaceae and Leuconostocaceae. The generic term 'lactobacilli' will remain useful to designate all organisms that were classified as Lactobacillaceae until 2020. This reclassification reflects the phylogenetic position of the micro-organisms, and groups lactobacilli into robust clades with shared ecological and metabolic properties, as exemplified for the emended genus Lactobacillus encompassing species adapted to vertebrates (such as Lactobacillus delbrueckii, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensensii, Lactobacillus johnsonii and Lactobacillus acidophilus) or invertebrates (such as Lactobacillus apis and Lactobacillus bombicola).

Genus-Wide Assessment of Antibiotic Resistance in Lactobacillus spp.

Lactobacillus species are widely used as probiotics and starter cultures for a variety of foods, supported by a long history of safe usage. Although more than 35 species meet the European Food Safety Authority (EFSA) criteria for qualified presumption of safety status, the safety of Lactobacillus species and their carriage of antibiotic resistance (AR) genes is under continuing ad hoc review. To comprehensively update the identification of AR in the genus Lactobacillus, we determined the antibiotic susceptibility patterns of 182 Lactobacillus type strains and compared these phenotypes to their genotypes based on genome-wide annotations of AR genes. Resistances to trimethoprim, vancomycin, and kanamycin were the most common phenotypes. A combination of homology-based screening and manual annotation identified genes encoding resistance to aminoglycosides (20 sequences), tetracycline (18), erythromycin (6), clindamycin (60), and chloramphenicol (42). In particular, the genes aac(3) and lsa, involved in resistance to aminoglycosides and clindamycin, respectively, were found in Lactobacillus spp. Acquired determinants predicted to code for tetracycline and erythromycin resistance were detected in Lactobacillus ingluviei, Lactobacillus amylophilus, and Lactobacillus amylotrophicus, flanked in the genome by mobile genetic elements with potential for horizontal transfer.IMPORTANCELactobacillus species are generally considered to be nonpathogenic and are used in a wide variety of foods and products for humans and animals. However, many of the species examined in this study have antibiotic resistance levels which exceed those recommended by the EFSA, suggesting that these cutoff values should be reexamined in light of the genetic basis for resistance discussed here. Our data provide evidence for rationally revising the regulatory guidelines for safety assessment of lactobacilli entering the food chain as starter cultures, food preservatives, or probiotics and will facilitate comprehensive genotype-based assessment of strains for safety screening.

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.

Effective identification of Lactobacillus casei group species: genome-based selection of the gene mutL as the target of a novel multiplex PCR assay.

Lactobacillus casei,Lactobacillus paracasei and Lactobacillusrhamnosus form a closely related taxonomic group (the L. casei group) within the facultatively heterofermentative lactobacilli. Strains of these species have been used for a long time as probiotics in a wide range of products, and they represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavour development. The close genetic relationship among those species, as well as the similarity of biochemical properties of the strains, hinders the development of an adequate selective method to identify these bacteria. Despite this being a hot topic, as demonstrated by the large amount of literature about it, the results of different proposed identification methods are often ambiguous and unsatisfactory. The aim of this study was to develop a more robust species-specific identification assay for differentiating the species of the L. casei group. A taxonomy-driven comparative genomic analysis was carried out to select the potential target genes whose similarity could better reflect genome-wide diversity. The gene mutL appeared to be the most promising one and, therefore, a novel species-specific multiplex PCR assay was developed to rapidly and effectively distinguish L. casei, L. paracasei and L. rhamnosus strains. The analysis of a collection of 76 wild dairy isolates, previously identified as members of the L. casei group combining the results of multiple approaches, revealed that the novel designed primers, especially in combination with already existing ones, were able to improve the discrimination power at the species level and reveal previously undiscovered intraspecific biodiversity.

Integrate genome-based assessment of safety for probiotic strains: Bacillus coagulans GBI-30, 6086 as a case study.

Probiotics are microorganisms that confer beneficial effects on the host; nevertheless, before being allowed for human consumption, their safety must be verified with accurate protocols. In the genomic era, such procedures should take into account the genomic-based approaches. This study aims at assessing the safety traits of Bacillus coagulans GBI-30, 6086 integrating the most updated genomics-based procedures and conventional phenotypic assays. Special attention was paid to putative virulence factors (VF), antibiotic resistance (AR) genes and genes encoding enzymes responsible for harmful metabolites (i.e. biogenic amines, BAs). This probiotic strain was phenotypically resistant to streptomycin and kanamycin, although the genome analysis suggested that the AR-related genes were not easily transferrable to other bacteria, and no other genes with potential safety risks, such as those related to VF or BA production, were retrieved. Furthermore, no unstable elements that could potentially lead to genomic rearrangements were detected. Moreover, a workflow is proposed to allow the proper taxonomic identification of a microbial strain and the accurate evaluation of risk-related gene traits, combining whole genome sequencing analysis with updated bioinformatics tools and standard phenotypic assays. The workflow presented can be generalized as a guideline for the safety investigation of novel probiotic strains to help stakeholders (from scientists to manufacturers and consumers) to meet regulatory requirements and avoid misleading information.

Complete PacBio Single-Molecule Real-Time Sequence of a Novel Probiotic-Like Bacterium, Rouxiella badensis subsp. acadiensis, Isolated from the Biota of Wild Blueberries in the Acadian Forest.

The PacBio whole-genome sequencing of the potential probiotic strain Canan SV-53T recovered from lowbush blueberries demonstrates high homology to Rouxiella badensis but with distinct enough clusters to propose the name Rouxiella badensis subsp. acadiensis. The sequencing also detected the presence of two native plasmids.

Glutathione production by non-Saccharomyces yeasts and its impact on winemaking: A review.

Glutathione (GSH) is a non-protein thiol naturally present in grape berries and produced by yeasts during fermentation. It has a strong antioxidant activity; thus, the addition of pure GSH during winemaking is recommended to limit the oxidative phenomena of wine, preserving sensory characteristics and stability, ultimately promoting a healthier product by reducing the need for SO2 addition. A promising alternative approach considers the use of yeast starter cultures high producers of this compound in situ, during the fermentation process, in substitution of external GSH addition. Recent research showed that multistarter fermentations with non-Saccharomyces yeasts produce even higher concentrations of GSH compared to single Saccharomyces cerevisiae. Accumulation of GSH in yeast cells is also considered valuable during the growth and dehydration of biomass for starter production, aiding strains to overcome the stressful conditions of industrial process. Moreover, a current trend in oenology is the use during fermentation of inactivated dry yeasts preparations as a source of nutrients, and many of them contain GSH-enriched cells. The aim of this review was to assess the significance of GSH production for the exploitation of wine-related non-Saccharomyces yeasts, both in starter biomass production and during fermentations, which were until now studied in detail for S. cerevisiae. This compendium highlights an interesting new feature of non-conventional yeasts and upgrade the strategy of multistarter fermentation as a valuable tool to positively modulate wine composition.

Reconstruction of Simplified Microbial Consortia to Modulate Sensory Quality of Kombucha Tea.

Kombucha is a fermented tea with a long history of production and consumption. It has been gaining popularity thanks to its refreshing taste and assumed beneficial properties. The microbial community responsible for tea fermentation-acetic acid bacteria (AAB), yeasts, and lactic acid bacteria (LAB)-is mainly found embedded in an extracellular cellulosic matrix located at the liquid-air interphase. To optimize the production process and investigate the contribution of individual strains, a collection of 26 unique strains was established from an artisanal-scale kombucha production; it included 13 AAB, 12 yeasts, and one LAB. Among these, distinctive strains, namely Novacetimonas hansenii T7SS-4G1, Brettanomyces bruxellensis T7SB-5W6, and Zygosaccharomyces parabailii T7SS-4W1, were used in mono- and co-culture fermentations. The monocultures highlighted important species-specific differences in the metabolism of sugars and organic acids, while binary co-cultures demonstrated the roles played by bacteria and yeasts in the production of cellulose and typical volatile acidity. Aroma complexity and sensory perception were comparable between reconstructed (with the three strains) and native microbial consortia. This study provided a broad picture of the strains' metabolic signatures, facilitating the standardization of kombucha production in order to obtain a product with desired characteristics by modulating strains presence or abundance.

Genomic diversity of Lactobacillus salivarius.

Strains of Lactobacillus salivarius are increasingly employed as probiotic agents for humans or animals. Despite the diversity of environmental sources from which they have been isolated, the genomic diversity of L. salivarius has been poorly characterized, and the implications of this diversity for strain selection have not been examined. To tackle this, we applied comparative genomic hybridization (CGH) and multilocus sequence typing (MLST) to 33 strains derived from humans, animals, or food. The CGH, based on total genome content, including small plasmids, identified 18 major regions of genomic variation, or hot spots for variation. Three major divisions were thus identified, with only a subset of the human isolates constituting an ecologically discernible group. Omission of the small plasmids from the CGH or analysis by MLST provided broadly concordant fine divisions and separated human-derived and animal-derived strains more clearly. The two gene clusters for exopolysaccharide (EPS) biosynthesis corresponded to regions of significant genomic diversity. The CGH-based groupings of these regions did not correlate with levels of production of bound or released EPS. Furthermore, EPS production was significantly modulated by available carbohydrate. In addition to proving difficult to predict from the gene content, EPS production levels correlated inversely with production of biofilms, a trait considered desirable in probiotic commensals. L. salivarius displays a high level of genomic diversity, and while selection of L. salivarius strains for probiotic use can be informed by CGH or MLST, it also requires pragmatic experimental validation of desired phenotypic traits.

Zygosaccharomyces gambellarensis sp. nov., an ascosporogenous yeast isolated from an Italian 'passito' style wine.

Yeast strains were isolated from Vin Santo of Gambellara, a sweet white wine with the specificity of Controlled Designation of Origin produced from off-vine overripened grapes in the Veneto region (Italy). Comparative sequence analysis of the 26S rRNA gene revealed that three representative strains (ZO03-5(T), CA06-8 and ME06-9) constitute a taxon related to, but distinct from, Zygosaccharomyces machadoi. Similarity between the 26S rRNA gene domain D1/D2 sequence of the three isolates and Z. machadoi was 97.9%; moreover, the morphological characteristics and the physiological behaviour also supported recognition of a novel taxon of osmophilic non-psychrophilic yeast showing a flower-like arrangement of budding cells that remain attached to each other. The name Zygosaccharomyces gambellarensis is proposed for the novel species, with ZO03-5(T) (=CBS 12191(T)=MUCL 53393(T)) as the type strain.

Reclassification of Lactobacillus catenaformis (Eggerth 1935) Moore and Holdeman 1970 and Lactobacillus vitulinus Sharpe et al. 1973 as Eggerthia catenaformis gen. nov., comb. nov. and Kandleria vitulina gen. nov., comb. nov., respectively.

The development of molecular tools and in particular the use of 16S rRNA gene sequencing has had a profound effect on the taxonomy of many bacterial groups. Gram-positive organisms that encompass the genera Lactobacillus and Clostridium within the Firmicutes are examples of taxa that have undergone major revisions based on phylogenetic information. A consequence of these reorganizations is that a number of organisms are now recognized as being misclassified. Previous studies have demonstrated that Lactobacillus catenaformis and Lactobacillus vitulinus are phylogenetically unrelated to Lactobacillus sensu stricto, being placed within the Clostridia rRNA cluster XVII. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, it is proposed that L. catenaformis and L. vitulinus be reclassified in two new genera, named respectively Eggerthia gen. nov., with the type species Eggerthia catenaformis gen. nov., comb. nov. (type strain DSM 20559(T) = ATCC 25536(T) = CCUG 48174(T) = CIP 104817(T) = JCM 1121(T)) and Kandleria gen. nov., with the type species Kandleria vitulina gen. nov., comb. nov. (type strain LMG 18931(T) = ATCC 27783(T) = CCUG 32236(T) = DSM 20405(T) = JCM 1143(T)).

Exploring Antibiotic Resistance Diversity in Leuconostoc spp. by a Genome-Based Approach: Focus on the lsaA Gene.

Leuconostoc spp. are environmental microorganisms commonly associated with fermented foods. Absence of antibiotic resistance (AR) in bacteria is a critical issue for global food safety. Herein, we updated the occurrence of AR genes in the Leuconostoc genus through in silico analyses of the genomes of 17 type strains. A total of 131 putative AR traits associated with the main clinically relevant antibiotics were detected. We found, for the first time, the lsaA gene in L. fallax ATCC 700006T and L. pseudomesenteroides NCDO 768T. Their amino acid sequences displayed high similarities (59.07% and 52.21%) with LsaA of Enterococcusfaecalis V583, involved in clindamycin (CLI) and quinupristin-dalfopristin (QUD) resistance. This trait has different distribution patterns in Leuconostoc nontype strains-i.e., L. pseudomesenteroides, L. lactis and L. falkenbergense isolates from fermented vegetables, cheeses, and starters. To better explore the role of lsaA, MIC for CLI and QUD were assessed in ATCC 700006T and NCDO 768T; both strains were resistant towards CLI, potentially linking lsaA to their resistant phenotype. Contrarily, NCDO 768T was sensitive towards QUD; however, expression of lsaA increased in presence of this antibiotic, indicating an active involvement of this trait and thus suggesting a revision of the QUD thresholds for this species.

Assessing Gut Microbiota in an Infant with Congenital Propionic Acidemia before and after Probiotic Supplementation.

Propionic Acidemia (PA) is a rare inherited metabolic disorder caused by the enzymatic block of propionyl-CoA carboxylase with the consequent accumulation of propionic acid, which is toxic for the brain and cardiac cells. Since a considerable amount of propionate is produced by intestinal bacteria, interest arose in the attempt to reduce propionate-producing bacteria through a monthly antibiotic treatment of metronidazole. In the present study, we investigated the gut microbiota structure of an infant diagnosed at 4 days of life through Expanded Newborn Screening (NBS) and treated the child following international guidelines with a special low-protein diet, specific medications and strict biochemical monitoring. Microbiota composition was assessed during the first month of life, and the presence of Bacteroides fragilis, known to be associated with propionate production, was effectively decreased by metronidazole treatment. After five antibiotic therapy cycles, at 4 months of age, the infant was supplemented with a daily mixture of three bifidobacterial strains, known not to be propionate producers. The supplementation increased the population of bifidobacteria, with Bifidobacterium breve as the dominating species; Ruminococcus gnavus, an acetate and formate producer, was also identified. Metabarcoding analysis, compared with low coverage whole metagenome sequencing, proved to capture all the microbial biodiversity and could be the elected tool for fast and cost-effective monitoring protocols to be implemented in the follow up of rare metabolic disorders such as PA. Data obtained could be a possible starting point to set up tailored microbiota modification treatment studies in the attempt to improve the quality of life of people affected by propionic acidemia.

Suitability of the Nisin Z-producer Lactococcus lactis subsp. lactis CBM 21 to be Used as an Adjunct Culture for Squacquerone Cheese Production.

This research investigated the technological and safety effects of the nisin Z producer Lactococcus lactis subsp. lactis CBM 21, tested as an adjunct culture for the making of Squacquerone cheese in a pilot-scale plant. The biocontrol agent remained at a high level throughout the cheese refrigerated storage, without having a negative influence on the viability of the conventional Streptococcus thermophilus starter. The inclusion of CBM 21 in Squacquerone cheesemaking proved to be more effective compared to the traditional one, to reduce total coliforms and Pseudomonas spp. Moreover, the novel/innovative adjunct culture tested did not negatively modify the proteolytic patterns of Squacquerone cheese, but it gave rise to products with specific volatile and texture profiles. The cheese produced with CBM 21 was more appreciated by the panelists with respect to the traditional one.

The Genomic Basis of Lactobacilli as Health-Promoting Organisms.

Lactobacilli occupy a unique position in human culture and scientific history. Like brewer's and baker's yeast, lactobacilli have been associated with food production and preservation for thousands of years. Lactobacillus species are used in mixed microbial cultures, such as the classical Lactobacillus bulgaricus/Streptococcus thermophilus inoculum for yogurt fermentation, or combinations of diverse lactobacilli/yeasts in kefir grains. The association of lactobacilli consumption with greater longevity and improved health formed the basis for developing lactobacilli as probiotics, whose market has exploded worldwide in the past 10 years. The decade that followed the determination of the first genome sequence of a food-associated species, Lactobacillus plantarum, saw the application to lactobacilli of a full range of functional genomics methods to identify the genes and gene products that govern their distinctive phenotypes and health associations. In this review, we will briefly remind the reader of the range of beneficial effects attributed to lactobacilli, and then explain the phylogenomic basis for the distribution of these traits across the genus. Recognizing the strain specificity of probiotic effects, we review studies of intraspecific genomic variation and their contributions to identifying probiotic traits. Finally we offer a perspective on classification of lactobacilli into new genera in a scheme that will make attributing probiotic properties to clades, taxa, and species more logical and more robust.

The Induction of Noble Rot (Botrytis cinerea) Infection during Postharvest Withering Changes the Metabolome of Grapevine Berries (Vitis vinifera L., cv. Garganega).

The natural or induced development of noble rot caused by the fungus Botrytis cinerea during the late stages of grapevine (Vitis vinifera L.) berry ripening is used in some traditional viticulture areas to produce high-quality wines such as Sauternes and Tokaji. In this research, we wanted to verify if by changing the environmental conditions during post-harvest withering we could induce the noble rot development on harvested berries in order to positively change the wine produced from withered Garganega berries. Therefore, we exposed the berries to postharvest withering under normal or artificially humid conditions, the latter to induce noble rot. The presence of noble rot symptoms was associated with the development of B. cinerea in the berries maintained under humid conditions. The composition of infected and non-infected berries was investigated by untargeted metabolomics using liquid chromatography/mass spectrometry. We also explored the effects of the two withering methods on the abundance of volatile organic compounds in wine by yeast-inoculated micro-fermentation followed by targeted gas chromatography/mass spectrometry. These experiments revealed significant metabolic differences between berries withered under normal and humid conditions, indicating that noble rot affects berry metabolism and composition. As well as well-known botrytization markers, we detected two novel lipids that have not been observed before in berries infected with noble rot. Unraveling the specific metabolic profile of berries infected with noble rot may help to determine the compounds responsible for the organoleptic quality traits of botrytized Garganega wines.