Anti-Biofilm Activity of Carnosic Acid from Salvia rosmarinus against Methicillin-Resistant Staphylococcus aureus.

The Salvia rosmarinus "Eretto Liguria" ecotype was studied as a source of valuable bioactive compounds. LC-MS analysis of the methanolic extract underlined the presence of diterpenoids, triterpenoids, polyphenolic acids, and flavonoids. The anti-virulence activity of carnosic acid along with the other most abundant compounds against methicillin-resistant Staphylococcus aureus (MRSA) was evaluated. Only carnosic acid induced a significant reduction in the expression of agrA and rnaIII genes, which encode the key components of quorum sensing (QS), an intracellular signaling mechanism controlling the virulence of MRSA. At a concentration of 0.05 mg/mL, carnosic acid inhibited biofilm formation by MRSA and the expression of genes involved in toxin production and made MRSA more susceptible to intracellular killing, with no toxic effects on eukaryotic cells. Carnosic acid did not affect biofilm formation by Pseudomonas aeruginosa, a human pathogen that often coexists with MRSA in complex infections. The selected ecotype showed a carnosic acid content of 94.3 ± 4.3 mg/g. In silico analysis highlighted that carnosic acid potentially interacts with the S. aureus AgrA response regulator. Our findings suggest that carnosic acid could be an anti-virulence agent against MRSA infections endowed with a species-specific activity useful in multi-microbial infections.

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.

Prevalence and virulence potential of Aeromonas spp. isolated from human diarrheal samples in North East Italy.

IMPORTANCE: In this work, we demonstrate the epidemiologic relevance of the Aeromonas genus as the cause of infective diarrhea in North East Italy, both in children and adult subjects, with the significative presence of highly pathogenic strains. Aeromonas strains possess a heterogeneous armamentarium of pathogenicity factors that allows the microbe to affect a wide range of human intestinal epithelial cell processes that justify the ability to induce diarrhea through different mechanisms and cause diseases of variable severity, as observed for other gastrointestinal pathogens. However, it remains to be determined whether specific genotype(s) are associated with clinical pictures of different severity to implement the diagnostic and therapeutic approaches for this relevant enteric pathogen.

Antibiotic Treatment Induces Long-Lasting Effects on Gut Microbiota and the Enteric Nervous System in Mice.

The side effects of antibiotic treatment directly correlate with intestinal dysbiosis. However, a balanced gut microbiota supports the integrity of the enteric nervous system (ENS), which controls gastrointestinal neuromuscular functions. In this study, we investigated the long-term effects of antibiotic-induced microbial dysbiosis on the ENS and the impact of the spontaneous re-establishment of the gut microbiota on gastrointestinal functions. C57BL/6J mice were treated daily for two weeks with antibiotics. After 0-6 weeks of antibiotics wash-out, we determined (a) gut microbiota composition, (b) gastrointestinal motility, (c) integrity of the ENS, (d) neurochemical code, and (e) inflammation. Two weeks of antibiotic treatment significantly altered gut microbial composition; the genera Clostridium, Lachnoclostridium, and Akkermansia did not regain their relative abundance following six weeks of antibiotic discontinuation. Mice treated with antibiotics experienced delayed gastrointestinal transit and altered expression of neuronal markers. The anomalies of the ENS persisted for up to 4 weeks after the antibiotic interruption; the expression of neuronal HuC/D, glial-derived neurotrophic factor (Gdnf), and nerve growth factor (Ngf) mRNA transcripts did not recover. In this study, we strengthened the idea that antibiotic-induced gastrointestinal dysmotility directly correlates with gut dysbiosis as well as structural and functional damage to the ENS.

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.

Labdane Diterpenoids from Salvia tingitana Etl. Synergize with Clindamycin against Methicillin-Resistant Staphylococcus aureus.

Quorum-sensing (QS) is a regulatory mechanism in bacterial communication, important for pathogenesis control. The search for small molecules active as quorum-sensing inhibitors (QSI) that can synergize with antibiotics is considered a good strategy to counteract the problem of antibiotic resistance. Here the antimicrobial labdane diterpenoids sclareol (1) and manool (2) extracted from Salvia tingitana were considered as potential QSI against methicillin-resistant Staphylococcus aureus. Only sclareol showed synergistic activity with clindamycin. The quantification of these compounds by LC-MS analysis in the organs and in the calli of S. tingitana showed that sclareol is most abundant in the flower spikes and is produced by calli, while manool is the major labdane of the roots, and is abundant also in the leaves. Other metabolites of the roots were abietane diterpenoids, common in Salvia species, and pentacyclic triterpenoids, bearing a γ-lactone moiety, previously undescribed in Salvia. Docking simulations suggested that 1 and 2 bind to key residues, involved in direct interactions with DNA. They may prevent accessory gene regulator A (AgrA) binding to DNA or AgrA activation upon phosphorylation, to suppress virulence factor expression. The antimicrobial activity of these two compounds probably achieves preventing upregulation of the accessory gene regulator (agr)-regulated genes.

Co-administration of vitamin D3 and Lacticaseibacillus paracasei DG increase 25-hydroxyvitamin D serum levels in mice.

PURPOSE: Subclinical vitamin D (vitD) deficiency enhances the predisposition to a myriad of acute and chronic pathologies in many people worldwide. Due to the scarcity of vitD-rich foods, the consumption of supplements or fortified foods can be required to maintain healthy serum levels of 25-hydroxyvitamin D [25(OH)D], and the major circulating form of vitD that is commonly measured in serum to determine the vitD status. Since the vitD absorption seems to resemble that of lipids, improved emulsification in the gut could favor vitD permeation through the enterocyte membrane. Contextually, we hypothesized that a microorganism with cholecalciferol (vitD3)-solubilization properties may potentially result in enhanced serum vitD levels. METHODS AND RESULTS: Six probiotic strains were screened for their ability to create a stable suspension of vitD3 in water: Lacticaseibacillus paracasei DG, L. paracasei LPC-S01, L. paracasei Shirota, L. rhamnosus GG, Limosilactobacillus reuteri DSM 17938, and Lactobacillus acidophilus LA5. The DG strain displayed the strongest vitD3 solubilization ability and, consequently, were used in an in vivo trial where a commercial preparation of vitD3 in refined olive oil was administered by gavage to CD-1 mice with or without the concurrent administration of L. paracasei DG. ELISA measurements showed that the DG strain significantly increased the serum levels of 25(OH) D when administered once a day for 1 week in association with the vitD3 supplement. CONCLUSION: This preliminary pre-clinical study suggests that the combined administration of L. paracasei DG with an oil-based cholecalciferol supplement could contribute to the maintenance of the adequate 25(OH) D serum levels in people at risk of vitD deficiency.

Characterization of PACs profile and bioactivity of a novel nutraceutical combining cranberry extracts with different PAC-A oligomers, D-mannose and ascorbic acid: An in vivo/ex vivo evaluation of dual mechanism of action on intestinal barrier and urinary epithelium.

In this paper, an A-type procyanidin (PAC)-rich cranberry extract (CB-B) was obtained mixing different extracts and was formulated with D-mannose and ascorbic acid to obtain a novel nutraceutical (URO-F) aimed at preventing non-complicated bacterial urinary tract infections (UTIs). To assess the bioactivity of CB-B and URO-F, urine samples collected from six healthy volunteers undergoing a 2-days oral consumption of 0.41 g/day of CB-B or 10 g/day of URO-F (corresponding to 72 mg/day of PACs) were tested against uropathogenic E. coli (UPEC) incubated on urinary bladder epithelial cells (T24). Urinary markers of CB-B and URO-F consumption were assessed in the same urine output by UPLC-QTOF-based untargeted metabolomics approach. CB-B and URO-F were evaluated for their ability to promote the intestinal barrier function by restoring the trans-epithelial electrical resistance (TEER) and to inhibit the production of inflammatory cytokines in intestinal epithelial Caco2 cells. CB-B was characterized by a high PAC-A content (70% of total PACs) and a broad distribution of different PACs polymers (dimers-hexamers). Urine from subjects consuming CB-B and URO-F showed a significant effect in reducing the adhesion of UPEC to urothelium in vitro, supporting their efficacy as anti-adhesive agents after oral intake. CB-B inhibited the release of cytokine IL-8, and both products were effective in restoring the TEER. Overall, our results show that the beneficial effects of CB-B and URO-F on UTIs are not only due to the antiadhesive activity of cranberry on UPEC in the urothelium, but also to a multi-target activity involving anti-inflammatory and permeability-enhancing effects on intestinal epithelium.

Polyphenol-Rich Larix decidua Bark Extract with Antimicrobial Activity against Respiratory-Tract Pathogens: A Novel Bioactive Ingredient with Potential Pharmaceutical and Nutraceutical Applications.

Larch (Larix decidua) bark is a sawmill waste, traditionally used for antiseptic, expectorant and dermatological (wound healing, eczema, psoriasis) purposes. In this work, we developed a food-grade dry larch bark extract (LBE) from sawmill by-products using hydro-alcoholic extraction. The antibacterial activity of LBE was evaluated against respiratory-tract pathogens, i.e., Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Haemophilus influenza, and it was compared to that of grapefruit seed extract (GSE), a commercially available raw material commonly proposed as antibacterial ingredient for over-the-counter products. Procyanidins (PACs) and other polyphenols contents in LBE were determined by HPLC-FLD-MS and HPLC-DAD-MSn, respectively. The antimicrobial activity of LBE and GSE was assessed using the micro-plate dilution technique in concentration range of 2-200 µg/mL, and the safety of these dosages was assessed in cellular and animal models. LBE showed considerable contents of PACs (15% w/w; especially B-type) and other polyphenols (3.8% w/w), among which the characteristic spiropolyphenols larixinol and epilarixinol were identified, together with the flavonoids isoquercitrin and rutin, already reported as growth inhibitors of different respiratory-tract pathogens. LBE showed higher antimicrobial activity compared to GSE, demonstrated by a growth inhibition range of 10-40% towards five of six strains tested, compared to 10-15% of GSE. These results suggest that LBE may represent a natural and sustainable source of active compounds with antibacterial activity for pharmaceutical and nutraceutical applications.

On the mechanism of tumor cell entry of aloe-emodin, a natural compound endowed with anticancer activity.

Aloe-emodin (1,8-dihydroxy-3-[hydroxymethyl]-anthraquinone), AE, is one of the active constituents of a number of plant species used in traditional medicine. We have previously identified, for the first time, AE as a new antitumor agent and shown that its selective in vitro and in vivo killing of neuroblastoma cells was promoted by a cell-specific drug uptake process. However, the molecular mechanism underlying the cell entry of AE has remained elusive as yet. In this report, we show that AE enters tumor cells via two of the five somatostatin receptors: SSTR2 and SSTR5. This observation was suggested by gene silencing, receptor competition, imaging and molecular modeling experiments. Furthermore, SSTR2 was expressed in all surgical neuroblastoma specimens we analyzed by immunohistochemistry. The above findings have strong implications for the clinical adoption of this natural anthraquinone molecule as an antitumor agent.

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.

The Biotherapeutic Potential of Lactobacillus reuteri Characterized Using a Target-Specific Selection Process.

A growing body of clinical and experimental data supports the view that the efficacy of probiotics is strain-specific and restricted to particular pathological conditions, which means that newly isolated probiotic strains need to be targeted to a specific disease. Following national and international guidelines, we used a conventional in vitro experimental approach to characterize a novel strain of Lactobacillus reuteri, LMG P-27481, for safety (sensitivity to antibiotics and genome analysis) and putative efficacy (resistance to gastro-intestinal transit, adhesiveness, induction of cytokines, and release of antimicrobial metabolites). In vitro assays, which were carried out to examine the probiotic's effect on diarrhea (lactose utilization, inhibition of pathogens such as bacteria and Rotavirus), showed that it was more efficacious with respect to well-known reference strains in antagonizing Clostridioides difficile (CD). Data confirming that the probiotic can effectively treat CD colitis was gained from in vivo trials involving mice conditioned with large spectrum antibiotics before they were subjected to CD challenge. Two out of the three antibiotic-treated groups received daily LMG P-27481 for different time durations in order to simulate a preventive approach (LMG P-27481 administered prior to CD challenge) or an antagonistic one (LMG P-27481 administered after CD challenge). Both approaches significantly reduced, with respect to the untreated controls, CD DNA concentrations in caecum and C. difficile toxin titers in the gut lumen. In addition, LMG P-27481 supplementation significantly mitigated body weight loss and the extent of inflammatory infiltrate and tissue damage. The study results, which need to be confirmed by in vivo clinical trials, have demonstrated that the L. reuteri LMG P-27481 strain is a promising probiotic candidate for the treatment of CD infection.

In Vitro Antimicrobial Activities of Commercially Available Tea Tree (Melaleuca alternifolia) Essential Oils.

Melaleuca alternifolia tea tree oil (TTO) is largely used in cutaneous infections. Clinical observations reported antibacterial, antifungal, and antiviral activities, whereas in vitro experiments ascribed most of biological properties to terpinen-4-ol. Since different plant chemotypes and storage conditions result in variations of chemical composition of commercially available TTO, in this study we investigated the antimicrobial activity and the chemical profile of ten commercially available TTO products. The antimicrobial activity was assessed against Candida glabrata, Herpes simplex virus type 1 (HSV-1), methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa grown in planktonic mode or biofilms. Only five out of ten TTO batches reported significant antimicrobial activity. The identified TTO products reduced bacterial survival in biofilms, generated oxidative damage in C. glabrata, and diminished HSV-1 infectivity. GC-MS analysis revealed that all the analyzed TTO batches fitted into the terpinen-4-ol chemotype even if we reported great variability in composition of nine major ISO-specified TTO components. Overall, we were not able to ascribe the antimicrobial activity to the content in terpinen-4-ol. We therefore conclude that the antimicrobial activity of TTO results from complex interaction among different components.

Synthesis and preliminary anti-inflammatory and anti-bacterial evaluation of some diflunisal aza-analogs.

Our aim was to identify new multi-target compounds endowed with both anti-inflammatory and anti-bacterial activities for treatment of human infections. Diflunisal, a nonsteroidal anti-inflammatory agent, has recently been repurposed for its anti-virulence properties against methicillin-resistant Staphylococcus aureus. Effective synthesis of some aza-analogs of the anti-inflammatory drug diflunisal was carried out following the route involving key oxazole intermediates to obtain o- and m-hydroxypyridinecarboxylic acid derivatives. The newly synthesized diflunisal aza-analogs did not exhibit cytotoxic activity up to 80 µM and some of them exhibited anti-inflammatory activities, decreasing the levels of pro-inflammatory cytokines and prostaglandins induced by bacterial lipopolysaccharide in human primary macrophages. Ten of the diflunisal aza-analogs were found to have interesting antibacterial activity, sensitizing S. aureus, Streptococcus pyogenes, Enterococcus faecium, and Pseudomonas aeruginosa to the antibacterial effects of beta-lactam antibiotics and protein synthesis inhibitors.