The reduction of the price of DNA sequencing has resulted in the emergence of large data sets to handle and analyze, especially in microbial ecosystems, which are characterized by high taxonomic and ...functional diversities. To assess the properties of these complex ecosystems, a conceptual background of the application of NGS technology and bioinformatics analysis to metagenomics is required. Accordingly, this article presents an overview of the evolution of knowledge of microbial ecology from traditional culture-dependent methods to culture-independent methods and the last frontier in knowledge, metagenomics. Topics that will be covered include sample preparation for NGS, starting with total DNA extraction and library preparation, followed by a brief discussion of the chemistry of NGS to help provide an understanding of which bioinformatics pipeline approach may be helpful for achieving a researcher’s goals. The importance of selecting appropriate sequencing coverage and depth parameters to obtain a suitable measure of microbial diversity is discussed. As all DNA sequencing processes produce base-calling errors that compromise data analysis, including genome assembly and microbial functional analysis, dedicated software is presented and conceptually discussed with regard to potential applications in the general microbial ecology field.
Metagenomic studies about cocoa fermentation have mainly reported on the analysis of short reads for determination of operational taxonomic units. However, it is also important to determine ...metagenome-assembled genomes (MAGs), which are genomes deriving from the assembly of metagenomics. For this research, all the cocoa metagenomes from public databases were downloaded, resulting in five data sets: one from Ghana and four from Brazil. In addition,
approaches were used to describe putative phenotypes and the metabolic potential of MAGs. A total of 17 high-quality MAGs were recovered from these microbiomes, as follows: (i) for fungi,
(
= 1); (ii) lactic acid bacteria, Limosilactobacillus fermentum (
= 5), Liquorilactobacillus cacaonum (
= 1), Liquorilactobacillus nagelli (
= 1), Leuconostoc pseudomesenteroides (
= 1), and Lactiplantibacillus plantarum subsp.
(
= 1); (iii) acetic acid bacteria, Acetobacter senegalensis (
= 2) and Kozakia baliensis (
= 1); and (iv) Bacillus subtilis (
= 1), Brevundimonas sp. (
= 2), and Pseudomonas sp. (
= 1). Medium-quality MAGs were also recovered from cocoa microbiomes, including some that, to our knowledge, were not previously detected in this environment (Liquorilactobacillus vini, Komagataeibacter saccharivorans, and Komagataeibacter maltaceti) and others previously described (Fructobacillus pseudoficulneus and Acetobacter pasteurianus). Taken together, the MAGs were useful for providing an additional description of the microbiome of cocoa fermentation, revealing previously overlooked microorganisms, with prediction of key phenotypes and biochemical pathways.
The production of chocolate starts with the harvesting of cocoa fruits and the spontaneous fermentation of the seeds in a microbial succession that depends on yeasts, lactic acid bacteria, and acetic acid bacteria in order to eliminate bitter and astringent compounds present in the raw material, which will be further roasted and grinded to originate the cocoa powder that will enter the food processing industry. The microbiota of cocoa fermentation is not completely known, and yet it advanced from culture-based studies to the advent of next-generation DNA sequencing, with the generation of a myriad of data that need bioinformatic approaches to be properly analyzed. Although the majority of metagenomic studies have been based on short reads (operational taxonomic units), it is also important to analyze entire genomes to determine more precisely possible ecological roles of different species. Metagenome-assembled genomes (MAGs) are very useful for this purpose; here, MAGs from cocoa fermentation microbiomes are described, and the possible implications of their phenotypic and metabolic potentials are discussed.
Objectives
The objective of this study was to evaluate the ability of
Lactobacillus curvatus
CRL705, CRL1532, and CRL1533 and
Lactobacillus sakei
CRL1613 to survive under simulated gastrointestinal ...conditions. Moreover, a microencapsulation approach was proposed to improve gastrointestinal survival. Finally, experiments were performed to demonstrate that
Lactobacill
us spp. can modulate the ability of
Listeria monocytogenes
FBUNT to adhere to and invade Caco-2 cells.
Results
Lactobacillus
strains were encapsulated in alginate beads to enhance the survival of bacteria under in vitro gastrointestinal conditions. All strains hydrolyzed bile salts using chenodeoxycholic acid as a substrate and adhered to Caco-2 cells. Cell-free supernatants (CFSs) showed antimicrobial activity against
L. monocytogenes
as demonstrated by agar diffusion assays. The average percentages of
L. monocytogenes
adhesion decreased from 67.74 to 41.75 and 38.7% in the presence of 50 and 90% (v/v), respectively, for all CFSs tested. The highest concentrations of CFSs completely inhibited the
L. monocytogenes
invasion of Caco-2 cells.
Conclusions
The studied
Lactobacillus
strains have protective effects against the adhesion and invasion of
L. monocytogenes
FBUNT. Alginate encapsulation of these bacteria improved gastrointestinal tolerance such that they could be further studied as potential probiotics against intestinal pathogenic bacteria.
Acetobacter senegalensis
belongs to the group of acetic acid bacteria (AAB) that present potential biotechnological applications, for production of D-gluconate, cellulose and acetic acid. AAB can ...overcome heat and acid stresses by using strategies involving the overexpression of heat-shock proteins and enzymes from the complex pyrroquinoline-ADH, besides alcohol dehydrogenases (ADH). Nonetheless, the isolation of
A. senegalensis
and other AAB from food may be challenging due to presence of viable but non-culturable (VBNC) cells and due to uncertainties about nutritional requirements. To contribute for a better understanding of the ecology of AAB, this paper reports on the pangenome analysis of five strains of
A. senegalensis
recently isolated from a Brazilian spontaneous cocoa fermentation. The results showed biosynthetic clusters exclusively found in some cocoa-related AAB, such as those related to terpene pathways, which are important for flavour development. Genes related to oxidative stress were conserved in all the genomes, with multiple clusters. Moreover, there were genes coding for ADH and putative ABC transporters distributed in core, shell and cloud genomes, while chaperonin-encoding genes were present only in the core and soft-core genomes. Regarding quorum sensing, a response regulator gene was in the shell genome, and the gene encoding for acyl-homoserine lactone efflux protein was in the soft-core genome. There were quorum quenching-related genes, mainly encoding for lactonases, but also for acylases. Moreover,
A. senegalensis
did not have determinants of virulence or antibiotic resistance, which are good traits for strains intended to be applied in food fermentation.
Worldwide efforts to tackle the nature of exotic nuclei comprise the construction of new-generation Radioactive Ion Beam facilities. The Italian community is deeply involved in the process and the ...construction of SPES at Legnaro National Laboratories (INFN) is progressing. This contribution describes the layout of SPES in all its flavours, from Nuclear Physics to Applications in Nuclear Medicine and Neutron Physics. In particular, the status of the SPES-β ISOL facility, together with some of the relevant physics cases and the associated equipment are described.
Microbial enumeration by serial dilution is one of the best resources to estimate cellular density for microbiological analysis. However, for metataxonomic analysis, it is not clear if serially ...diluted samples may accurately be used for metataxonomic analysis to represent species composition in beef samples. In this study, the effect of sampling preparation of beef samples on the bacterial composition was evaluated by the comparison of dilution and exudate. Based on the obtained results, data obtained from the exudate of the samples were more robust in terms of number of generated reads, but no significant differences in terms of biological diversity were observed (P < .05, Wicoxon Test). Besides, both sample preparation procedures evidenced equivalent results of bacterial composition as well as its relative abundances. In conclusion, the use of exudate allows bacterial enumeration and metataxonomic analysis, which is interesting for the point of view of food microbiologists as cellular loads and microbial composition of culturable and unculturable bacteria could be compared.
To determine the ability of probiotic lactobacilli to improve the treatment of vulvovaginal candidiasis (VVC) using a randomized, double-blind and placebo-controlled trial. Fifty-five women diagnosed ...with VVC by vaginal discharge positive for Candida spp. (according to culture method) associated with at least one of the symptoms (itching and burning vaginal feeling, dyspareunia and dysuria), were treated with single dose of fluconazole (150 mg) supplemented every morning for the following 4 weeks with two placebo or two probiotic capsules (containing Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14). At 4 weeks, the probiotic treated group showed significantly less vaginal discharge associated with any of the above mentioned symptoms (10·3%vs 34·6%; P = 0·03) and lower presence of yeast detected by culture (10·3%vs 38·5%; P = 0·014). This study has shown that probiotic lactobacilli can increase the effectiveness of an anti-fungal pharmaceutical agent in curing disease. This novel finding of probiotic lactobacilli augmenting the cure rate of yeast vaginitis, not only offers an alternative approach to a highly prevalent condition that adversely affects the quality of life of women around the world, but also raises the question of how this combination works.
In pig nutrition, antibiotics are used to promote growth and/or to treat diseases in order to improve animal performance. However, due to the potential risk of cross selective pressure for antibiotic ...resistance among bacterial pathogens, the development of new nutritional additives is needed. Among them, probiotics are of great interest since they could improve the immune response, maintain animal intestinal health, and improve nutritional efficiency. Studies with probiotics have also demonstrated their antimicrobial effects on several pathogenic strains, emphasizing that the form of administration can enhance the beneficial effects. In view of the promising advances in probiotic research, it is opportune to highlight their capacity to modulate health and improve performance at all stages of pig production. Therefore, in this review, we will discuss the benefits of probiotics on physiological, immunological, and clinical aspects during different stages of the pig’s life cycle. Specifically, probiotics improve performance during pregnancy, parturition and lactation in sows, they can improve immunohematological parameters and defenses in the growing phase, they can influence the quality of meat in the finishing phase and can also help in the reduction of environmental pollutants.
•The use of probiotics has different effects on each life stage of pigs.•In sows, probiotics improve performance in pregnancy, parturition and lactation.•Growing phase: improvement in immunohematological parameters and defenses.•Finishing stage: better meat quality and reduction of environmental pollutants.
Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence ...of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes
esp,
gel,
ace,
as,
efaA,
hyl and
cylA) and antibiotic resistance was checked only for
Enterococcus faecium and
Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated.
E. faecium was the predominant species, followed by
E. faecalis,
E. casseliflavus and
Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were
β-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in
E. faecalis than in
E. faecium. Three
E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of
E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant
E. faecalis and
E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic
E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety.
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