Search

The purpose of this study was to correlate the chemical composition of four commercial concentrated glycerine macerates (C-GMs), produced through the same extraction method, with their in vitro antimicrobial, antioxidant, and immunomodulatory properties, in order to evaluate their potential for healing upper airway diseases. C-GMs of Carpinus betulus (CB), Ficus carica (FC), Alnus glutinosa (AG) and Ribes nigrum (RN) were studied. The quality was evaluated using HPLC and IM-SPME/GC-MS systems, anti-oxidant and anti-microbial activities were assessed by the respective DPPH test, and micro-broth dilution test performed against 10 strains of Streptococcus pyogenes and 10 probiotic strains. ELISA and MTT tests were used to assess the immunomodulatory activity and the cytotoxicity of C-GMs, respectively. A significant correlation was found between the number of active compounds and the in vitro C-GMs effectiveness. Furthermore, the C-GMs of AG showed the best anti-microbial activity on pathological strains and, together with CB, the best anti-oxidant activity. The ELISA test exhibited a good immunomodulatory activity of RN. In vitro data support the integrated use of C-GMs of CB, AG, and RN in presence of airway diseases, and highlight the importance of standard procedures in cultivation, harvest and post-harvest treatments, as a premise for C-GMs with consistent characteristics.

The hydrolate, or hydrosol, is the residue of essential oil (EO) distillation traditionally used in perfumery. Evidence that also this product exerts anti-microbial activity promoted a study on Monarda citriodora EO and hydrolate against thirty representative strains of bacteria and fungi. Additionally, the sole hydrolate was tested in four moulds responsible for the spoilage of paper artworks. The micro-broth dilution test served to assess EO (concentrations between 0.0078% and 4%) and hydrolate (concentrations between 12.5% and 50%) suppressiveness vs untreated control. EO and hydrolate were analysed (GC–MS), resulting in thymol being the major terpenic compound of both products (19.6% and 66.4% in the respective spectra). EO suppressed all micro-organisms, although resistant Pseudomonas aeruginosa strains required up to 4% EO as minimum inhibitory concentration (MIC). Hydrolate suppressed all micro-organisms, except three P. aeruginosa strains where even the highest concentration (50% hydrolate) failed to reach the MIC. In each strain, the concentration needed to obtain 50% inhibition (IC50) was calculated by means of an exponential decay function. Median IC50 was 0.06% (EO) and 7.35% (hydrolate), while six IC50 EO data were shown to be outliers. In non-outlier data, IC50 hydrolate and EO were significantly correlated (r = 0.73**), and IC50 hydrolate was approximately a hundred times higher than IC50 EO. Despite higher concentration needed for the same inhibitory effect, the amount of terpenes supplied with hydrolate was lower than with EO. This means a higher hydrolate effectiveness that was likely due to the hydrophilic environment promoting higher terpene availability. Lastly, hydrolate exhibited promising results in the control of fungal growth on paper artworks, suppressing the four tested strains at concentrations of 25–50%.

Antimicrobial resistance (AMR) represents a serious threat to human health, as well as to food-producing animals and the environment. AMR is a growing phenomenon, while antibiotic efficacy against human and zoonotic infections is decreasing [1]. Salmonellosis is the 2nd most common zoonosis in Europe, and recent studies highlight multidrug resistance (MDR) of numerous Salmonella strains to the most commonly used antimicrobial agents [2]. EOs could meet some of the strategic objectives outlined in the WHO's Global Action Plan on AMR. Salmonella is the most frequently isolated bacterial agent in food-borne infections. Food-producing animals are the main reservoirs of infection, with animal-derived foods (meat, eggs and milk) sources of transmission. The aim of the study was to evaluate antibacterial efficacy of single EOs (Lavandula intermedia and Origanum vulgare) and of GR-OLI (25% commercial solution of an unknown mixture of Eucalyptus globulus, Satureja montana, Citrus aurantium var. dulcis, Thymus vulgaris, Melaleuca alternifolia, Citrus limon, Lavanda hybrida, Melaleuca cajeputi, Thymus capitatus) against Salmonella (29 strains) isolated from swine and poultry farms and against beneficial microorganisms used as probiotics, including Saccharomyces cerevisiae, S. boulardii, Enterococcus faecium and Bifidobacterium thermoacidophilum (6 strains). EO efficacy was evaluated through MIC analysis according to EUCAST guidelines [3]. The action of scalar concentrations between 2% v / v and 0.06% v / v for OEs and between 8% v / v and 0.25% v / v for GR-OLI was evaluated. Moreover, the EOs with sub-MIC values capable of reducing bacterial growth were tested in CaCo-2 cell adhesion and biofilm experiments using Salmonella (12 strains). GR-OLI and O. vulgare proved more effective than L. intermedia against multidrug-resistant Salmonella strains. The EOs evaluated had higher MIC values against probiotics than against Salmonella. GR-OLI also decreased biofilm formation and reduced adhesion of Salmonella strains to CaCo-2 cells. GR-OLI showed higher efficacy against multidrug-resistant Salmonella strains than against drug-sensitive Salmonella strains. Our data on the evaluated EOs require further investigation and confirmation, also exploring the possibility of resistance development. However, sub-MIC doses of GR-OLI could prove to be an interesting strategy for reducing Salmonella presence in intensive swine and poultry farms [1] Singer A.C. et al. Review of antimicrobial resistance in the environment and its relevance to environmental regulators, Front Microbiol., 7:1-22, 2016. [2] Santos F.S., Novales M.G.M. Essential oils from aromatic herbs as antimicrobial agents, Curr. Opin. Biotechnol., 23:136–141, 2012. [3] EUCAST, 2017. European Committee for Antimicrobial Susceptibility Testing. Version 7.1. www.eucast.org.

1. Introduction. As known, one of the major problems linked to the wound care is the highest probability of infection. Until now, many studies have been developed to modify gauzes made with hydrogel, with the only function to make the wound moist, giving it also an antimicrobial action. However, articles published to date show gauzes modified with the addition of antibiotics or chemical compounds with antimicrobial action or at most essential oils like Melaleuca alternifolia (Tea Tree Oil). Our studies focused on the antimicrobial action of a hydrogel, like the gellan gel, when modified with the addition of hydrolates (Hys), natural waste products of the essential oils production method. 2. Materials and Methods. On seven fungal strains (4 clinical isolates and 3 ATCC strains) and 11 GRAM+ strains both sensible and resistant to antibiotics (6 clinical isolates and 5 ATCC strains), micro-broth dilution test, in line with EUCAT guidelines, was performed. We studied the cytocidal action of 1 commercial Hy (Citrus aurantium var amara - CA), and 4 Hys produced at the DISTAL department of the University of Bologna (Monarda citriodora, M. didyma, M. fistulosa, Lavandula vera). The same test was performed to evaluate the antimicrobial action of preservatives present in the commercial Hy. The cytocidal action of a prototype of hydrogel-impregnated gauze (HIG) modified with 50% v/v of the Hy of CA was evaluated. Ten microL of a suspension of each strain were adsorbed on HIG and incubated overnight at 37°C. Subsequently, the HIGs were printed on Muller Hilton agar medium that was further incubated at 37°C for 24h. To assess the in vivo efficacy, preliminary study of subcutaneous S. aureus infections were developed in 8 Wistar rats by inoculating 100 microL of 1x107 cfu/mL suspension and bandaging only the treated group (n=4) with HIG. Every day, for 5 days, the HIG was changed and the rat was monitored for the development of subcutaneous abscess. 3. Results. Among all the investigated Hys, CA was the most effective (MIC90 ≤3.125% v/v). Tests carried out on preservatives alone did not show any cytocidal action (MIC >50% v/v); while, when associated with the Hy, they enhance the action of hydrolate alone (MIC90=12.5% v/v). Tests performed on HIG showed a cytocidal action on all tested strains. Preliminary in vivo data showed a reduction of about 70% of volume of abscess in treated rats compared to those in control group. 4. Discussion and Conclusions. Our data showed a cytocidal action of commercial Hy of CA vs fungal and bacterial strains potential skin pathogens. Although further in vivo studies are needed to confirm this activity, our study points out to a possible use of the Hy of CA in pre-medicated gauze for the treatment of skin wounds.

Identification and quantification of bacteria affecting human life, directly causing diseases and indirectly contaminating natural ecosystems, are mostly carried out by expensive and time-consuming methods. There is the need for rapid and economic ways for detecting bacteria in the environment and in clinics. We propose the use of engineered liposomes for detecting bacteria in drinking water. Our approach exploits cationic liposomes functionalized with a surface potential-sensitive fluorophore, 4-heptadecylumbelliferone (C17-HC). The interaction between liposomes and bacteria involves a change in the surface potential experienced by C17-HC and switches on an optical signal. We investigated, by DLS, zeta-potential and fluorescence experiments, a large number of cationic liposomes formulated with a natural phospholipid 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC) or 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), C17-HC, and one of three synthetic cationic components, differing from each other for the number of unsaturations on the polar ammonium head, two of which ad hoc synthesized. Then we evaluated the ability of liposomes to produce a fluorescent signal upon interaction with three bacterial strains, Staphylococcus aureus , Escherichia coli and Enterococcus faecalis ; moreover, we analyzed the fluorescent response of each liposome formulation in the presence of the three bacterial strains at the same time, in order to simulate a real scenario. We found that interaction with bacteria triggers an optical signal in six of the evaluated formulations, resulting responsive down to 10 2 CFU/mL of bacteria suspended in pipeline water coming from the water main of Rome (Italy).

Background Epicardial adipose tissue (EAT) has a close functional and anatomic relationship with epicardial coronary arteries. Accumulating evidence suggests that host microbiome alterations may play a role in several inflammatory/immune disorders, triggering a robust proinflammatory response also involving interleukin-1β (IL-1β) and the NALP3 inflammasome. In the current study, we explore the hypothesis that in patients with non-ST elevation acute coronary syndrome (ACS), EAT contains potentially pro-atherosclerotic bacteria that might elicit inflammasome activation. Methods EAT samples were obtained during coronary artery bypass grafting from ACS ( n =18) and effort stable angina (SA; n =16) patients, and as controls, from patients with angiographically normal coronary arteries undergoing surgery for mitral insufficiency (MVD; n =13). In all patients, NALP3 and proIL-1β mRNA expressions were evaluated with qRT-PCR. In 3 patients from each group, EAT microbiota composition was determined using next-generation sequencing technologies. Results In EAT, mRNA expression of both NALP3 and pro-IL1β was significantly higher in ACS than in SA and MVD ( P =0.028 and P =0.005, respectively). A broad range of bacterial species ( n =76) was identified in both ACS and SA, with different predominant species. In contrast, microbial DNA was barely observed in MVD. Conclusions Our study demonstrated the presence of bacterial DNA directly into EAT, surrounding diseased coronary arteries, of patients with ACS. Furthermore, ACS is associated with NALP3/inflammasome pathway activation in EAT. Our data suggest that the EAT environment is susceptible to microbial colonization that might stimulate a proinflammatory response. These findings add new elements to the pathogenesis of ACS and suggest novel therapeutic targets.

Background: Irritable bowel syndrome (IBS) is a functional disorder without any pathological alteration, in which the alterations of the Candida/Saccharomyces ratio of the gut microbiota, the balance of pro and anti-inflammatory cytokines and the brain-gut-microbiome axis are important for the development and progression of IBS. The aim of the study was to identify natural products, including essential oils or hydrolates, which were contextually harmless for the gut beneficial strains (e.g. Saccharomyces spp.) but inhibitory for the pathogenic ones (Candida spp.). Methods: The effectiveness of 6 essential oils and 2 hydrolates was evaluated using microbiological tests, carried out on 50 clinical isolates (Candida, Saccharomyces and Galattomyces species) and 9 probiotic strains (Saccharomyces cerevisiae, Lactobacillus species, Akkermansia muciniphila and Faecalibacterium prausnitzii) and immunological and antioxidant assays. Results: The study led to a mixture based on a 1/100 ratio of Citrus aurantium var. amara essential oil / Vitis vinifera cv Italia hydrolate able to contextually reduce, in a concentration-dependent manner, the ability of Candida species to form hyphal filaments and have an interesting immunomodulatory and anti-oxidant action. This mixture can potentially be useful in the IBS treatment promoting the restoration of the intestinal microbial and immunological balance.

Salmonella spp. represent a public health concern for humans and animals due to the increase of antibiotic resistances. In this scenario, the use of essential oils (EOs) could be a valid tool against Salmonella contamination of meat. This work compares the in vitro effectiveness of an Italian mixture of feed additives based on EOs (GR-OLI) with EO of Origanum vulgare L., recently admitted by European Food Safety Authority (EFSA) for animal use. Twenty-nine Salmonella serotypes isolated from poultry and pig farms were used to assess GR-OLI and O. vulgare EO antimicrobial propeties. O. vulgare EO was active on the disaggregation of mature biofilm, while GR-OLI was capable of inhibiting biofilm formation and disaggregating preformed biofilm. Furthermore, GR-OLI inhibited bacterial adhesion to Caco-2 cells in a dose-dependent manner. Both products showed inhibition of bacterial growth at all time points tested. Finally, the synergistic action of GR-OLI with commonly used antibiotics against resistant strains was investigated. In conclusion, the mixture could be used both to reduce the meat contamination of Salmonella spp. before slaughter, and in synergy with low doses of ciprofloxacin against resistant strains. Although EOs as feed additives are already used in animal husbandry, no scientific study has ever highlighted their real antimicrobial potential.

Background : Among the opportunistic fungi, Candida species represent one of the most common causes of nosocomial bloodstream infections. The large use of antifungal agents, most of them launched on the market more than twenty years ago, led to the selection of drug-resistant or even multidrug-resistant fungi. In the last years, we described a novel class of antifungal macrocyclic compounds bearing an amidinourea moiety, highly active against various azole-resistant Candida strains. Objective : In this study, one representative of this family, compound BM1, has been investigated on its in vitro activity against various Candida species, including C. auris isolates, its interaction with the ABC transporter CDR6 and its in vivo distribution and safety. Methods : In vitro assays (CYP inhibition, microsomal stability, permeability, spot assays) have been used to collect chemical and biological data; animal models (rat) paired with LC-MS analysis have been exploited to evaluate in vivo toxicology, pharmacokinetics, and distribution. Results : Our research highlights the low in vivo toxicity profile of BM1, its affinity for the renal system in rats and its good absorption, distribution, metabolism, and excretion (ADME) features. Our compound preserves a potent activity also against azole-resistant fungal strains, including C. auris isolates and CDR6-overexpressing strains. Conclusions : We confirmed low MICs against several Candida species, including preliminary data versus C. auris. The good ADME and biochemical characteristics make BM1 suitable and safe for daily administration and particularly indicated to treat renal infections. These data make BM1 and its derivatives a novel promising antifungal class.

The clinical challenge on surface engineering of medical devices to prevent microorganisms adhesion and biofilm formation, has become an essential aspect for medical implants. Antibacterial properties of Graphene Oxide (GO) have been demonstrated across a broad spectrum of bacteria, and the different mechanisms of action with which this nanomaterial interacts with the microbial surface have been elucidated in detail. Innovative protective coatings based on graphene film and hydrogel could represent an innovative solution for the prevention of nosocomial pathogens colonization on implantable device. This brief review mainly focuses on the applications of graphene in nanomedicine with a particular deepening on the antibacterial properties of GO and GO-based nanomaterials. In order to evaluate the possible future applications of GO as an anti-biofilm coating material for medical devices, studies on the ability of graphene coated surface to prevent microbial adhesion are also discussed. A concise review on in vitro toxicity and in vivo safety is also presented.

Background Probiotics are defined as live, nonpathogenic bacteria that confer health benefits beyond their nutritional value. In particular, VSL#3 exhibits demonstrated efficacy in the management of diseases characterized by an increased intestinal permeability. Our study aimed to understand how VSL#3 promotes gut health by secreting bioactive factors and identify which human pathways are modulated by secretome derived from the VSL#3 formula. Methods Two different lots of VSL#3 were used, and Caco-2 cell line was treated with conditioned media (CM) prepared using 1 g of the probiotic formula. We evaluated the effects of the probiotics on cellular proliferation and apoptosis by cytometry and the expression of tight junction proteins by western blotting. A proteomics analysis of both culture media and the whole proteome of Caco-2 cells treated with VSL#3-CM was performed by nano-ultra performance liquid chromatography - tandem mass (nUPLC MS/MS) spectrometry. Results The probiotic formula increased cell proliferation, decreased cellular apoptosis cells, and increased re-epithelialization in the scratch assay. Several peptides specifically synthetized by all the species within the probiotic preparation were recognized in the proteomics analysis. Human proteins synthesized by CaCo-2 cells were also identified. Conclusions To our knowledge, this manuscript describes the first evaluation of the probiotic secretome, and the results showed that the improvement in intestinal barrier functions induced by probiotics seems to be accompanied by the modulation of some human cellular pathways.

Overexpression of P-glycoprotein (P-gp) and other ATP-binding cassette (ABC) transporters in multidrug resistant (MDR) cancer cells is responsible for the reduction of intracellular drug accumulation, thus decreasing the efficacy of chemotherapeutics. P-gp is also found at endothelial cells’ membrane of the blood-brain barrier, where it limits drug delivery to central nervous system (CNS) tumors. We have previously developed a set of pyrazolo[3,4-d]pyrimidines and their prodrugs as novel Src tyrosine kinase inhibitors (TKIs), showing a significant activity against CNS tumors in in vivo. Here we investigated the interaction of the most promising pair of drug/prodrug with P-gp at the cellular level. The tested compounds were found to increase the intracellular accumulation of Rho 123, and to enhance the efficacy of paclitaxel in P-gp overexpressing cells. Encouraging pharmacokinetics properties and tolerability in vivo were also observed. Our findings revealed a novel role of pyrazolo[3,4-d]pyrimidines which may be useful for developing a new effective therapy in MDR cancer treatment, particularly against glioblastoma.

I sierotipi di Salmonella spp, un genere patogeno di Enterobacteriaceae, sono responsabili di infezioni in animali da sub-cliniche a severe, nonchè di febbre tifoide e diarrea severa negli uomini (Foley et al, 2008). Questi batteri Gram negativi sono una delle maggiori cause di preoccupazione per l’industria veterinaria. Nel particolare, i polli e i maiali sono noti come i maggiori veicoli di Salmonella. Grandi sforzi sono rivolti al controllo della colonizzazione di Salmonella nei reservoir di maiali e polli poiché questi animali, se infetti, generalmente ospitano il batterio in modo asintomatico nelle tonsille, nell’intestino e nel tessuto linfoide associato all’intestino (GALT) (Boyer et al 2008). Per questo anche la European Food Safety Authority (EFSA) considera i sierotipi di Salmonella isolati da maiali e da polli un pericolo per la salute pubblica (EFSA, 2006) tanto da istituire misure di controllo atte alla riduzione della prevalenza della Salmonella spp specialmente negli allevamenti in cui la produzione di polli e maiali è aumentata negli anni (Evangelopoulou et al, 2015 Foley 2008). Il primo scopo del nostro studio è stato quello di testare l’efficacia degli oli essenziali (OE) di Lavandula intermedia e Coridothymus capitatus e di GR-OLI, una formulazione veterinaria a base di OE (25% del totale caratterizzato da: Eucalyptus globulus, Satureja Montana, Citrus aurantium, Thymus vulgaris, Melaleuca alternifolia, Citrus lemon, Lavandula intermedia, Melaleuca cajeputi, Coridothymus capitatus), su 20 ceppi di Salmonella spp isolati da allevamenti di polli e maiali, 2 ceppi di Saccharomyces cerevisiae e 2 ceppi di Saccharomyces boulardii appartenenti alla collezione microbica dell’Università di Bologna. Il secondo scopo è stato quello di individuare le dosi sub-MIC in grado di inibire la formazione di biofilm e la capacità adesiva di ceppi di S. typhimurium sensibili e resistenti agli antibiotici. Il primo scopo è stato raggiunto eseguendo test di micro-brodo diluizione in linea con le linee guida internazionali EUCAST. Il secondo scopo è stato raggiunto coltivando biofilm di S. typhimurium o valutando la loro adesione sulle cellule intestinali umane CaCo-2 in presenza di diluizioni sub-MIC degli OE e del GR-OLI. Inoltre è stata valutata l’azione delle sub-MIC studiando, in tempo reale, le curve di crescita dei ceppi batterici. I nostri risultati mostrano la maggiore efficacia dell’OE di C. capitatus (MIC90 = 0.5% v/v) e GR-OLI (MIC90=1%v/v) se paragonata a quella dell’OE di L. intermedia EO (MIC90≥2% v/v). Inoltre, le MIC di questi OE vs i ceppi probiotici sono più alte (MIC uguale a 1% - 2% e >2% v/v, rispettivamente per C. capitatus, GR-OLI e L. intermedia EO). I dati preliminari sull’inibizione della formazione del biofilm e sull’adesione dei ceppi alle cellule, mostra come il GR-OLI sia efficace quando usato a concentrazioni sub-MIC comprese tra 0.25% e 0.03% v/v. Le curve di crescita di S. typhimurium mostrano l’efficacia del C. capitatus e GR-OLI sia sui ceppi sensibili che resistenti agli antibiotici, mentre l’OE di L. intermedia inibisce solo i ceppi sensibili. I nostri dati preliminari mostrano come sia possibile usare concentrazioni sub-MIC di OE per rendere la Salmonella meno virulenta senza interferire con la vitalità dei ceppi appartenenti al microbiota intestinale. Ulteriori studi devono essere fatti per confermare i nostri dati e studiare l’eventuale, anche se improbabile, sviluppo di resistenze agli OE. Salmonella spp., a pathogenic genus of Enterobacteriaceae, has serovars causing subclinical to severe clinical infections in animals, and typhoid fever and severe diarrhoea in humans (Foley, 2008). These Gram negative bacteria are one of the major causes of concern for the veterinary industry. Particularly, chickens and pigs are recognized as a major vehicle for Salmonella. Recently, great importance has been given to practices aimed at controlling the colonization of Salmonella in pig and chicken reservoirs: in fact infected animals, such as pigs, generally carry these bacteria asymptomatically in the tonsils, the intestines and the gut-associated lymphoid tissue (GALT) (Boyer 2008). For these reasons, strains isolated from pigs and chickens are considered a hazard for public health also by the European Food Safety Authority (EFSA) (EFSA, 2006); thisis a real incentive for some nations in undertaking control measures aiming at their reduction in farms where pigs and chickens breeding has increased over the years (Evangelopoulou et al, 2015 Foley 2008). The first aim of our study was to test the effectiveness of both Lavandula intermedia and Coridothymus capitatus essential oils (EOs) and GR-OLI, a veterinary formulation based on EOs (25% of a total characterised by Eucalyptus globulus, Satureja Montana, Citrus aurantium, Thymus vulgaris, Melaleuca alternifolia, Citrus lemon, Lavandula intermedia, Melaleuca cajeputi, Coridothymus capitatus), on 20 strains of Salmonella spp isolated from poultry and pig farms, 2 probiotic strains of Saccharomyces cerevisiae and 2 of Saccharomyces boulardii belonging to the University of Bologna microbial collection. The second aim was to identify sub-MIC doses that are able to inhibit the biofilm formation and the adhesion capacity of S. typhimurium strains, both sensitive and resistant to antibiotics. The first aim was reached using the micro-broth dilution test in line with EUCAST international guidelines to identify MIC values. The second aim was achieved growing S. typhimurium biofilm or evaluating its adhesion to CaCo-2 intestinal human cells in presence of sub-MIC dilutions of both EOs and GR-OLI. Furthermore, the activity of sub-MICs on strains growth was evaluated by studying, in real time, their growth curves. Our results show the major effectiveness of the C. capitatus EO (MIC90 = 0.5% v/v) and GR-OLI formulation (MIC90=1%v/v) if compared to L. intermedia EO (MIC90≥2% v/v). However, the effectiveness of these ones vs probiotic strains is observed at higher concentrations (MIC equal to 1% - 2% and >2% v/v, respectively of C. capitatus EO, GR-OLI formulation and L. intermedia EO). Furthermore, preliminary data on inhibition of both biofilm formation and cell adhesion show the effectiveness of GR-OLI when used at sub-MIC doses between 0.25% and 0.03% v/v. Data from growth curves show the effectiveness of C. capitatus EO and GR-OLI on both antibiotic sensitive and resistant strains, while L. intermedia EO is able to inhibit the growth of only antibiotic sensitive strains. Our preliminary data show that it is possible to use sub-MIC concentrations of EOs to make Salmonella spp., if present, less virulent than in normal conditions, without interfering with the viability of strains belonging to the intestinal microbiota. Further studies need to be done in order to confirm our data and study the possibility, although unlikely, to develop strain resistance vs EOs.

It is now established that the gastric pathogen Helicobacter pylori has the ability to form biofilms in vitro as well as on the human gastric mucosa. The aim of this study is to evaluate the antimicrobial effects of Clarithromycin on H. pylori biofilm and to enhance the effects of this antibiotic by combining it with Alginate Lyase, an enzyme degrading the polysaccharides present in the extracellular polymeric matrix forming the biofilm. We evaluated the Clarithromycin minimum inhibition concentration (MIC) on in vitro preformed biofilm of a H. pylori. Then the synergic effect of Clarithromycin and Alginate Lyase treatment has been quantified by using the Fractional Inhibitory Concentration index, measured by checkerboard microdilution assay. To clarify the mechanisms behind the effectiveness of this antibiofilm therapeutic combination, we used Atomic Force Microscopy to analyze modifications of bacterial morphology, percentage of bacillary or coccoid shaped bacteria cells and to quantify biofilm properties. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1584-1591, 2016.

The huge resources that had gone into Human Immunodeficiency virus (HIV) research led to the development of potent antivirals able to suppress viral load in the majority of treated patients, thus dramatically increasing the life expectancy of people living with HIV. However, life-long treatments could result in the emergence of drug-resistant viruses that can progressively reduce the number of therapeutic options, facilitating the progression of the disease. In this scenario, we previously demonstrated that inhibitors of the human DDX3X helicase can represent an innovative approach for the simultaneous treatment of HIV and other viral infections such as Hepatitis c virus (HCV). We reported herein 6b, a novel DDX3X inhibitor that thanks to its distinct target of action is effective against HIV-1 strains resistant to currently approved drugs. Its improved in vitro ADME properties allowed us to perform preliminary in vivo studies in mice, which highlighted optimal biocompatibility and an improved bioavailability. These results represent a significant advancement in the development of DDX3X inhibitors as a novel class of broad spectrum and safe anti-HIV-1 drugs.

The increasing resistance to conventional antibiotics is an urgent problem that can be addressed by the discovery of new antimicrobial drugs such as antimicrobial peptides (AMPs). AMPs are components of innate immune system of eukaryotes and are not prone to the conventional mechanisms that are responsible of drug resistance. Fish are an important source of AMPs and, recently, we have isolated and characterized a new 22 amino acid residues peptide, the chionodracine (Cnd), from the Antarctic icefish Chionodraco hamatus. In this paper we focused on a new Cnd-derived mutant peptide, namely Cnd-m3a, designed to improve the selectivity against prokaryotic cells and the antimicrobial activity against human pathogens of the initial Cnd template. Cnd-m3a was used for immunization of rabbits, which gave rise to a polyclonal antibody able to detect the peptide. The interaction kinetic of Cnd-m3a with the Antarctic bacterium Psychrobacter sp. (TAD1) was imaged using a transmission electron microscopy (TEM) immunogold method. Initially the peptide was associated with the plasma membrane, but after 180 min of incubation, it was found in the cytoplasm interacting with a DNA target inside the bacterial cells. Using fluorescent probes we showed that the newly designed mutant can create pores in the outer membrane of the bacteria E. coli and Psychrobacter sp. (TAD1), confirming the results of TEM analysis. Moreover, in vitro assays demonstrated that Cnd-m3a is able to bind lipid vesicles of different compositions with a preference toward negatively charged ones, which mimics the prokaryotic cell. The Cnd-m3a peptide showed quite low hemolytic activity and weak cytotoxic effect against human primary and tumor cell lines, but high antimicrobial activity against selected Gram – human pathogens. These results highlighted the high potential of the Cnd-m3a peptide as a starting point for developing a new human therapeutic agent.

The clinical challenge on surface engineering of medical devices to prevent microorganisms adhesion and biofilm formation, has become an essential aspect for medical implants. Antibacterial properties of Graphene Oxide (GO) have been demonstrated across a broad spectrum of bacteria, and the different mechanisms of action with which this nanomaterial interacts with the microbial surface have been elucidated in detail. Innovative protective coatings based on graphene film and hydrogel could represent an innovative solution for the prevention of nosocomial pathogens colonization on implantable device. This brief review mainly focuses on the applications of graphene in nanomedicine with a particular deepening on the antibacterial properties of GO and GO-based nanomaterials. In order to evaluate the possible future applications of GO as an anti-biofilm coating material for medical devices, studies on the ability of graphene coated surface to prevent microbial adhesion are also discussed. A concise review on in vitro toxicity and in vivo safety is also presented.

Enterococcus faecium is an important health care-associated pathogen that is difficult to treat due to the high level of antibiotic resistance of clinical isolates. The identification of new potential therapeutic targets or vaccination strategies is therefore urgently needed. In this regard, we carried out a transcriptomic analysis of the E. faecium vancomycin-resistant strain AUS0004, comparing the gene expression of bacteria grown under laboratory conditions and bacteria isolated from an infection site. This analysis highlighted more than 360 genes potentially induced under infection conditions. Owing to their expression profiles, four LysM domain-containing proteins were characterized in more detail. The EFAU004_01059, 1150 and 494 proteins are highly homologous, whereas EFAU004_01209 has a unique domain-architecture and sequence. The analysis of corresponding mutants showed that all LysM proteins played relevant roles in the infection process of E. faecium in mice. The EFAU004_01209 mutant also displayed profound morphological modifications, suggesting it has a role in cell wall synthesis or cell division. Furthermore, the adhesion to kidney cells and growth of the mutant was affected in human urine. All these phenotypes and the surface exposure of EFAU004_01209 identify this protein as an interesting new drug target in E. faecium.

Aim: Fabrication of graphene oxide (GO)-based medical devices coatings that limit adhesion of Candida albicans, a main issue of healthcare-associated infections. Methods: The GO composites noncovalently functionalized with curcumin (CU), a hydrophobic molecule with active antimicrobial action, polyethylene glycol (PEG) that hinders the absorption of biomolecules or a combination of CU and PEG (GO–CU–PEG) were drop-casted on surfaces and antifungal efficacy was assessed. Results: We demonstrate that GO–CU–PEG coatings can reduce fungal adhesion, proliferation and biofilm formation. Furthermore, in an aqueous environment, surfaces release curcumin–PEG nanocomposites that have a minimum inhibitory concentration of 9.25 μg/ml against C. albicans. Conclusion: Prevention of early cell adhesion and creation of a proximal environment unfavorable for growth make these GO-supported biomaterials attractive for innovative medical device manufacturing. [Formula: see text]

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for serious hospital infections worldwide and represents a global public health problem. Curcumin, the major constituent of turmeric, is effective against MRSA but only at cytotoxic concentrations or in combination with antibiotics. The major issue in curcumin-based therapies is the poor solubility of this hydrophobic compound and the cytotoxicity at high doses. In this paper, we describe the efficacy of a composite nanoparticle made of curcumin (CU) and graphene oxide (GO), hereafter GOCU, in MRSA infection treatment. GO is a nanomaterial with a large surface area and high drug-loading capacity. GO has also antibacterial properties due mainly to a mechanical cutting of the bacterial membranes. For this physical mechanism of action, microorganisms are unlikely to develop resistance against this nanomaterial. In this work, we report the capacity of GO to support and stabilize curcumin molecules in a water environment and we demonstrate the efficacy of GOCU against MRSA at a concentration below 2 µg ml −1 . Further, GOCU displays low toxicity on fibroblasts cells and avoids haemolysis of red blood cells. Our results indicate that GOCU is a promising nanomaterial against antibiotic-resistant MRSA.

Graphene and Graphene oxide (GO) are capable of inducing stem cells differentiation into bone tissue with variable efficacy depending on reductive state of 362a Monday, February 19, 2018 the material. Thus, controlling the oxidative state of the GO it is possible, in principle, to modulate the osteogenic process and the bone mineral density distribution. In this study, we laser-printed GO surfaces in order to obtain both a local photo/thermal GO reduction and the formation of nano-wrinkles along precise geometric pattern. Calvarial-derived Mesenchimal Stem cells (CMSC) were isolated in primary culture from skull calvarial fragments and their behaviour was characterized on GO patterns. Initially, after cells adhesion on the surface, stem cells migrated and accumulated on the reduced/wrinkled surface. When the local density of the stem cells on the reduced stripes was high, cells started to proliferate and occupy the oxidized/flat area. The designed surfaces morphology guided stem cell orientation and the reduction accelerated differentiation. Furthermore, the reduced nano-wrinkles greatly inhibited the growth of S. aureus. We propose a promising laser-printing technology for tissue engineering and regenerative medicine, based on graphene to enhance cell growth and differentiation in a controlled way. We show that by adjusting the laser emission is possible to finely tune the GO reduction and shape substrate landscape to spatially control bone regeneration and microbial infection. This strategy can offer a revolution in present and future trends of medicine and surgery

Today, liposomes are an advanced technology of drug carriers with a dozen drugs in clinical practice and many more in clinical trials. A bottleneck associated with the clinical translation of liposomes has long been ‘opsonization’, i.e. the adsorption of plasma proteins at the liposome surface resulting in their rapid clearance from circulation. For decades, the most popular way to avoid opsonization has been grafting polyethylene glycol (PEG) onto the liposome surface. Recent studies have clarified that grafting PEG onto the liposome surface reduces, but does not completely prevent protein binding. In this work, we employed dynamic light scattering, zeta-potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), semi-quantitative densitometry and cell imaging to explore the bio-nano-interactions between human plasma (HP) and Onivyde, a PEGylated liposomal drug that has recently been approved by the Food and Drug Administration (FDA) for the treatment of metastatic pancreatic ductal adenocarcinoma (PDAC). To properly evaluate the role of PEGylation, an unPEGylated variant of Onivyde was used as a reference. Collectively, our findings suggest that: (i) although PEGylated, Onivyde is not “stealth” in HP; (ii) surface chemistry is more important than PEGylation in controlling the bio-nano-interactions between Onivyde and plasma components. Of note is that the PC was found to boost the cellular uptake of Onivyde in the pancreas ductal adenocarcinoma cell line (PANC-1) thus suggesting its prominent role in its indication for PDAC treatment. Relevant implications for drug delivery and drug design are discussed.

The opportunistic pathogenic mould Aspergillus fumigatus (A. fumigatus) is an increasing cause of morbidity and mortality in immunocompromised and in part immunocompetent patients. A. fumigatus can grow in multicellular communities by the formation of a hyphal network embedded in an extracellular matrix (ECM) meanly composed by polysaccharides, melanin, proteins. Because adhesion properties is one primary factor affecting the balance between growth, detachment and biofilm formation, its quantification is essential in understanding, predicting, and modelling biofilm development. Atomic force microscopy (AFM) imaging and force spectroscopy have recently opened a range of novel applications in microbiology including the imaging and manipulation of membrane proteins at the subnanometer level, the observation of the surface of living cells at high resolution, the mapping of local properties such as surface charges, the measurement of elastic properties of cell-surface constituents and the probing of cellular interactions using functionalized probes. Nevertheless, the principal disadvantage of this approach is the relatively slow acquisition rate that makes AFM is not able to detect fast dynamics. In this study we demonstrated that digital pulsed force mode (DPFM) atomic force microscopy can be used to obtain high-resolution topographical images and to quantify the adhesion properties of the A. fumigatus biofilm with an high acquisition rate. Here we show by means of DPFM-AFM that Alginate Lyase (AlgL), an enzyme known to reduce negatively charged alginate levels in microbial biofilm, is able to reduce the biofilm adhesion forces forming several nano-fractures in the ECM. These results suggest that the AlgL could used to enhance the antifungal drugs transit through the ECM.

Antibacterial items are one of the major queries from the medical community in the fight against medical infections. Indeed, bacteria are resistant and their multiplication and biofilm formation on devises are one of the major causes of infections. Finding antibacterial surfaces, which are biocompatible, cost-effective, not toxic, and spreadable over large and irregular surfaces, is not easy. However, we created an antibacterial cloak by laser printing of Graphene Oxide (GO) hydrogels by mimicking the Cancer Pagurus carapace. This surface provides up to 90% reduction of bacteria cells through a bacteriostatic and bactericidal effect. Indeed, Laser treating allows GO sheets gel to cut and wrap microorganisms. Our findings are confirmed by a theoretical active matter model. This new technology based on antibiotic-free biomimetic Graphene Oxide gels opens untrodden roads to the fight against infections in biomedical applications and chirurgical equipment.

Background Preeclampsia (PE) is a major cause of maternal and neonatal morbidity and mortality. Epidemiological association between Helicobacter pylori (Hp) infection and PE onset has been widely shown. The aim of this study was to analyze a possible correlation between Hp infection and the severity of clinical presentation of PE and to identify an immunologic mechanism triggered by Hp infection potentially contributing to the pathogenesis of PE. Materials and Methods Sera from 93 preeclamptic women and 87 healthy pregnant women were tested for Hp infection by immunoassay and for anti-CagA antibodies by Western blot assay. The serologic results were correlated with the clinical features of PE. The functional effect of serum IgG fractions, positive or negative for Hp, from preeclamptic women or controls were tested on trophoblast and endothelial cell cultures and in a murine model of angiogenesis. Results Preeclamptic women showed higher seroprevalence of Hp infection (57.0%) compared to controls (33.3%) (P

E. faecalis and E. faecium cause urinary tract infections highly resistant to therapies due to a protective extracellular matrix. To exploit a new strategy able to treat infections without increasing antibiotic doses, we used enzymes targeting specific biofilm matrix components in combination with Vancomycin. We investigated the activity of Vancomycin combined with two matrix-degrading enzymes, Alginate Lyase (AlgL) and Deoxyribonuclease I (DNase I) against in vitro biofilm of E. faecalis and E. faecium clinical isolates. The heterogeneity of matrix composition leads to defined physiological responses of biofilm communities to their environment: we demonstrated that the use of DNase I and AlgL enzymes affects biofilm structure, cell viability and reduces MBEC values of Vancomycin in E. faecalis and E. faecium, respectively.

By nonlinear optical supercavitation we realize an antibacterial cloak on graphene-oxide (GO) based hydrogel. By combining the GO antimicrobial effects with a multiscale bio-inspired pattern, we observe up to 90% reduction of bacteria cells.

Aspergillus fumigatus biofilms represent a problematic clinical entity, especially because of their recalcitrance to antifungal drugs, which poses a number of therapeutic implications for invasive aspergillosis, the most difficult-to-treat Aspergillus -related disease. While the antibiofilm activities of amphotericin B (AMB) deoxycholate and its lipid formulations (e.g., liposomal AMB [LAMB]) are well documented, the effectiveness of these drugs in combination with nonantifungal agents is poorly understood. In the present study, in vitro interactions between polyene antifungals (AMB and LAMB) and alginate lyase (AlgL), an enzyme degrading the polysaccharides produced as extracellular polymeric substances (EPSs) within the biofilm matrix, against A. fumigatus biofilms were evaluated by using the checkerboard microdilution and the time-kill assays. Furthermore, atomic force microscopy (AFM) was used to image and quantify the effects of AlgL-antifungal combinations on biofilm-growing hyphal cells. On the basis of fractional inhibitory concentration index values, synergy was found between both AMB formulations and AlgL, and this finding was also confirmed by the time-kill test. Finally, AFM analysis showed that when A. fumigatus biofilms were treated with AlgL or polyene alone, as well as with their combination, both a reduction of hyphal thicknesses and an increase of adhesive forces were observed compared to the findings for untreated controls, probably owing to the different action by the enzyme or the antifungal compounds. Interestingly, marked physical changes were noticed in A. fumigatus biofilms exposed to the AlgL-antifungal combinations compared with the physical characteristics detected after exposure to the antifungals alone, indicating that AlgL may enhance the antibiofilm activity of both AMB and LAMB, perhaps by disrupting the hypha-embedding EPSs and thus facilitating the drugs to reach biofilm cells. Taken together, our results suggest that a combination of AlgL and a polyene antifungal may prove to be a new therapeutic strategy for invasive aspergillosis, while reinforcing the EPS as a valuable antibiofilm drug target.

Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA- or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections.

Background: This study aims to evaluate, in patients on chronic hemodialysis (PHD), the levels of endotoxin through a chemiluminescent bioassay based on the oxidative burst reaction of activated neutrophils to complement coated LPS-IgM immune complexes and define the variables possibly correlated. Methods: In 61 PHD, we measured serum endotoxin activity (EA) with the Endotoxin Activity Assay (EAA™) and we defined the possible association with demographic, clinical and laboratory variables. Results: Mean serum EA was 0.43 ± 0.26 UI. EA was low (0.60) in 18 (29.5%). A significant exponential relationship was detected between EA and serum interleukin-6 (IL-6) levels (r = 0.871). At the multiple regression analysis, intermediate-high EA was directly associated only with serum IL-6 levels. In a second model of multiple regression analysis without the variable serum IL-6 levels, intermediate-high EA was directly associated with constipation and serum troponin levels and inversely associated with serum albumin and the monthly number of sevelamer tablets. Conclusions: A high percentage of PHD has intermediate or high EA. Intermediate-high EA is significantly associated with serum IL-6 levels.

Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.

In the modern view of selective drug delivery of bioactive molecules, the attention is moving onto the setup of the perfect carrier more than in the optimization of the active compound. In this respect, virus-like particles constitute bioinspired nanodevices with the intrinsic ability to transport a large class of molecules, ranging from smart drugs to small interfering RNAs. In this work, we demonstrate the efficacy of a novel construct obtained by fusing a self-assembling protein from the human Rotavirus A, VP6, with the Small Ubiquitin Modifier domain, which maintains the ability to form nanoparticles and nanotubes and is able to be used as a drug carrier, even without specific targeting epitopes. The high expression and purification yield, combined with low toxicity of the empty particles, clearly indicate a good candidate for future studies of selective drug delivery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2769, 2019.

Alpha-latrotoxin (alpha-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the alpha-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca(2+) uptake assay, we isolated a Fab fragment (FM1) able to bind the alpha-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since alpha-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.

Graphene and its derivatives are versatile materials promising candidates for important biomedical applications. Graphene is composed of sp2-hybridized carbon atoms hexagonally arranged in a two-dimensional structure, resulting in a large surface area on both sides of the planar axis. Among the materials of the graphene family, graphene oxide (GO), because of the formation of hydrogen bonds between polar functional groups on its surface and water molecules, is stable in solution and has more advantages for potential biomedical applications. However studies concerning antimicrobial effects of GO evidenced contradictory results and toxic and nontoxic effects were simultaneously observed. Goal of this work is the precise analysis of size, buffer and concentration dependence of GO antimicrobial effects on important human pathogens, such as E. Coli, C. Albicans, E. Faecalis and S. Aureus. Different sizes of GO nanosheets have been produced by sonication and characterized by Dynamic Light Scattering and Atomic Force Microscopy. Subsequently GO sheets having size comprised between 1 μm and 50 nm have been incubated with pathogens in ddH20, saline solution or in LB Broth, i.e. in different ionic strength conditions, to follow the growth kinetics in presence of GO. We analysed in detail how the GO aggregation influences experimental results and optimized an experimental protocol and analysis method. Moreover, since high GO concentration caused the formation of complexes between pathogens and GO nanosheets, we characterized the surface covering of the bacteria and the leakage of genetic material, membrane damaging and biofilm formation. These results allow the optimization GO-based nanomaterial design for a new “green” antimicrobial therapy.

Besides being part of anti- Helicobacter pylori treatment regimens, proton pump inhibitors (PPIs) are increasingly being used to treat dyspepsia. However, little is known about the effects of PPIs on the human gastric microbiota, especially those related to H. pylori infection. The goal of this study was to characterize the stomach microbial communities in patients with dyspepsia and to investigate their relationships with PPI use and H. pylori status. Using 16S rRNA gene pyrosequencing, we analyzed the mucosa-associated microbial populations of 24 patients, of whom 12 were treated with the PPI omeprazole and 9 (5 treated and 4 untreated) were positive for H. pylori infection. The Proteobacteria , Firmicutes , Bacteroidetes , Fusobacteria , and Actinobacteria phyla accounted for 98% of all of the sequences, with Helicobacter , Streptococcus , and Prevotella ranking among the 10 most abundant genera. H. pylori infection or PPI treatment did not significantly influence gastric microbial species composition in dyspeptic patients. Principal-coordinate analysis of weighted UniFrac distances in these communities revealed clear but significant separation according to H. pylori status only. However, in PPI-treated patients, Firmicutes , particularly Streptococcaceae , were significantly increased in relative abundance compared to those in untreated patients. Consistently, Streptococcus was also found to significantly increase in relation to PPI treatment, and this increase seemed to occur independently of H. pylori infection. Our results suggest that Streptococcus may be a key indicator of PPI-induced gastric microbial composition changes in dyspeptic patients. Whether the gastric microbiota alteration contributes to dyspepsia needs further investigation. IMPORTANCE Although PPIs have become a popular treatment choice, a growing number of dyspeptic patients may be treated unnecessarily. We found that patients treated with omeprazole showed gastric microbial communities that were different from those of untreated patients. These differences regarded the abundances of specific taxa. By understanding the relationships between PPIs and members of the gastric microbiota, it will be possible to envisage new strategies for better managing patients with dyspepsia.

We recently identified a novel family of macrocyclic amidinoureas showing potent antifungal activity against Candida spp. In this study, we demonstrate the fungicidal effect of these compounds as well as their killing activity in a dose-dependent manner. Transcriptional analysis data indicate that our molecules induce a significant change in the transcriptome involving ATP binding cassette (ABC) transporter genes. Notably, experiments against Candida albicans mutants lacking those genes showed resistance to the compound, suggesting the involvement of ABC transporters in the uptake or intracellular accumulation of the molecule. To probe the mode of action, we performed fluorescence microscopy experiments on fungal cells treated with an ad-hoc synthesized fluorescent derivative. Fluorescence microscopy images confirm the ability of the compound to cross the membrane and show a consistent accumulation within the cytoplasm. Finally, we provide data supporting the in vivo efficacy in a systemic infection murine model setup with a drug-resistant strain of C. albicans.

Background Mechanisms underlying the transition from commensalism to virulence in Enterococcus faecalis are not fully understood. We previously identified the enterococcal leucine-rich protein A (ElrA) as a virulence factor of E. faecalis. The elrA gene is part of an operon that comprises four other ORFs encoding putative surface proteins of unknown function. Results In this work, we compared the susceptibility to phagocytosis of three E. faecalis strains, including a wild-type (WT), a ΔelrA strain, and a strain overexpressing the whole elr operon in order to understand the role of this operon in E. faecalis virulence. While both WT and ΔelrA strains were efficiently phagocytized by RAW 264.7 mouse macrophages, the elr operon-overexpressing strain showed a decreased capability to be internalized by the phagocytic cells. Consistently, the strain overexpressing elr operon was less adherent to macrophages than the WT strain, suggesting that overexpression of the elr operon could confer E. faecalis with additional anti-adhesion properties. In addition, increased virulence of the elr operon-overexpressing strain was shown in a mouse peritonitis model. Conclusions Altogether, our results indicate that overexpression of the elr operon facilitates the E. faecalis escape from host immune defenses. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0448-y) contains supplementary material, which is available to authorized users.

156a Sunday, February 12, 2017 has been conducted. Despite substantial efforts, results are controversial and a framework clarifying all effects reported in literature against bacteria still has to be defined. In our work, we demonstrate that by modulating the stability of GO in solution, the antibacterial or growth enhancement effect can been controlled both on S. aureus and E. coli bacteria. We expose these two species to concentrations of GO ranging from 3 to 200 ug/ml, in different incubation conditions (ultrapure water, PBS, NaCl, MgCl2 ...

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for serious hospital infections worldwide and represents a global public health problem. Curcumin, the major constituent of turmeric, is effective against MRSA but at high and cytotoxic concentrations or in combination with antibiotics. The major issue in curcumin-based therapies is the poor solubility of this hydrophobic compound and its cytotoxicity at high doses. In this work, we describe the efficacy of GOCU, a composite made of curcumin and Graphene Oxide (GO) in the MRSA infections treatment. GO is a nanomaterial with a large surface area and high drug loading capacity. GO has also antibacterial properties due mainly to a mechanical cutting of the bacterial membranes. We investigated GOCU properties by means of Dynamic Light Scattering, Electron and Atomic Force Microscopy and Zeta Potential. We analysed growth curves and Minimal Inhibitory and Bactericidal concentrations (MIC and MBC), and cytotoxic effects on cells by MTT assay. We report the capacity of GO to support and stabilize curcumin molecules in water environment. Curcumin creates a corona around Graphene Oxide sheets and modifies surface charge towards more positive values. We demonstrate the efficacy of GOCU against MRSA at concentration down to 2m g/ml, while GO alone is effective only at concentrations higher than 100m g/ml. However, GOCU displays low toxicity on fibroblasts cells in respect to bare GO. The low MIC of GOCU against MRSA and absence of cytotoxicity open new applications of GOCU nanomaterial in the global health challenge of the antibiotic resistant infections treatment.

High proteolytic degradation and poor absorption through epithelial barriers are major challenges to successful oral delivery of therapeutics. Nanoparticle platforms can enhance drug stability and extend the residence time in gastrointestinal (GI) tract. However, drug delivery systems are often inactivated in acidic environment of stomach or suffer poor absorption from intestinal cells due to the mucus layer. To overcome these issues we developed a drug delivery system constituted by a protein construct made by a Rotavirus capsid protein (VP6) and the small ubiquitin-like modifier SUMO. This chimeric construct allows specificity towards intestinal cells, the Rotavirus natural target, combined by an enhanced stability given by the eukaryotic protein transporter SUMO. Furthermore SUMO can act as a molecular switch that facilitates import/export of its ligand to the nucleus, the hypersensitive subcellular site target of many cell killing therapies. In this paper we show that SUMO-VP6 constructs self-assembly into stable nanocarriers. SUMO-VP6 nanocarriers display ideal features for drug delivery: a small size and high monodispersity, a high stability in different pH conditions and a high uptake in the nuclear and cytoplasmic compartment of intestinal cells. These features make SUMO-VP6 nanocarriers a promising novel system for oral delivery of poorly soluble drugs.

Graphene and graphene oxide (GO) are capable of inducing stem cells differentiation into bone tissue with variable efficacy depending on reductive state of the material. Thus, modulation of osteogenic process and of bone mineral density distribution is theoretically possible by controlling the GO oxidative state. In this study, we laser-printed GO surfaces in order to obtain both a local photo-thermal GO reduction and the formation of nano-wrinkles along precise geometric pattern. Initially, after cells adhered on the surface, stem cells migrated and accumulated on the reduced and wrinkled surface. When the local density of the stem cells on the reduced stripes was high, cells started to proliferate and occupy the oxidized/flat area. The designed surfaces morphology guided stem cell orientation and the reduction accelerated differentiation. Furthermore the reduced sharp nano-wrinkles were able to enhance the GO antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), a common cause of prosthetic joints infections. This strategy can offer a revolution in present and future trends of scaffolds design for regenerative medicine.

We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001 , renamed pmvE (for p olyamine m etabolism and v irulence of E. faecalis ). PmvE shares strong homologies with N 1 -spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535- pmvE (V19/p3535- pmvE ), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N -acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis , likely through its involvement in the polyamine metabolism.

Francesca Bugli,1 Valeria Caprettini,2 Margherita Cacaci,1 Cecilia Martini,1 Francesco Paroni Sterbini,1 Riccardo Torelli,1 Stefano Della Longa,3 Massimiliano Papi,4 Valentina Palmieri,4 Bruno Giardina,5 Brunella Posteraro,1 Maurizio Sanguinetti,1 Alessandro Arcovito5 1Istituto di Microbiologia, Università Cattolica del Sacro Cuore, 2Dipartimento di Fisica, Sapienza Università di Roma, Rome, 3Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze della Vita e dell’Ambiente, Università dell’Aquila, L’Aquila, 4Istituto di Fisica, 5Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy Abstract: In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6sequence, VP6fused to thioredoxin, and VP6obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6protein. Moreover, the VP6protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6trimers,60nm spherical virus-like particles, or nanotubes a few micron long. This different quaternary organization of VP6shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures – opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, as well as represents a top-quality biomaterial to be further modified for biotechnological purposes. Keywords: Virus-like particles, protein-based nanotubes, SUMO fusion tag, human rotavirus vaccine

Clinical and experimental evidence indicates that the hepatitis C virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate for the development of an effective anti-HCV vaccine. Identification of the human epitopes that are conserved among isolates and are able to elicit protective antibodies would constitute a significant step forward. This work describes the mapping of the B-cell epitopes present on the surface of HCV/E2, as recognized by the immune system during infection, by the analysis of the reciprocal interactions of a panel of human recombinant Fabs derived from an HCV-infected patient. Three unrelated epitopes recognized by antibodies with no neutralization-of-binding (NOB) activity were identified; a fourth, major epitope was defined as a clustering of minor epitopes recognized by Fabs endowed with strong NOB activity.

Evidence from clinical and experimental studies indicates that hepatitis C virus E2 (HCV/E2) glycoprotein is the major target of a putatively protective immune response. However, even in the presence of a vigorous production of anti-HCV/E2 antibodies, reinfection can occur. Dissection of the human immune response against HCV/E2 indicated that blocking of binding of HCV/E2 to target cells [neutralization of binding (NOB) activity] varies widely among antibody clones. Moreover, in vivo, simultaneous binding of antibodies to distinct epitopes can induce conformational changes and synergies that may be relevant to understanding the anti-HCV immune response. In this study, human recombinant Fabs were generated by affinity-selecting a phage display repertoire library with antibody-coated HCV/E2. These Fabs, which share the same complementarity-determining region DNA sequences, had higher affinity than other anti-HCV/E2 Fabs but showed no NOB activity even at the highest concentrations. Binding of Fabs to HCV/E2 caused conformational changes modifying Fab-binding patterns and reducing, with a negative synergistic effect, Fab-mediated NOB activity. These data suggest that some antibody clones have the potential to modify HCV/E2 conformation and that, in this state, binding of this glycoprotein to its cellular target is less prone to inhibition by some antibody clones. This can explain why high anti-HCV/E2 antibody titers do not directly correlate with protection from infection. Information on the interactions among different antibody clones can contribute to understanding virus–host interplay and developing more effective vaccines.

The increased incidence of invasive candidiasis and of patients at risk requires early diagnosis and treatment to improve prognosis and survival. The aim of this study was to set up a ten-protein array-based immunoassay to assess the IgG antibody responses against ten well-known immunogenic C. albicans proteins (Bgl2, Eno1, Pgk1, Pdc11, Fba1, Adh1, Als3, Hwp1, Hsp90 and Grp2) in 51 patients with invasive candidiasis (IC) and in 38 culture-negative controls (non-IC). Antibody levels were higher against Bgl2, Eno1, Pgk1, Als3, Hwp1 and Grp2, than against Adh1, Pdc11, Fba1 and Hsp90, irrespectively of the patient group considered. Moreover, the IgG levels against Bgl2, Eno1, Pgk1 and Grp2 were significantly higher in IC than in non-IC patients. Furthermore, the ROC curves generated by the analysis of the antibody responses against Bgl2, Grp2 and Pgk1 displayed AUC values above 0.7, thus discriminating IC and non-IC patients. According to these results, the employment of the microarray immunoassay (a rapid, sensitive and multiparametric system), in parallel with conventional diagnostics, can help to spot IC patients. This ultimately will allow to initiate an early, focused and optimized antifungal therapy.

Gliotoxin (GT) belongs to the epipolythiodioxopiperazine class of toxins secreted from certain fungi including Aspergillus fumigatus, which is the most prolific producer of this secondary metabolite. Recently, enhanced amounts of GT were found in in vitro biofilm-grown A. fumigatus mycelium. To further correlate the A. fumigatus biofilm growth phenotype with the enhanced secretion of GT, a polyclonal antibody (pAb) was produced by immunizing mice against GT. By an indirect immunofluorescent assay, pAb was then able to recognize specifically GT onto A. fumigatus Af293 biofilm formed on human pulmonary epithelial cells. Then, treating Af293 biofilms with a compound which reduces the GT disulfide bonds provoked shutdown of the GT-specific immunofluorescence (IF) signals along the hyphae. To explore the potential of GT for diagnostic use, pAb was shown to react with GT on hyphae into Aspergillus culture-positive respiratory tract specimens from patients with probable invasive aspergillosis (IA) and into tissue specimens from the lungs of patients with proven IA. As the presence of fungal hyphae in clinical specimens strongly indicates the in vivo A. fumigatus growth as a biofilm, anti-GT antibodies could be a specific and sensitive diagnostic tool for detecting A. fumigatus biofilm-associated clinical infections.