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The engineering of the surface of nanomaterials with bioactive molecules allows controlling their biological identity thus accessing functional materials with tuned physicochemical and biological profiles suited for specific applications. Then, the manufacturing process, by which the nanomaterial surface is grafted, has a significant impact on their development and innovation. In this regard, we report herein the grafting of sugar headgroups on a graphene oxide (GO) surface by exploiting a green manufacturing process that relies on the use of vibrational ball mills, a grinding apparatus in which the energy is transferred to the reacting species through collision with agate spheres inside a closed and vibrating vessel. The chemical composition and the morphology of the resulting glyco-graphene oxide conjugates (glyco-GO) are assessed by the combination of a series of complementary advanced techniques (

The aim of this systematic review is to collect clinical trials conducted using essential oils (EOs) in obstetric symptoms by evaluating if and in which context the aromatherapy practice is effective in obstetrics. The research was conducted by using the databases of EMBASE, Medline, Biosis and Toxcenter, PubMed, and Google Scholar search engine, selecting articles from January 2004 to July 2020. This study was performed according to the MOOSE and PRISMA guidelines. Only the randomized clinical trials were considered, and in cases of multiple publications, it was considered the most up to date information. Biases were highlighted. In the presence of homogeneous data, pooling statistics and meta-analysis were applied. The research led to 71 articles, 17 of which were eligible. Among the trials selected, eight investigated the effectiveness of EOs on anxiety, depression, and stress. Two concerned the treatment of nausea and vomiting, six evaluated the application of EOs on labor for pain treatment, and two showed the effectiveness in the treatment of episiotomy. The heterogeneity of works carried out so far has made it possible to develop a meta-analysis only in the field of pain treatment during childbirth, identifying the effectiveness of the EOs Lavandula spp. and Rosa damascena.

Antibiotic resistance is a serious health and social problem that will have a substantial impact in the coming years on the world health and economy. Thus, the increasing demand for innovative antibiotics, has prompted many researchers in the medical, microbiological, and biochemical fields to exploit the properties of antimicrobial peptides (AMPs). When properly used, designed, and conveyed, AMPs can really represent a valid alternative to conventional drugs especially in situations that are particularly difficult to treat such as chronic infections found in Cystic Fibrosis (CF) patients. In this review we focused on the applications of AMPs in the specific field of CF, illustrating different types of peptides from natural, naturally modified, synthetic as well as the different strategies used to overcome the barriers, and the physiological conditions in which AMPs must operate.

Sugar headgroups have been grafted on the surface of graphene oxide (GO) by exploiting vibrational ball mills. The resulting glyco-GO conjugates significantly disrupt an already established P. aeruginosa biofilm.

This letter takes a cue from a recently published study, which looked at diagnostic tests currently available for SARS-CoV-2 infection, to present and discuss the results of a re-evaluation of 122 positive serum samples from COVID-19 patients using an in-house serological assay for SARSCoV-2 antibodies detection. While serology targeting the SARS-CoV-2 S protein should be preferable, IgA testing is proposed in all situations for which serology is the solely practicable diagnostic strategy for SARSCoV-2 infection.

Glycerol (Gly) can be dissimilated by two pathways in bacteria. Either this sugar alcohol is first oxidized to dihydroxyacetone (DHA) and then phosphorylated or it is first phosphorylated to glycerol-3-phosphate (GlyP) followed by oxidation. Oxidation of GlyP can be achieved by NAD-dependent dehydrogenases or by a GlyP oxidase. In both cases, dihydroxyacetone phosphate is the product. Genomic analysis showed that Enterococcus faecium harbors numerous genes annotated to encode activities for the two pathways. However, our physiological analyses of growth on glycerol showed that dissimilation is limited to aerobic conditions and that despite the presence of genes encoding presumed GlyP dehydrogenases, the GlyP oxidase is essential in this process. Although E. faecium contains an operon encoding the phosphotransfer protein DhaM and DHA kinase, which are required for DHA phosphorylation, it is unable to grow on DHA. This operon is highly expressed in stationary phase but its physiological role remains unknown. Finally, data obtained from sequencing of a transposon mutant bank of E. faecium grown on BHI revealed that the GlyP dehydrogenases and a major intrinsic family protein have important but hitherto unknown physiological functions.

Background Hospitalized patients with COVID-19 admitted to the intensive care unit (ICU) and requiring mechanical ventilation are at risk of ventilator-associated bacterial infections secondary to SARS-CoV-2 infection. Our study aimed to investigate clinical features of Staphylococcus aureus ventilator-associated pneumonia (SA-VAP) and, if bronchoalveolar lavage samples were available, lung bacterial community features in ICU patients with or without COVID-19. Methods We prospectively included hospitalized patients with COVID-19 across two medical ICUs of the Fondazione Policlinico Universitario A. Gemelli IRCCS (Rome, Italy), who developed SA-VAP between 20 March 2020 and 30 October 2020 (thereafter referred to as cases). After 1:2 matching based on the simplified acute physiology score II (SAPS II) and the sequential organ failure assessment (SOFA) score, cases were compared with SA-VAP patients without COVID-19 (controls). Clinical, microbiological, and lung microbiota data were analyzed. Results We studied two groups of patients (40 COVID-19 and 80 non-COVID-19). COVID-19 patients had a higher rate of late-onset (87.5% versus 63.8%; p = 0.01), methicillin-resistant (65.0% vs 27.5%; p p p = 0.12), clinical cure (p = 0.20) and microbiological eradication (p = 0.31). On multivariable logistic regression analysis, SAPS II and initial inappropriate antimicrobial therapy were independently associated with ICU mortality. Then, lung microbiota characterization in 10 COVID-19 and 16 non-COVID-19 patients revealed that the overall microbial community composition was significantly different between the patient groups (unweighted UniFrac distance, R2 0.15349; p p S. aureus (log2 fold change, 29.5), Streptococcus anginosus subspecies anginosus (log2 fold change, 24.9), and Olsenella (log2 fold change, 25.7) were significantly enriched in the COVID-19 group compared to the non–COVID-19 group of SA-VAP patients. Conclusions In our study population, COVID-19 seemed to significantly affect microbiological and clinical features of SA-VAP as well as to be associated with a peculiar lung microbiota composition.

The development of new pharmacological strategies that evade bacterial resistance has become a compelling worldwide challenge. Graphene oxide (GO) can represent the nanotechnology answer being economical and easy to produce and to degrade and having multitarget specificity against bacteria. Several groups tried to define the interaction between GO sheets and human pathogens. Unfortunately, controversial results from inhibition to bacterial growth enhancement have been reported. The main difference among all experimental evidence relies on the environmental conditions adopted to study the bacteria-GO interaction. Indeed GO, stable in deionized water, undergoes a rapid and salt-specific DLVO-like aggregation that influences antimicrobial effects. Considering this phenomenon, the interaction of bacteria with GO aggregates having different sizes, morphologies, and surface potential can create a complex scenario that explains the contrasting results reported so far. In this article, we demonstrate that by modulating the GO stability in solution, the antibacterial or growth enhancement effect can be controlled on S. aureus and E. coli. GO at low concentration cuts microorganism membranes and at high concentration forms complexes with pathogens and inhibits or enhances bacterial growth in a surface potential-dependent manner. With the framework defined in this study, the clinical application of GO gets closer, and controversial results in literature can be explained.

The custom functionalization of a graphene surface allows access to engineered nanomaterials with improved colloidal stability and tailored specific properties, which are available to be employed in a wide range of applications ranging from materials to life science. The high surface area and their intrinsic physical and biological properties make reduced graphene oxide and graphene oxide unique materials for the custom functionalization with bioactive molecules by exploiting different surface chemistries. In this work, preparation (on the gram scale) of reduced graphene oxide and graphene oxide derivatives functionalized with the well-known antibacterial agent salicylic acid is reported. The salicylic acid functionalities offered a stable colloidal dispersion and, in addition, homogeneous absorption on a sample of textile manufacture (i.e., cotton fabrics), as shown by a Raman spectroscopy study, thus providing nanoengineered materials with significant antibacterial activity toward different strains of microorganisms. Surprisingly, graphene surface functionalization also ensured resistance to detergent washing treatments as verified on a model system using the quartz crystal microbalance technique. Therefore, our findings paved the way for the development of antibacterial additives for cotton fabrics in the absence of metal components, thus limiting undesirable side effects.

Despite the advent of echinocandins and newer triazoles (voriconazole, posaconazole, and isavuconazole) (1), fluconazole (FLZ) remains an important component of today’s antifungal arsenal, particularly for treatment of Candida infections (2, 3).…

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.

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.

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.

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 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.

Bactericidal antibiotics, including β-lactams, have been proposed to induce perturbations of cellular physiology, including respiration, leading to the formation of reactive oxygen species (ROS). Although first widely accepted, recent contrasting data, obtained mainly in Escherichia coli, have challenged the model. Here we show that β-lactams trigger a strong increase in ROS production leading to hydrogen peroxide accumulation in Enterococcus faecalis cultures under aerobic but not under anaerobic conditions. Interestingly, both amoxicillin for which the bacterium is susceptible and cefotaxime for which it is resistant triggered this response. ROS formation depends mainly on demethylmenaquinone, a component of its respiratory chain but, in contrast to E. coli, is observed only in the absence of aerobic or non-oxidative respiration. Moreover, the possibility that the E. faecalis increased ROS production might provide a mechanistical explanation for the higher incidence of colorectal cancers in patients frequently treated with β-lactams is discussed.

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.

The role of the veterinarian as a public health officer is intrinsic to the history and the culture of veterinary organization in Italy. The Veterinary service being part of the Health administration since the birth of the Italian State in the XIX Century. In the second half of the last century the birth of the Italian National Health Service confirmed that the function of the Italian veterinary service was to analyze and reduce the risks for the human population connected to the relationship man-animal-environment, animal health, food safety and security. The Italian Veterinary Medicine School curricula, reflected this "model" of veterinarian as well. In the majority of countries in the world, Veterinary Services are organized within the Agriculture Administration with the main function to assure animal health and wellbeing. After the so-called "Mad-cow crisis" the awareness of the direct and essential role of veterinary services in the prevention of human illness has been officially recognized and in the third millennium the old concept of "one health" and "human-animal interface" has gained popularity worldwide.The concept of Veterinary Public Health, has evolved at International level and has incorporated the more than a century old vision of the Italian Veterinary medicine and it is defined as "the sum of the contributions to the physical, mental and social development of people through the knowledge and application of veterinary science" (WHO, Future trends in veterinary public health. Gruppo di lavoro OMS: TE, Italy, 1999, Available from: http://www.who.int/zoonoses/vph/en/ . Last visited 16 Feb 2016, 1999).On the subject of Cooperation, Sustainability and Public Health, the EXPO 2015 event and the activities of international organizations WHO, FAO and World Organization for Animal Health are refocusing at present their worldwide mandate to protect human health and the economy of both the poorest Countries and the developed countries, according to the "new" concept of Veterinary Public Health.Focus of Italian Veterinary Services activity is connected to research, diagnosis and epidemiological analysis of infectious diseases, including zoonosis, food safety as well as food security.

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.

Enterococcus faecium has emerged as a major opportunistic pathogen for 2 decades with the spread of hospital-adapted multidrug-resistant clones. As members of the intestinal microbiota, they are subjected to numerous bacterial stresses, including antibiotics at subinhibitory concentrations (SICs). Since fluoroquinolones are extensively prescribed, SICs are very likely to occur in vivo , with potential effects on bacterial metabolism with subsequent modulation of opportunistic traits. The aim of this study was to evaluate globally the impact of SICs of ciprofloxacin on antimicrobial resistance and pathogenicity of E. faecium . Transcriptomic analysis was performed by RNA sequencing (RNA-seq) (HiSeq 2500; Illumina) using the vanB -positive reference strain E. faecium Aus0004 in the absence or presence of ciprofloxacin SIC (0.38 mg/liter, i.e., 1/8 of the MIC). Several genetic and phenotypic tests were used for validation. In the presence of ciprofloxacin SIC, 196 genes were significantly induced, whereas 286 genes were significantly repressed, meaning that 16.8% of the E. faecium genome was altered. Among upregulated genes, EFAU004_02294 (fold change, 14.3) encoded a protein (Qnr of E. faecium [EfmQnr]) homologue of Qnr proteins involved in quinolone resistance in Gram-negative bacilli. Its implication in intrinsic and adaptive fluoroquinolone (FQ) resistance in E. faecium was experimentally ascertained. Moreover, EFAU004_02292, coding for the collagen adhesin Acm, was also induced by the SIC of ciprofloxacin (fold change, 8.2), and higher adhesion capabilities were demonstrated phenotypically. Both EfmQnr and Acm determinants may play an important role in the transition from a commensal to a pathogenic state of E. faecium that resides in the gut of patients receiving fluoroquinolone therapy.

The role of the veterinarian as a public health officer is intrinsic to the history and the culture of veterinary organization in Italy. The Veterinary service being part of the Health administration since the birth of the Italian State in the XIX Century. In the second half of the last century the birth of the Italian National Health Service confirmed that the function of the Italian veterinary service was to analyze and reduce the risks for the human population connected to the relationship man-animal-environment, animal health, food safety and security. The Italian Veterinary Medicine School curricula, reflected this "model" of veterinarian as well. In the majority of countries in the world, Veterinary Services are organized within the Agriculture Administration with the main function to assure animal health and wellbeing. After the so-called "Mad-cow crisis" the awareness of the direct and essential role of veterinary services in the prevention of human illness has been officially recognized and in the third millennium the old concept of "one health" and "human-animal interface" has gained popularity worldwide.The concept of Veterinary Public Health, has evolved at International level and has incorporated the more than a century old vision of the Italian Veterinary medicine and it is defined as "the sum of the contributions to the physical, mental and social development of people through the knowledge and application of veterinary science" (WHO, Future trends in veterinary public health. Gruppo di lavoro OMS: TE, Italy, 1999, Available from: http://www.who.int/zoonoses/vph/en/ . Last visited 16 Feb 2016, 1999).On the subject of Cooperation, Sustainability and Public Health, the EXPO 2015 event and the activities of international organizations WHO, FAO and World Organization for Animal Health are refocusing at present their worldwide mandate to protect human health and the economy of both the poorest Countries and the developed countries, according to the "new" concept of Veterinary Public Health.Focus of Italian Veterinary Services activity is connected to research, diagnosis and epidemiological analysis of infectious diseases, including zoonosis, food safety as well as food security.

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.

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.

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

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.