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. IMPORTANCE Lactobacillus 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.

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.

In spite of a global concern on the transfer of antibiotic resistances (AR) via the food chain, limited information exists on this issue in species of Leuconostoc and Weissella, adjunct cultures used as aroma producers in fermented foods. In this work, the minimum inhibitory concentration was determined for 16 antibiotics in 34 strains of dairy origin, belonging to Leuconostoc mesenteroides (18), Leuconostoc citreum (11), Leuconostoc lactis (2), Weissella hellenica (2), and Leuconostoc carnosum (1). Atypical resistances were found for kanamycin (17 strains), tetracycline and chloramphenicol (two strains each), and erythromycin, clindamycin, virginiamycin, ciprofloxacin, and rifampicin (one strain each). Surprisingly, L. mesenteroides subsp. mesenteroides LbE16, showed resistance to four antibiotics, kanamycin, streptomycin, tetracycline and virginiamycin. PCR analysis identified tet(S) as responsible for tetracycline resistance in LbE16, but no gene was detected in a second tetracycline-resistant strain, L. mesenteroides subsp. cremoris LbT16. In Leuconostoc mesenteroides subsp. dextranicum LbE15, erythromycin and clindamycin resistant, an erm(B) gene was amplified. Hybridization experiments proved erm(B) and tet(S) to be associated to a plasmid of ≈35 kbp and to the chromosome of LbE15 and LbE16, respectively. The complete genome sequence of LbE15 and LbE16 was used to get further insights on the makeup and genetic organization of AR genes. Genome analysis confirmed the presence and location of erm(B) and tet(S), but genes providing tetracycline resistance in LbT16 were again not identified. In the genome of the multi-resistant strain LbE16, genes that might be involved in aminoglycoside (aadE, aphA-3, sat4) and virginiamycin [vat(E)] resistance were further found. The erm(B) gene but not tet(S) was transferred from Leuconostoc to Enterococcus faecalis both under laboratory conditions and in cheese. This study contributes to the characterization of AR in the Leuconostoc-Weissella group, provides evidence of the genetic basis of atypical resistances, and demonstrates the inter-species transfer of erythromycin resistance.

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.

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.