Nutraceutical formulation for immune system modulation: Active constituents, in vitro antibacterial and immunomodulatory activity, and metabolomics analysis.

There is a large demand for nutraceuticals in the market and studies related to their action are needed. In this paper, the antimicrobial activity and the immunomodulatory effect of a nutraceutical formulation containing 14.39% of ascorbic acid, 7.17% of coenzyme Q10, 1.33% of Echinacea polyphenols, 0.99% of pine flavan-3-ols, 0.69% of resveratrol and 0.023% of Echinacea alkylamides were studied using in vitro assays and cell-based metabolomics. Chromatographic analysis allowed us to study the nutraceutical composition. The antibacterial activity was evaluated on S. aureus, K. pneumoniae, P. aeruginosa, E. coli, H. influenzae, S. pyogenes, S. pneumoniae and M. catarrhalis. The immunomodulatory activity was assessed on human macrophages and dendritic cells. The production of IL-1ß, IL-12p70, IL-10 and IL-8 was evaluated on culture medium by ELISA and the activation/maturation of dendritic cells with cytofluorimetric analysis. Treated and untreated macrophages and dendritic cell lysates were analysed by liquid chromatography coupled with high-resolution mass spectrometry, and results were compared using multivariate data analysis to identify biological markers related to the treatment with the food supplement. The food supplement decreased K. pneumoniae, P. aeruginosa, E. coli, Methicillin-resistant Staphylococcus aureus (MRSA) and M. catharralis growth, reduced the inflammatory response in macrophages exposed to lipopolysaccharide (LPS) and modulated the activation and maturation of the dendritic cells. Oxidized phospholipids were identified as the main biological markers of treated cell lysates, compared with controls.

Exploiting Conserved Quorum Sensing Signals in Streptococcus mutans and Streptococcus pneumoniae

Bacterial species of the Streptococcus genera are considered either commensal bacteria or potential pathogens, according to their metabolic evolution and production of quorum sensing (QS)-controlled virulence factors. S. mutans, in particular, has become one of the best-studied examples of bacteria that are able to get along or cheat commensal species, even of the same genera. S. mutans and S. pneumoniae share homolog QS pathways and a competence stimulating peptide (CSP) for regulating bacteriocin production. Intriguingly, the abundance of S. pneumoniae and S. mutans alternates in complex microbial communities, thus opening the role for the fratricide communication of homolog QS systems. Since the inhibition of the QS has been proposed in treating bacterial infections, in this study, we designed and synthesized analogs of S. pneumoniae CSP with precise residual modifications. We reported that S. pneumoniae CSP analogs reduced the expression of genes involved in the QS of S. mutans and biofilm formation without affecting bacterial growth. The CSP analogs inhibited bacteriocin production in S. mutans, as reported by co-cultures with commensal bacteria of the oral cavity. The peptide CSP1AA, bearing substitutions in the residues involved in QS receptor recognition and activation, reported the most significant quorum-quenching activities. Our findings provide new insights into specific chemical drivers in the CSP sequences controlling the interconnection between S. mutans and S. pneumoniae. We think that the results reported in this study open the way for new therapeutic interventions in controlling the virulence factors in complex microbial communities such as the oral microbiota.

A novel phenolic derivative inhibits AHL-dependent quorum sensing signaling in Pseudomonas aeruginosa.

Increasing antibiotic resistance and the decline in the pharmaceutical industry's investments have amplified the need for novel treatments for multidrug-resistant bacteria. Quorum sensing (QS) inhibitors reduce pathogens' virulence without selective pressure on bacteria and provide an alternative to conventional antibiotic-based therapies. P. aeruginosa uses complex QS signaling to control virulence and biofilm formation. We aimed to identify inhibitors of P. aeruginosa QS acting on acyl-homoserine lactones (AHL)-mediated circuits. Bioluminescence and qRT-PCR assays were employed to screen a library of 81 small phenolic derivatives to reduce AHL-dependent signaling. We identified GM-50 as the most active compound inhibiting the expression of AHL-regulated genes but devoid of cytotoxic activity in human epithelial cells and biocidal effects on bacteria. GM-50 reduces virulence factors such as rhamnolipids, pyocyanin, elastase secretion, and swarming motility in P. aeruginosa PAO1 laboratory strain. By molecular docking, we provide evidence that GM-50 highly interacts with RhlR. GM-50 significantly improved aztreonam-mediated biofilm disruption. Moreover, GM-50 prevents adhesion of PAO1 and inflammatory damage in the human A549 cell line and protects Galleria mellonella from PAO1-mediated killing. GM-50 significantly reduces virulence factors in 20 P. aeruginosa clinical isolates from patients with respiratory tract infections. In conclusion, GM-50 inhibits AHL-signaling, reduces virulence factors, enhances the anti-biofilm activity of aztreonam, and protects G. mellonella larvae from damage induced by P. aeruginosa. Since GM-50 is active on clinical strains, it represents a starting point for identifying and developing new phenolic derivatives acting as QS-inhibitors in P. aeruginosa infections.

Anti-Inflammatory Activities of Euglena gracilis Extracts.

Dietary supplementation with nutrients able to control intestinal and systemic inflammation is of marketable interest. Indeed, gastrointestinal homeostasis plays a significant role in maintaining human health. In this setting, E. gracilis may sustain or promote human health, but the effects on the intestinal inflammatory milieu are not clear. In this study, we investigated the anti-inflammatory activity of E. gracilis and inferred possible mechanisms. Paramylon, crude, and fractionated extracts were obtained from E. gracilis grown in vitro. Phytoconstituents of the extracts were characterized using TLC and HPLC UV-Vis. The anti-inflammatory and antioxidant activities were investigated in primary human macrophages and an intestinal epithelial cell line (HT-29). The analysis of the extracts led to identifying ß-carotene, neoxanthin, diadinoxanthin, canthaxanthin, and breakdown products such as pheophytins and pheophorbides. E. gracilis fractionated extracts reduced the production of tumor necrosis factor-α triggered by bacterial lipopolysaccharide (LPS) in the short and long terms. Pheophytin a and b and canthaxanthin increased the intracellular reducing potential and dampened the production of LPS-induced reactive oxygen species and lipid peroxidation, intracellular events usually involved in the perpetuation of chronic inflammatory disorders. This study rationalizes the role of specific extract fractions of E. gracilis in controlling LPS-driven intestinal inflammation.

A Novel Aza-Derivative Inhibits agr Quorum Sensing Signaling and Synergizes Methicillin-Resistant Staphylococcus aureus to Clindamycin.

Increasing antibiotic resistance and diminishing pharmaceutical industry investments have increased the need for molecules that can treat infections caused by dangerous pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Quorum Sensing (QS) is a signaling mechanism that regulates bacterial virulence in pathogens. A report demonstrating that the anti-inflammatory drug Diflunisal reduces MRSA virulence factors' expression prompted us to design, synthesize and test 16 aza-analogs as inhibitors of S. aureus virulence factors controlled by the accessory gene regulator (agr) QS system. At first, we evaluated by qRT-PCR the activity of compounds on rnaIII expression, a QS related gene. Azan-7 was the most active molecule tested and it did not show cytotoxic activity in human cell lines. Moreover, we demonstrated that it did not affect bacterial proliferation. Regulation of MRSA virulence genes by Azan-7 was investigated using qRT-PCR and RNAseq. Azan-7 significantly reduced hla, psmα, hysA, agrA, cap1A, and cap1C gene expression. In silico docking demonstrated that Azan-7 binds the response regulator AgrA. This data was confirmed by electrophoretic mobility shift assay (EMSA) reporting that Azan-7 binding to AgrA protein strongly reduced the AgrA-DNA complex formation at the P3 promoter region involved in the regulation of rnaIII transcription. Azan-7 inhibited MRSA-mediated haemolysis, reduced survival of the pathogen at low pH levels, and increased macrophage killing. In addition, Azan-7 enhanced MRSA susceptibility to clindamycin both in planktonic growth and biofilm. Azan-7 did not induce resistance over 10 days in culture. It was equally active against all the AgrA MRSA subtypes encountered among clinical isolates, but it was not active against Staphylococcus epidermidis, although the AgrA proteins show an approximate 80% homology. These results demonstrate that Azan-7 inhibits the expression of MRSA virulence factors by interfering in the QS and synergizes MRSA biofilm with clindamycin, indicating the compound as a promising candidate for the treatment of MRSA infections.

Changes of Physical Activity and Ultra-Processed Food Consumption in Adolescents from Different Countries during Covid-19 Pandemic: An Observational Study.

AIM: to describe physical activity and ultra-processed foods consumption, their changes and sociodemographic predictors among adolescents from countries in Europe (Italy and Spain) and Latin America (Brazil, Chile, and Colombia) during the SARS-CoV-2-pandemic period. METHODS: Cross-sectional study via web survey. International Physical Activity Questionnaire (IPAQ) and weekly ultra-processed food consumption data were used. To compare the frequencies of physical activity status with sociodemographic variables, a multinomial logistic and a multiple logistic regression for habitual ultra-processed foods was performed. In final models, p < 0.05 was considered significant. RESULTS: Sample of 726 adolescents, mostly females (59.6%) aged 16-19 years old (54.3%). Adolescents from Latin America presented odds ratio (OR) 2.98 (CI 95% 1.80-4.94) of being inactive and those whose mothers had higher level of education were less active during lockdown [OR 0.40 (CI 95% 0.20-0.84)]. The habitual ultra-processed consumption was also high during this period in all countries, and more prevalent in Latin America. CONCLUSION: A higher prevalence of inactivity was observed in this population, but reductions of physical activity and habitual ultra-processed consumption during the pandemic were more pronounced in Latin America. Our findings reinforce the importance of promoting a healthy lifestyle, i.e., exercise and diet, during periods of social isolation.

Covid-19 Confinement and Changes of Adolescent's Dietary Trends in Italy, Spain, Chile, Colombia and Brazil.

Confinement due to the COVID-19 pandemic can influence dietary profiles, especially those of adolescents, who are highly susceptible to acquiring bad eating habits. Adolescents' poor dietary habits increase their subsequent risk of degenerative diseases such as obesity, diabetes, cardiovascular pathologies, etc. Our aim was to study nutritional modifications during COVID-19 confinement in adolescents aged 10 to 19 years, compare them with their usual diet and dietary guidelines, and identify variables that may have influenced changes. Data were collected by an anonymous online questionnaire on food intake among 820 adolescents from Spain, Italy, Brazil, Colombia, and Chile. The results show that COVID-19 confinement did influence their dietary habits. In particular, we recorded modified consumption of fried food, sweet food, legumes, vegetables, and fruits. Moreover, gender, family members at home, watching TV during mealtime, country of residence, and maternal education were diversely correlated with adequate nutrition during COVID-19 confinement. Understanding the adolescents' nutrition behavior during COVID-19 lockdown will help public health authorities reshape future policies on their nutritional recommendations, in preparation for future pandemics.