MALDI-TOF as a powerful tool for identifying and differentiating closely related microorganisms: the strange case of three reference strains of Paenibacillus polymyxa

Accurate identification and typing of microbes are crucial steps in gaining an awareness of the biological heterogeneity and reliability of microbial material within any proprietary or public collection. Paenibacillus polymyxa is a bacterial species of great agricultural and industrial importance due to its plant growth-promoting activities and production of several relevant secondary metabolites. In recent years, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used as an alternative rapid tool for identifying, typing, and differentiating closely related strains. In this study, we investigated the diversity of three P. polymyxa strains. The mass spectra of ATCC 842(T), DSM 292, and DSM 365 were obtained, analysed, and compared to select discriminant peaks using ClinProTools software and generate classification models. MALDI-TOF MS analysis showed inconsistent results in identifying DSM 292 and DSM 365 as belonging to P. polimixa species, and comparative analysis of mass spectra revealed the presence of highly discriminatory biomarkers among the three strains. 16S rRNA sequencing and Average Nucleotide Identity (ANI) confirmed the discrepancies found in the proteomic analysis. The case study presented here suggests the enormous potential of the proteomic-based approach, combined with statistical tools, to predict and explore differences between closely related strains in large microbial datasets

Bifidobacterium breve PRL2020: antibiotic-resistant profile and genomic detection of antibiotic resistance determinants

Antibiotics are one of the greatest scientific achievements of modern medicine, but excessive use is creating challenges for the future of medicine. Antibiotic resistance (AR) is thought to cause changes in bowel habits and an increased risk of gastroenteritis, but it may also increase the risk of overweight, obesity, autoimmune and atopic diseases, and a low response to vaccines and cancer, likely mediated by antibiotic-induced gut dysbiosis. Probiotic add-on therapy could partially prevent antibiotic-induced gut dysbiosis, but their antibiotic sensitivity features likely limits this potential. The EFSA (European Food Safety Authority) guidelines consider the use of probiotics whose antibiotic-resistant profile could be transferable an important hazard. Recently, a strain of B. breve (PRL2020) has shown to be resistant to amoxicillin and amoxicillin-clavulanate (AC) by applying the microdilution protocol according EFSA guidelines. After verifying that horizontal gene transfer is unlikely to take place, this feature suggests its concomitant use with these specific antibiotics. The results of our tests demonstrated that the strain PRL2020 is indeed endowed with amoxicillin- and AC-resistant properties and that it is also insensitive to ampicillin. In-depth analysis of the annotated genome sequence of B. breve PRL2020 was employed to query the Comprehensive Antibiotic Resistance Database (CARD) using Resistance Gene Identifier (RGI) software (version 5.2.1). The similarity among the AR determinants found was studied through nucleotide sequence alignment, and it was possible to verify not only the absence of genes explaining these features in the flanking regions but also the presence of genetic sequences (rpoB and erm(X)) putatively responsible for rifampicin and erythromycin resistance. Both features are not phenotypically expressed, and for these antibiotics, the strain is within the EFSA limits. Analysis of the flanking regions of these genes revealed possible mobile elements upstream and downstream only in the case of the erm(X) gene, but the features of the Insertion Sequences (IS) are described as not to cause horizontal transfer. Our findings on strain PRL2020 demonstrate that its AR profile is compatible with antibiotics when taken with the aim of reducing the risk of dysbiosis

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

Development and validation of a multiplex PCR-based DNA microarray hybridisation method for detecting bacterial antibiotic resistance genes in cheese

The aim of this study was to develop a method for detecting antibiotic resistance (AR) genes in cheese based on a combination of multiplex PCR and a DNA microarray hybridisation system. Twenty oligonucleotide probes were designed targeting 10 common AR genes, namely aac(6′)-Ie-aph(2″)-Ia, aadE, aphA-3, ermB, tet(L), tet(M), tet(O), tet(S), vanA and vanB. Specificity of the probes was tested by hybridising against DNA from Enterococcus strains harbouring known AR genes. DNA was labelled through two multiplex PCR reactions with fluorescent nucleotides and specific primers flanking the probe sequences. Sensitivity of the microarray was assessed by contamination of a cheese with an Enterococcus faecium strain carrying vanA gene. Two tetracycline resistance genes, tet(M) and tet(S), proved to be present in a series of retail cheeses, while genes aadE, aphA3, ermB, tet(L) and tet(O) were occasionally detected. This method is envisioned as a valuable tool for identification of AR genes in foods. © 2010 Elsevier Ltd.This research was partly supported by the Joint Project 2005 “DEVICE” funded jointly by the Università degli Studi di Verona and Biodiversity S.P.A., and by a Spain-Italy bilateral collaboration program (references IT2009-0080 and IT105MD12L). S. Delgado was granted with a postdoctoral fellowship from Fundación Alonso Martín Escudero (Spain).Peer Reviewe

La biodiversit\ue0 come risorsa: studio della variabilit\ue0 intraspecifica nella specie Oenococcus oeni, per la valorizzazione di tratti fenotipici peculiari

I ceppi della specie Oenococcus oeni sono i principali agenti microbici responsabili della fermentazione malolattica (FML) e sono perfettamente adattati a sopravvivere in un ambiente ostile come il vino. Oenococcus oeni, infatti, \ue8 in grado di crescere in carenza di nutrienti, a pH molto bassi e tollera concentrazioni di etanolo molto elevate. In funzione del ceppo \ue8 anche in grado di garantire al vino note aromatiche tipiche. Grazie a queste caratteristiche risulta il microrganismo pi\uf9 idoneo ad essere utilizzato come starter per indurre la FML. L\u2019enorme interesse per questa specie non deriva solamente da motivi economici, ma anche da studi di base che hanno portato alcuni autori a proporre O. oeni come specie modello sia per lo studio del comportamento acidofilo nei batteri lattici, sia per l\u2019analisi dei meccanismi evolutivi che plasmano il genoma. Infatti, la specie O. oeni \ue8 prona ad accumulare mutazioni in quanto non possiede due componenti fondamentali del sistema di riparazione MMR (MisMatch Repairing) utilizzato per correggere gli errori di replicazione nel DNA. Questo la rende suscettibile sia all\u2019accumulo di mutazioni puntiformi disperse nel genoma, sia all\u2019acquisizione di DNA esogeno, aumentandone la variabilit\ue0 intraspecifica. Tuttavia, data l\u2019importanza tecnologica ed economica di O. oeni, l\u2019analisi della biodiversit\ue0 interna alla specie si \ue8 spesso concentrata solamente su aspetti con ricadute immediate nel settore enologico. Per tutti questi motivi risulta evidente che il quadro delle potenzialit\ue0 metaboliche e fisiologiche della specie O. oeni non \ue8 ancora completo. Questo lavoro di tesi \ue8 stato preceduto da diversi studi ecologici che hanno avuto come obiettivo l\u2019analisi della diffusione e della variabilit\ue0 della specie O. oeni in vini di pregio della Provincia di Verona, in particolar modo nel vino Amarone. Nel corso di tali studi sono stati isolati molti ceppi con propriet\ue0 enologiche differenti. Alcuni ceppi hanno mostrato potenzialit\ue0 tecnologiche interessanti, mentre altri si sono rivelati inadatti all\u2019applicazione nel settore enologico. Preso atto della variabilit\ue0 interna alla specie O. oeni e delle diverse potenzialit\ue0 enologiche dei ceppi presenti nella collezione del Dipartimento di Biotecnologie, in questa Tesi di Dottorato il lavoro si \ue8 concentrato su un numero ridotto di ceppi che presentavano caratteristiche singolari, con l\u2019obiettivo di approfondire le conoscenze riguardanti ciascun ceppo; di mettere a punto procedure atte a differenziarli in modo efficace; di valorizzare gli aspetti peculiari di alcuni ceppi per rivalutarne il potenziale tecnologico in vista di possibili applicazioni enologiche. Il lavoro di tesi \ue8 diviso in due parti. Nella prima parte della tesi \ue8 stata caratterizzata dal punto di vista fenotipico e genotipico una collezione modello di dieci ceppi di O. oeni, rappresentativi della variabilit\ue0 riscontrata nei lavori precedenti. Successivamente \ue8 stata dimostrata a livello fisiologico l\u2019esistenza di una correlazione tra risposta ad elevate concentrazioni di etanolo e origine di isolamento, tramite analisi dei cambiamenti nella fluidit\ue0 di membrana in risposta a diverse concentrazioni di alcol. Infine, \ue8 stata considerata la possibilit\ue0 di differenziare i ceppi in base all\u2019espressione genica di marcatori ubiquitari coinvolti nella risposta agli stress e grazie alla presenza di specifici loci correlati con una maggiore resistenza. Nella seconda parte, il lavoro si \ue8 concentrato sulla caratterizzazione del ceppo Br10, che mostra la capacit\ue0 di sviluppare in aggregati (pellicole), mai descritta prima nella specie O. oeni ed \ue8 caratterizzato da una buona resistenza a diversi stress ambientali. In particolare, \ue8 stata determinata tramite saggi enzimatici e colorimetrici la natura polisaccaridica della matrice extracellulare ed \ue8 stata analizzata l\u2019influenza del mezzo di coltura sulla capacit\ue0 fermentativa in vino e sulla formazione di aggregati. Infine, \ue8 stata valutata con successo la possibilit\ue0 di sfruttare questa sua peculiarit\ue0 in applicazioni del settore enologico come la fermentazione di vini difficili (dopo opportuno preadattamento) e la pratica del co-inoculo di lieviti e batteri in mosto. In conclusione, lo studio della biodiversit\ue0 ha dimostrato che essa \ue8 un\u2019importante risorsa, non solo dal punto di vista puramente scientifico, ma anche da quello tecnologico e applicativo. Il continuo isolamento di nuovi ceppi e lo studio approfondito delle loro caratteristiche fenotipiche e genotipiche rappresenta, infatti, il prerequisito fondamentale per continuare ad innovare i processi fermentativi e migliorare la qualit\ue0 del prodotto finale.The strains belonging to the species Oenococcus oeni are the main microbial agents responsible for malolactic fermentation (MLF), as they are perfectly adapted to survive in a harsh environment such as wine. Oenococcus oeni is indeed able to grow in starving conditions, at low pH values, and it tolerates very high concentrations of ethanol. Depending on the strain, O. oeni gives to wine also typical aromatic notes. Thanks to these characteristics, strains of O. oeni are the most suitable microorganisms to be used as MLF starter. This species is very interesting not only for economic and technological reasons, but also for basic studies. Oenococcus oeni has indeed been suggested by some authors as a model species both to study the behaviour of lactic acid bacteria in acidic environments, and to analyse the evolutionary mechanisms of microbial genome. In fact, O. oeni is prone to accumulate mutations as it lacks two components of the MMR (MisMatch Repairing) system, necessary to correct DNA replication errors. This leads O. oeni to increase variability by accumulating both point mutations in the genome, and the acquisition of exogenous DNA by horizontal gene transfer. Nonetheless, the analysis of the microbial biodiversity has often focused on few topics with the purpose to ensure certain outcomes for starter producing companies and winemakers. Therefore the framework of metabolic and physiological potential of the O. oeni species is not yet complete. The present research was preceded by numerous ecological studies to analyse the diffusion and the biodiversity of O. oeni strains in Verona wines, especially the Amarone wine. During those researches several strains with different features has been isolated: some strains showed interesting technological properties, while many of them were unsuitable in winemaking trials. Considering the variability showed by the species O. oeni and the different aptitudes of the strains present in the collection of the Department of Biotechnology to carry out MLF in winemaking processes, in this PhD thesis the attention was focused on a small number of strains with unique characteristics. The final aims were to deepen the knowledge about the strains; to develop procedures for their effective differentiation and to exploit the distinctive features of some strains re-evaluating their potential in view of possible applications in winemaking processes. The study was divided into two parts. In the first part, a model collection of ten O. oeni strains, representing the variability observed in previous research was phenotypically and genotypically characterized. Then a correlation between the response to high concentrations of ethanol and the isolation source was demonstrated, through the analysis of membrane fluidity changes in response to different concentrations of alcohol. Finally, the possibility to differentiate strains by the expression patterns of ubiquitous marker genes involved in stress response was evaluated. Moreover, the correlation between the presence in the genome of specific loci associated with an increased resistance and the real properties of the strains was assessed. In the second part, the research focused on the characterization of the strain Br10, which develops in aggregates (pellicles or biofilms), a property never described before for the O. oeni species, and also characterized by a good resistance to various environmental stresses. More in detail, the presence of polysaccharides in the extracellular matrix was determined by colorimetric and enzymatic assays. Moreover, the influence of the culture medium on the capacity to form the aggregates and to carry out MLF in wine was analysed. The possibility to exploit the phenotypic peculiarity showed by the strain Br10 was successfully evaluated, first inoculating the pellicles in difficult wine (after appropriate preadaptation), then performing co-inoculation of yeasts and aggregative bacteria in grape juice. In conclusion, biodiversity represents an important resource, not only from a purely scientific point of view, but also for technological aspects. The constant isolation of new strains and the analysis of their phenotypic and genotypic characteristics are, indeed, the prerequisites to innovate the fermentation processes and to improve the quality of the final products

HIGHLY POLYMORPHIC AND MODULAR EXTRAGENIC (H.P.M.E.) MARKERS WITHIN SPECIFIC TAXA OF MICROORGANISMS AND USE THEREOF FOR THEIR DIFFERENTIATION, IDENTIFICATION AND QUANTIFICATION

The present invention concerns the field of nucleic acid based methods suitable for the generation of tools for biomedical research and biotechnological applications, which allow to differentiate, identify and quantify microorganisms and viruses

Il controllo microbiologico in vinificazione: i batteri

La composizione e la qualità del vino sono influenzate da innumerevoli variabili intrinseche ed estrinseche, molte delle quali sono mediate da microrganismi. Un ruolo preminente viene svolto dai lieviti, in particolare Saccharomyces cerevisiae, che conducono la fermentazione alcolica, e dai batteri lattici, che effettuano la fermentazione malolattica (FML). Durante la FML, Oenoccocus oeni riduce l’acidità fissa, contribuisce alla complessità aromatica e migliora la stabilità microbica del vino. Tuttavia, non tutti i batteri sono positivi. Diversi batteri possono alterare il vino in vari modi e devono quindi essere attentamente controllati. Lo stesso O. oeni è causa di difetti quando sviluppa in vini dove la FML non è desiderata. Nel presente articolo sarà analizzata la componente batterica del microbiota associato alla filiera produttiva del vino, considerando sia i batteri positivi sia quelli causa di alterazioni, descrivendo alcuni metodi per il loro rilevamento e controllo

Use of Saccharomyces and non-Saccharomyces yeasts for the management and optimization of the Vin Santo of Gambellara fermentation process

Vin Santo of Gambellara is a typical sweet white wine produced in the Veneto region (Italy) with the specificity of Controlled Designation of Origin. Its winemaking procedure includes a withering step, where Garganega grapes are partially dried in attics for five-six months, a slow fermentation phase, and finally an aging period of two or more years in oak barrels. The high sugar levels of the must (up to 350 g/L), the presence of inhibitory substances and the poor control of fermentation parameters, as the indigenous microbial population and the temperature, can cause frequent sluggish and stuck fermentations or high production of volatile acidity. To avoid all possible risks which can occur during vinification and preserve the typical sensory properties of Vin Santo of Gambellara, the aim of the present study was the development of a protocol to provide indigenous yeast starter cultures to better overcome the stressing conditions of the alcoholic fermentation. A large collection of Saccharomyces and non-Saccharomyces yeasts from spontaneous fermentation of musts and wines aged in barrels was constituted. The isolates showed a high interspecific biodiversity as depicted by molecular methods, and they were found to belong mainly to the species S. cerevisiae, Zygosaccharomyces rouxii, Z. bailii, and Z. gambellarensis. Strains were screened for several oenological properties and the most promising ones were employed in pilot-scale single and sequential fermentation trials. The combination of S. cerevisiae with Zygosaccharomyces spp. strains prevented the risk of stuck or sluggish fermentations. Furthermore, wines obtained were characterized by the presence of positive metabolites, as fruity esters and higher alcohols, and by low levels of acetic acid and ethyl acetate with respect to the spontaneous fermentations. In conclusion, the present study suggests that the use of the newly selected indigenous Saccharomyces and Zygosaccharomyces cultures represents a valuable strategy for the management and optimization of the fermentation process of Vin Santo of Gambellara, minimizing the production of spoilage metabolites and to preserve the complexity of the wine aroma composition