A polyphasic molecular approach to characterize a collection of grapevine endophytic bacteria with bioprotective potential.

AIMS: The work presented here was conducted to characterize the biodiversity of a collection of bacterial isolates, mainly wood endophytes, as part of a research project focused on exploring their bioprotective potential for postharvest biological control of fruits. METHODS AND RESULTS: This work was the basis for the development of a tailored method combining 16S rDNA sequencing and Rep-PCR to differentiate the isolates and identify them to genus level or below. More than one hundred isolates obtained from wood and roots of different grapevine genotypes were cultured on appropriate growth media and then subjected to the specified multistep molecular identification. CONCLUSIONS: We have obtained good dereplication for grapevine-endophytic bacteria, together with reliable genetic identification. Both are essential prerequisites to properly characterize a biome bank and, at the same time, beneficial prerequisites to subsequently perform a correct bioprotection assessment.

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 842T, 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.

Isolation and Sequencing of the First Case of Symptomatic MDR Salmonella enterica Subsp. Enterica ST40 in Human in Italy, July 2020.

Strains of drug-resistant nontyphoidal Salmonella spp. are emerging in livestock worldwide. We describe the first case of symptomatic multidrug-resistant (MDR) Salmonella enterica subsp. enterica in human and the genetic mechanisms at the basis of its antibiotic resistance. To control outbreaks, rapid identification and sequencing are necessary. Proactive research and notification are needed to evaluate the routes of transmission from livestock to humans and risk-management strategies of MDR Salmonella strains.

Bifidobacteria Strain Typing by Fourier Transform Infrared Spectroscopy.

Fourier transform infrared (FTIR) spectroscopy, a technology traditionally used in chemistry to determine the molecular composition of a wide range of sample types, has gained growing interest in microbial typing. It is based on the different vibrational modes of the covalent bonds between atoms of a given sample, as bacterial cells, induced by the absorption of infrared radiation. This technique has been largely used for the study of pathogenic species, especially in the clinical field, and has been proposed also for the typing at different subspecies levels. The high throughput, speed, low cost, and simplicity make FTIR spectroscopy an attractive technique also for industrial applications, in particular, for probiotics. The aim of this study was to compare FTIR spectroscopy with established genotyping methods, pulsed-field gel electrophoresis (PFGE), whole-genome sequencing (WGS), and multilocus sequence typing (MLST), in order to highlight the FTIR spectroscopy potential discriminatory power at strain level. Our study focused on bifidobacteria, an important group of intestinal commensals generally recognized as probiotics. For their properties in promoting and maintaining health, bifidobacteria are largely marketed by the pharmaceutical, food, and dairy industries. Strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium animalis subsp. lactis were taken into consideration together with some additional type strains. For B. longum subsp. longum, it was possible to discriminate the strains with all the methods used. Although two isolates were shown to be strictly phylogenetically related, constituting a unique cluster, based on PFGE, WGS, and MLST, no clustering was observed with FTIR. For B. animalis subsp. lactis group, PFGE, WGS, and MLST were non-discriminatory, and only one strain was easily distinguished. On the other hand, FTIR discriminated all the isolates one by one, and no clustering was observed. According to these results, FTIR analysis is not only equivalent to PFGE, WGS, and MLST, but also for some strains, in particular, for B. animalis subsp. lactis group, more informative, being able to differentiate strains not discernible with the other two methods based on phenotypic variations likely deriving from certain genetic changes. Fourier transform infrared spectroscopy has highlighted the possibility of using the cell surface as a kind of barcode making tracing strains possible, representing an important aspect in probiotic applications. Furthermore, this work constitutes the first investigation on bifidobacterial strain typing using FTIR spectroscopy.

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.

A DNA array based assay for the characterization of microbial community in raw milk.

An oligonucleotide array based on PCR method containing a combination of probes for taxonomic markers and species-specific virulence genes was developed for the simultaneous identification of 14 Lactic Acid Bacteria (LAB) and food-borne pathogenic bacteria in raw milk. The hypervariable regions V3 and V6 of 16S, together with a variable region of 23S rRNA genes and several genes specific for virulence factors were selected. Universal primers and multiplex PCR were used for the rapid differentiation of bacterial species and low concentration of specific pathogenic and spoilage bacteria were detected in milk. The dominant species such as Streptococcus thermophilus, Enterococcus faecalis, Lactococcus lactis, andLeuconostoc lactis were identified by indirect-labelling reactions based upon incorporation of amino-allyl dUTPs. The results regarding food-borne pathogens detection showed highly specific hybridisation patterns with the genomic DNA from Campylobacter jejuni, Escherichia coli, Salmonella thyphimurium, Listeria monocytogenes and Yersinia enterocolitica. A clear differentiation of dominant species present in a complex microbial community such as raw milk was achieved by the application of short oligonucleotide probes which discriminate sequences differing by few nucleotides.