The Influence of Protein Secretomes of Enterococcus durans on ex vivo Human Gut Microbiome.

The gut microbiome plays a critical role to all animals and humans health. Methods based on ex vivo cultures are time and cost-effective solutions for rapid evaluation of probiotic effects on microbiomes. In this study, we assessed whether the protein secretome from the potential probiotic Enterococcus durans LAB18S grown on fructoligosaccharides (FOS) and galactoligosaccharides (GOS) had specific effects on ex vivo cultured intestinal microbiome obtained from a healthy individual. Metaproteomics was used to evaluate changes in microbial communities of the human intestinal microbiome. Hierarchical clustering analysis revealed 654 differentially abundant proteins from the metaproteome samples, showing that gut microbial protein expression varied on the presence of different E. durans secretomes. Increased amount of Bacteroidetes phylum was observed in treatments with secretomes from E. durans cultures on FOS, GOS and albumin, resulting in a decrease of the Firmicutes to Bacteroidetes (F/B) ratio. The most functionally abundant bacterial taxa were Roseburia, Bacteroides, Alistipes and Faecalibacterium. The results suggest that the secretome of E. durans may have favorable effects on the intestinal microbial composition, stimulating growth and different protein expression of beneficial bacteria. These findings suggest that proteins secreted by E. durans growing on FOS and GOS have different effects on the modulation of gut microbiota functional activities during cultivation.

Genomic analysis of Enterococcus durans LAB18S, a potential probiotic strain isolated from cheese.

Gut microbiota exerts a fundamental role in human health and increased evidence supports the beneficial role of probiotic microorganisms in the maintenance of intestinal health. Enterococcus durans LAB18S was previously isolated from soft cheese and showed some desirable in vitro probiotic properties, for that reason its genome was sequenced and evaluated for genes that can be relevant for probiotic activity and are involved in selenium metabolism. Genome sequencing was performed using the Illumina MiSeq System. A variety of genes potentially associated with probiotic properties, including adhesion capability, viability at low pH, bile salt resistance, antimicrobial activity, and utilization of prebiotic fructooligosaccharides (FOS) were identified. The strain showed tolerance to acid pH and bile salts, exhibited antimicrobial activity and thrived on prebiotic oligosaccharides. Six genes involved in selenium metabolism were predicted. Analysis of the SECIS element showed twelve known selenoprotein candidates. E. durans LAB18S was the only food isolate showing absence of plasmids, virulence and antimicrobial resistance genes, when compared with other 30 E. durans genomes. The results of this study provide evidence supporting the potential of E. durans LAB18S as alternative for probiotic formulations.

Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection. OBJECTIVES: In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples. METHODS AND FINDINGS: We evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the AllplexTM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity. MAIN CONCLUSIONS: We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples.

Emergence of the novel SARS-CoV-2 lineage VUI-NP13L and massive spread of P.2 in South Brazil.

In this study, we analyzed 340 whole genomes of SARS-CoV-2, which were sampled between April and November 2020 in 33 cities of Rio Grande do Sul, South Brazil. We demonstrated the circulation of two novel emergent lineages, VUI-NP13L and VUI-NP13L-like, and five major lineages that had already been assigned (B.1.1.33, B.1.1.28, P.2, B.1.91, B.1.195). P.2 and VUI-NP13L demonstrated a massive spread in October 2020. Constant and consistent genomic surveillance is crucial to identify newly emerging SARS-CoV-2 lineages in Brazil and to guide decision making in the Brazilian Public Healthcare System.

Proteomic analysis of Listeria monocytogenes exposed to free and nanostructured antimicrobial lipopeptides.

In this work, the effect of antimicrobial lipopeptide P34 on Listeria monocytogenes was evaluated for the first time through a proteomics approach. Bacteria were treated with sub-lethal doses of peptide P34 (F-P34) and P34 encapsulated into nanoliposomes (N-P34), while empty nanoliposomes (NE) and fresh buffer were used as controls. The proteomic analysis allowed the detection of one group of proteins commonly differentially represented in response to free and encapsulated P34 exposure. A second group of proteins was found to be exclusively differentially represented after exposure with encapsulated P34 only. The antimicrobial peptide P34 caused a significant downregulation of proteins associated with the transport of manganese and the over-representation of proteins related with iron transport in L. monocytogenes. In addition, reduction of stress tolerance proteins related to the σB and VirR regulons, together with the modulation of phosphoenolpyruvate phosphotransferase systems (PTS) for sugar transport were observed. The sugar and oligopeptide transporters regulated by antimicrobial action may influence the key virulence factor PrfA, reducing the pathogenicity of this microorganism.

Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load

BACKGROUND Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection. OBJECTIVES In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples. METHODS AND FINDINGS We evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the AllplexTM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity. MAIN CONCLUSIONS We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples.

Comparative proteomic analysis reveals metabolic variability of probiotic Enterococcus durans during aerobic and anaerobic cultivation.

The variation in the bioavailability of oxygen constitutes the environmental conditions found by bacteria in their passage through the host gastro-intestinal tract. Given the importance of oxygen in the defense mechanism of bacteria, it is important to understand how bacteria respond to this stress at a metabolic level. The probiotic strain Enterococcus durans LAB18S was cultivated under aerobic and anaerobic conditions using prebiotic oligosaccharides as carbon source. The whole cell proteome and secretome were analyzed through label-free quantitative proteomics approach. The results showed that the LAB18S isolate when grown with fructo-oligosacchrides (FOS) showed a higher number of differentially expressed proteins compared to samples with galacto-oligosaccharides (GOS) or glucose. Clinically important enzymes for the treatment of cancer, L-asparaginase and arginine deiminase, were overexpressed when the isolate was cultured in FOS. In addition, the absence of oxygen induced the strain to produce proteins related to cell multiplication, cell wall integrity and resistance, and H2O2 detoxification. This study showed that E. durans LAB18S growing on FOS was stimulated to produce clinically important biomolecules, including proteins that have been investigated as potential antineoplastic agents. Significance: The probiotic strain E. durans LAB18S produce clinically relevant enzymes for the treatment of cancer when cultivated in symbiosis with fructo-oligosacchrides (FOS). In addition, proteins associated with cellular multiplication, cell wall integrity and resistance, and H2O2 detoxification were induced under anaerobic growth. These characteristics could be relevant to support maintenance of intestinal health.

Proteomic study of Enterococcus durans LAB18S growing on prebiotic oligosaccharides.

This study evaluates the influence of prebiotic carbohydrates, namely fructooligosaccharides (FOS) and galactooligosaccharides (GOS), on the protein expression of Enterococcus durans LAB18S. The strain was cultivated in 10 g L-1 FOS, GOS or glucose (control) and cellular proteins were extracted for mass spectrometry analysis. A total of 771 proteins were identified and 135 E. durans proteins were validated by the Scaffold algorithm. The proteins were functionally categorized according to Gene Ontology terms. Both FOS and GOS were used as carbon source by E. durans LAB18S, upregulating the production of proteins that may be associated with intestinal mucosa adhesion, carbohydrate and nitrogen metabolism, and stress response. Cells grown with GOS showed an increased expression of the cell division protein divIVA, EF-Tu and glyceraldehyde 3-phosphate dehydrogenase that have been associated with epithelial cell adhesion. The use of FOS stimulated the production of proteins related to amino acid metabolism and energy conversion, and ClpX protein, which plays an important role in protein turnover. The results of this study indicate that FOS and GOS can be metabolized by E. durans and stimulate the microorganism to produce proteins related to some desirable characteristics for a probiotic strain.

Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin.

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.

Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.