Raspberry leaves, despite their potential health benefits, often face wastage on a global scale, leading to their classification as bio-waste. However, recent advancements have shed light on the significant bioactive compounds present in these leaves. Studies have unveiled their high phenolic content and potent antioxidant activity, sparking interest across various industries. Indeed, the present study delves into recent advancements and applications concerning raspberry leaves, focusing on their dermatological, antioxidant, antimicrobial, and antibiofilm activities. The primary aims are to explore the potential dermatological benefits of raspberry, antioxidant capacity, antimicrobial efficacy against relevant skin pathogens, and their ability to hinder biofilm formation. The findings reveal notable properties: a total antioxidant activity of 93.5%±0.12, a complete inhibition of hyaluronidase activity (100%), 69.4%±2.7 suppression of elastase, and 30.0%±5.9 attenuation of tyrosinase. Moreover, the antibiofilm results exhibit substantial inhibitory effects, with a mass quantification inhibition rate of 51 % against Cutibacterium acnes, and 42% against Staphylococcus aureus, accompanied by 41 % and 41 % suppression of their respective metabolic activities.

This study underscores the remarkable potential of raspberry leaves for dermatological applications, emphasizing their antioxidative, antimicrobial, and antibiofilm properties. These findings suggest their promising role in skincare and cosmetic formulations

Multidrug-resistant bacteria represent a growing concern and complex challenge in healthcare [1]. The presence of these microorganisms in diabetic foot ulcers (DFU) is responsible for high hospitalization and amputation rates [2]. Honey has demonstrated effectiveness in DFU treatment due to its anti-inflammatory and antibacterial properties, namely against bacterial biofilms and multidrug-resistant bacteria [3]. However, the variety of physical-chemical characteristics among different types of honey, such as the type of pollen, might confer different biological properties, potentially leading to differences in antimicrobial response [4].This study aimed to assess the effect of different types and concentrations of honey against bacterial biofilm. Seven different types of honey from the region of Trás-os-Montes (Portugal) were tested at three concentrations: i) 1xMinimum Inhibitory Concentration (MIC), ii) 5xMIC, and iii) 10xMIC, against biofilms of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans. For Candida albicans, honey type-3, and type-4 removed 80.2% and 78.4% of biofilms, respectively, which was significantly higher than type-5 (8.87%), type-8 (27.5%), and type-9 (21.5%). Similarly, biofilm removal promoted by the honey type-2 (64.1%) and type-4 (57.2%) was greater than type-10 (28.5%) for E. coli. Principal Component Analysis suggested correlations between different pollen content and antimicrobial activity. Principal regression analyses were significant suggesting negative correlations of biofilm removal with pollen from Erica sp. and Sedum sp., and positive correlations with pollens from Corrigiola telephiifolia, and Jasione montana. Differences in bacterial responses may be due to variations in honey's pollen content and bacterial strain sensitivity.

Nontuberculous mycobacteria (NTM) are responsible for increased pulmonary infections in immunocompromised individuals and patients with underlying pulmonary diseases like cystic fibrosis. The treatment is a very long multidrug regimen associated with severe side effects and increased antibiotic resistance, which urges the discovery of new anti-NTM drugs. However, there is a significant discrepancy between in vitro antibiotic susceptibility and clinical effectiveness. Therefore, new antimycobacterial compounds must be tested in in vitro setups able to simulate the in vivo complexity of infection as accurately as possible. In this context, we are developing new strains of Mycobacterium abscessus, which simultaneously express the gene for a fluorescent protein, mScarlet, and the gene for the light-generating enzyme, luciferase. The fluorescent signal can be used as a marker of bacterial load, and the bioluminescent signal to monitor bacterial metabolism. We characterized these new double-reporter strains by comparing their growth profile with the non-transformed strain and by evaluating their susceptibility to antibiotics already used in the clinic to treat M. abscessus infections. We also assessed their ability to form biofilms to infect host cells, such as murine macrophages and human organoid-derived alveolar cells, and validated them for in vivo infection using bioluminescence imaging technology. The results show that these new double-reporter strains can be an essential tool to aid in the discovery of new drugs against mycobacterial infections.

This work is financed by portuguese national funds through FCT – Fundação para a Ciência e a Tecnologia, I.P, within the project PTDC/BIA-MIC/3458/2020 and PhD fellowships UI/BD/150830/2021 to CMB and 2021.07335.BD to GSO and by FWO – Research Foundation Flanders, grant n° 1S68720N.

The genus Aspergillus with more than 300 species includes several opportunistic pathogenic fungi (Aspergillus fumigatus), toxin-producing fungi (Aspergillus flavus) and species used in the food industry (Aspergillus niger). Aspergillus species produce small spores called conidia with an average size of 2-3.5 µm, easily dispersed into the air, where they can remain for long periods of time, ending up being inhaled by humans and other animals, and causing aspergillosis.

In the present work extracts from marine Actinobacteria (n=30) were screened against three Aspergillus species: Aspergillus flavus ATCC 204304, Aspergillus fumigatus ATCC 204305 and Aspergillus brasiliensis ATCC 16404. For that, the disk diffusion method (DD) was performed, following the CLSI guidelines. Among the three species, the most susceptible to the extracts was A. brasiliensis. After DD, and for the best extracts, the values of minimum inhibitory/fungicide concentrations (respectively MIC and MFC) were determined. For A. flavus and A. fumigatus and for all extracts, both MIC and MCF were higher than 250 μg/mL. For A. brasiliensis only two extracts stood out, one from Actinobacteria isolated from deep-sea sponge and one from the macroalgae Laminaria ochroleuca, with MIC and MFC around 15.62 μg/mL. These two extracts were selected to evaluate their impact on spore germination at the concentrations, ½ MIC, MIC and 2*MIC, but the extracts did not show any impact on conidia germination. The tested actinobacterial extracts exhibited some efficacy against A. brasiliensis. The other species (A. fumigatus and A. flavus) were less susceptible to the tested extracts.

The human gut is sheltered by overwhelming number of complex microorganisms, including mucin utilizers that play a significant role in host health and diseases. It is speculated that only 0.9% of faecal flora participates in the degradation of mucin molecules, and less than 100 such bacterial strains have been identified and a few were reported as probiotic candidate. The knowledge of the bacterial population that facilitates mucin degradation is still in infancy, as a significant proportion of the microbiota in mucin neither been cultured nor fully elucidated till now. In present investigation, different human derived mucolytic bacteria have been isolated and characterized. The data exhibited different mucus utilizing strains viz. Enterococcus (3), Priestia (2), Shigella (3) and Escherichia (1) via meta-genomic technique. The amido black assay implies that these isolates produce a halo around the colony that manifests the release of mucin degrading enzyme. Further, the findings of decrease in carbohydrate concentration in the range of 45-77% and protein content of 4-24% in mucin enriched spent medium confirmed the ability of strains to degrade mucin. Additionally, the enzymatic data reveal our isolated secrete inconsiderable amount of sialidase, α- galactosidase, β-galactosidase, α- glucosidase and β-glucosidase, that cleave mucin oligosaccharide chains. Further, the cell hydrophobicity results revealed all culture show low hydrophobicity (2-30%) and antibiotic susceptibility data manifested variation in antibiotic resistance profile. Safety analysis parameters such as haemolytic assay, gelatinase and, antibiotic susceptibility showed that the E. gallinarium KS4 and P. flexa KS1 lack virulence traits. Also, better acclimatization of E. gallinarium KS4 and P. flexa KS1 strain under in vitro gut conditions suggests they could act as a prominent probiotic candidate for human and amend intestinal integrity.

Introduction: In the last years, the North-Est region of Italy, in particular Veneto and Emilia-Romagna [1], has been characterized by a significant increase of West Nile Virus (WNV) infection rate. Neuroinvasive WNV viral infection may be linked epidemiologically and mechanistically to neurodegeneration, which have been associated with a significant prevalence of sequelae such as memory loss, confusion, and fatigue years later.

Non-structural protein 1 (NS1) is a highly conserved protein among Flaviviruses, which is actively secreted by infected cells and detected in the serum between days 3 and 8 post-infection, peaking on day 5, the day prior to the onset of clinical disease. Extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, facilitating WNV dissemination to the brain and affecting disease outcomes.

Aim: Focusing on the recently discovered antimicrobial roles of amyloid beta [2], we connected WNV late pathology to overlapping features encountered in neurodegenerative diseases such as Alzheimer’s disease. We aimed to investigate the possible effect of soluble NS1 on neurodegenerative and dysfunctional biomarkers (e.g. amyloid beta (Aβ), total and phosphorylated tau protein (p-tau), alpha-synuclein (α-syn), transactive response (TAR) DNA-binding protein 43 (TDP-43), prion protein (PrPSc), neurofilament light chains (NFL)) expression in neuronal cells (neurons, oligodendrocytes, and microglia), to clarify the mechanism underlying the CNS sequelae associated to WNV infection.

Methods: 2D cultures and 3D neuronal model were obtained with iPS (Induced Pluripotent Stem) cells and treated with purified WNV NS1. The mRNA and proteomic profiles were evaluated.

Results: We observed the ability of soluble NS1 to affect the expression of neurodegenerative and dysfunctional biomarkers. In particular, NS1 induced Aβ altered expression via TLR3, an endosomal Pathogen Pattern Receptors (PPRs) involved in RNA viruses sensing [3].

Conclusion: Our preliminary results suggest a possible role of soluble NS1 on CNS damage associated to WNV infection. Interestingly, TLR3 increased expression has been found associated to Aβ plaque in AD brains [4] and Aβ itself stimulates TLRs expression, prompting the neurodegeneration [5]. NS1 released by WNV infected cells might participate in CNS neurodegenerative process by altering TLR3 signaling and Aβ expression, suggesting a novel pathogenetic role.

References

1. Ricco, M., et al., West Nile Virus Infection: A Cross-Sectional Study on Italian Medical Professionals during Summer Season 2022. Trop Med Infect Dis, 2022. 7(12).
2. Bortolotti, D., et al., HHV-6A infection induces amyloid-beta expression and activation of microglial cells. Alzheimers Res Ther, 2019. 11(1): p. 104.
3. Wang, T., et al., Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis. Nat Med, 2004. 10(12): p. 1366-73.
4. Walker, D.G., T.M. Tang, and L.F. Lue, Increased expression of toll-like receptor 3, an anti-viral signaling molecule, and related genes in Alzheimer's disease brains. Exp Neurol, 2018. 309: p. 91-106.
5. Caldeira, C., et al., Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation. Front Aging Neurosci, 2017. 9: p. 277.

Enterococcus spp. are ubiquitous bacteria that inhabit the normal intestinal microbiota of most animals and are widespread in the environment. Enterococci have emerged as opportunistic pathogens, due to rapid adaptation features and ability to acquire virulence and antibiotic resistance genes. Considering the low numbers of investigations on the characterization of enterococcal isolates from animals and animal-related samples, the goal of the present study was to isolate enterococci from those settings and evaluate the associated antibiotic susceptibility.

Briefly, enterococci were isolated from faecal samples of stabled animals (horses, cows, and goats), healthy domestic cats and dogs, and from surface swabs and dogs’ anal exudates at a veterinary teaching hospital. Each sample was spread-plated in enterococcal selective media, Slanetz and Bartley agar, with/without vancomycin supplementation. Subsequently, 20% of the characteristic colonies were selected, submitted to DNA extraction and genetic comparison using RAPD-PCR. RAPD-pattern analysis and dendrogram construction allowed the selection of 110 genomically distinct representative isolates, further characterized for antibiotic susceptibility.

Regarding the results, around 3% were found to be vancomycin-resistant (two from goat and one from a domestic cat), while 10% were considered multidrug-resistant (isolates from one goat, one dogs’ scale, 1 tickets’ machine and 8 dogs’ anal exudates). Most enterococci recovered from stabled animals, and some from domestic dogs/cats, were classified as susceptible to all tested antibiotics.

Overall, relevant antibiotic resistances were detected, and resistance phenotypes were found to be disseminated among distinct sample sources.

Protected Designation of Origin (PDO) labels are attributed to foods produced at a given geographic area. Traditional PDO-cheeses harbor autochthonous microbiota involved in natural fermentation/maturation processes, contributing to specific organoleptic features. Among microorganisms with technological potential, the genus Enterococcus has been extensively researched. On the other hand, enterococci are known opportunistic pathogens that exhibit resistance to several clinically relevant antibiotics, contributing to the persistence of the disease. Hence, evaluating antimicrobial resistance of food-related bacteria is crucial to assess associated risks. In the present study, PDO-cheese samples from Portugal were collected over six years (2016-2022) and submitted to microbial isolation procedures; genomically distinct enterococci were further characterized regarding antibiotic susceptibility.

Overall, a total of 73 enterococci (selected based on RAPD-patterns from a larger microbial collection) were screened by susceptibility testing, using various antibiotics representing distinct drug classes and bacterial targets.

Resistance levels among the cheese enterococci were as follows: quinupristin/dalfopristin (92%), streptomycin (74%), and tetracycline (71%), while lower levels of resistance (<10%) were observed for ampicillin, erythromycin, chloramphenicol, ciprofloxacin, norfloxacin, and vancomycin. All the isolates were sensitive to gentamicin and linezolid, but approximately 16% showed a multidrug-resistant (MDR) phenotype. No statistically significant differences were observed in antibiotic resistance patterns among enterococci recovered from cheeses produced in different years (p<0.05).

In conclusion, our results revealed that the resistance level of the isolates recovered from PDO-cheeses was maintained over six years. However, the presence of MDR-enterococci constitutes a matter of concern and further emphasizes the need for active surveillance procedures.

Background: As other European countries, Italy launched its SARS-CoV-2 vaccination campaign on 27 December 2020 (1). The analysis of SARS-CoV-2 vaccination impact on host immune system of healthy subjects might elucidate the potential impact on COVID-19 outcomes. It is known that the presence of high affinity and persistent protective antibody responses indicate an efficient humoral immune response to vaccination (2,3). The aim of this study is to evaluate whether mRNA-based anti-SARS-CoV-2 vaccination (Comirnaty) elicited a robust protective innate immune response.

Methods: PBMC were obtained form whole blood obtained by donors who received three doses of mRNA-based anti-SARS-CoV-2 vaccination (Comirnaty). NK (Natural Killer) cells immunophenotype and cytotoxicity have been tested after stimulation with SARS-CoV-2 spike antigen (Wuhan, Alpha B.1.1.7, Delta B.1.617.2, Omicron B1.1.529 variants) by FACS assay and related with the anti-SARS-CoV-2 antibody production.

Results: We reported the presence of specific effector cytotoxic CD56dim, characterized by high levels of CD107a and granzyme production, and memory-like CD57+NKG2C+CD56dim phenotype of NK cells exposed to SARS-CoV-2 spike antigen (Wuhan, Alpha B.1.1.7, Delta B.1.617.2, Omicron B1.1.529 variants), in association with specific anti-SARS-CoV-2 antibody production, especially after the booster dose (4).

Conclusions and Discussions: We found that the booster dose caused early NK CD56dim subset activation and memory-like phenotype, confirming the relevance of innate immune response in the efficacy of SARS-CoV-2 vaccination.

References:

1. Olliaro P, Torreele E, et al. COVID-19 vaccine efficacy and effectiveness-the elephant (not) in the room. Lancet Microbe. 2021; 2: e279-e280.
2. Collier AY, McMahan K, et al. Immunogenicity of COVID-19 mRNA Vaccines in Pregnant and Lactating Women. JAMA. 2021; 325: 2370-2380.
3. Rizzo R, Bortolotti D, et al. Humoral and adaptive immune responses to the SARS-CoV-2 vaccine. Int J Infect Dis. 2022, 412-414.
4. Gentili V, Bortolotti D, et al. Natural Killer Cells in SARS-CoV-2-Vaccinated Subjects with Increased Effector Cytotoxic CD56dim Cells and Memory-Like CD57+NKG2C+CD56dim Cells. Front. Biosci. (Landmark Ed) 2023, 28(7), 156.

Gram-positive bacteria Streptococcus mutans and Staphylococcus aureus are important pathogens responsible for infections associated with dental caries and other medical implants. Sodium hypochlorite (NaOCl) is the most commonly used solution in root canal treatments, due to its low-cost and its high antimicrobial activity against microorganisms that infect root canals. The asepsis protocols drawn up by the Italian Society of Endodontics (SIE) recommend the use of a 5% NaOCl and 80% ethanol disinfectant solution for 2min to minimize the risk of contamination of the root canal system during endodontic treatments and for root canal disinfections (1). However, if this solution comes into contact with adjacent tissues, can cause complications, with tissue reactions resulting in life-threatening type I and IV hypersensitivity reactions (2,3). Due to its cytotoxic characteristics, the effects of which are directly proportional to the concentration of NaOCl used, rapid treatment must be carried out to prevent long-term sequelae (4, 5). In light of this, the aim of this work was to evaluate the use of Thyme Essential Oil (TEO, Thymus vulgaris) in synergistic combinations with NaOCl in order to reduce the concentrations of NaOCl in oral therapies, to improve the antiseptic efficacy, and to reduce side effects.

The microbroth dilution method, validated by Vanegas et al. (6), was used to determine the antimicrobial activity of TEO (9,28mg/mL) combined with NaOCl (1%), in the presence of organic material (6% sheep blood) against reference strains, S. mutans (ATCC70061) and S. aureus (ATCC43300). Based on previous data demonstrating the antibacterial efficacy of TEO after a few minutes of contact (7), the activity of the compound (TEO and NaOCl) was evaluated against Gram + and Gram -, after 1min, 3min and 5min of contact with bacterial strains at room temperature conditions. Aliquots of each suspension were diluted (ten-fold dilutions), cultured into Plate Count Agar (PCA) plates and incubated at 37°C for 24h and 48h to monitor bacterial growth. The combination of TEO and NaOCl has demonstrated total effectiveness in inhibiting microbial growth even in the presence of organic material. Using NaOCl in combination with TEO would allow to reduce the concentration of NaOCl currently used for dental procedures by at least 4 times. Our data therefore demonstrates that essential oils may play a role in the development of new dental treatments.