Growth ability, carbon source utilization and biochemical features of the new specie Zalaria obscura.

This research investigated the characteristics of Zalaria obscura LS31012019 in terms of growth ability in different media (SDB, YPD and TSB) and temperatures (22, 25 and 37 °C), utilization of several carbon sources (Glucose, Fructose, Lactose, Sucrose, Xylose, Glycerol and Mannitol at 5, 2 and 1%) and several biochemical features (total protein content, Glutathione, pigments), in comparison with those of the phylogenetically related Aureobasidium pullulans ATCC 15233. The best growth of Z. obscura LS31012019 was obtained in YPD at 25 °C with the highest OD value (0.45) after 144 h of incubation, similar to that of A. pullulans ATCC 15233 (0.48). Glucose resulted the preferred carbon source for both the considered yeasts but also sucrose resulted in efficacy supporting the growth of Z. obscura LS31012019 and A. pullulans ATCC 15233, for their ability in converting sucrose to glucose and fructose and the latter into glucose. Interestingly, Z. obscura LS31012019 utilized also glycerol and mannitol. The biochemical analysis showed the similarity of protein profile in Z. obscura LS31012019 and A. pullulans ATCC 15233 (from 90 to 20 kDa) and a reduced GSH content (0.321 and 0.233 µmol/mg). The pigments extraction with hexane generated a yellow oleaginous pellet in both the strains, while a yellow solid matrix more intensely coloured in A. pullulans ATTC 15233 was visible with the following solvent extractions. Overall, our data showed that Z. obscura LS31012019 can grow in different media and temperatures and utilize carbon sources apart from glucose and sucrose, shifting to a non-fermentative metabolism. These results improve the information regarding the characteristics of Z. obscura, opening a new field of investigation for the possible application of new species of black yeasts in human application.

Marine bisindole alkaloid 2,2-bis(6-bromo-3-indolyl)ethylamine to control and prevent fungal growth on building material: a potential antifungal agent.

The potential antifungal activity of the marine alkaloid 2,2-bis(6-bromo-3-indolyl)ethylamine (URB 1204) was firstly assessed by minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) against different fungi. Then, URB 1204 was applied to a building material experimentally contaminated with selected fungi, in single and mixed species, for determining its potential application in preventing fungal growth. In addition, the over-time protection efficacy of URB 1204 was verified, subjecting the treated building surfaces to natural fungal contamination for 6 weeks. URB 1204 showed different antifungal activity, with the lowest MIC value (16 µg/mL) observed against Aspergillus flavus IDRA01, Cladosporium cladosporioides ATCC 16022 and Mucor circinelloides EHS03, and the highest MIC (128 µg/mL) against the dermatophytes strains. The growth Alternaria alternata BC01, Penicillium citrinum LS1, and C. cladosporioides ATCC 16022 on building material treated with URB 1204 water solution (64 µg/mL) was remarkably reduced with an effect time-dependent and related to the examined fungi. In terms of over-time efficacy, the samples treated with URB 1204 showed a delay of fungal growth comparable with that of a commercial antifungal product. These findings evidenced not only the ability of 2,2-bis(6-bromo-3-indolyl)ethylamine to limit the growth of different fungal species on building material but also to provide long-term protection against mold growth and proliferation, opening new perspectives for URB 1204 as preventive agent.

Inactivation of pathogenic dermatophytes by ultraviolet irradiation in swimming pool thermal water.

The effect of ultraviolet (UV) radiation from low-pressure mercury lamp against some pathogenic dermatophytes species such as Epidermophyton floccosum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton tonsurans and Trichophyton violaceum suspended in thermal water was evaluated in laboratory-scale condition at various times. The main results showed that within 120 s of exposure, all species of dermatophytes are completely inactivated, which was evidenced by the absence of fungal regrowth, while after 60 s only T. tonsurans was recovered, with a reduction of 3.28 log. Shorter exposure times were not enough to completely inactivate all dermatophytes species. The samples treated with UV radiation for 120 s did not give evidence of fungal regrowth indicating that this disinfectant action is persistent over time. In conclusion, UV radiation can be proposed to reduce the risk of infection by dermatophytes eventually present in swimming pools that use thermal water.

An atypical, pigment-producing Metschnikowia strain from a leukaemia patient.

A yeast strain was isolated from the sputum sample of a leukaemia patient in the Spirito Santo Hospital of Pescara, Italy. The fungus produced a pigment that formed a reddish halo around colonies, and was identified and deposited as a Metschnikowia spp. (accession number IHEM 25107-GenBank accession number JQ921016) in the BCCM/IHEM collection of biomedical fungi and yeasts (Bruxelles, Belgium). Although the physiology of the strain was close to that of Metschnikowia sinensis, the D1/D2 sequence did not correspond to any previously described Metschnikowia species. Phylogeny of the genus Metschnikowia is complex and requires far more analysis. We present the first non-M. pulcherrima Metschnikowia spp. isolate recovered from a human, and emphasize the role of man as a transient carrier of environmental yeasts, the pathogenicity of which still needs to be defined.

Synthesis and Biological Evaluation of 6-O-Sucrose Monoester Glycolipids as Possible New Antifungal Agents.

A small library of 6-O-sucrose monoester surfactants has been synthesized and tested against various microorganisms. The synthetic procedure involved a modified Mitsunobu reaction, which showed improved results compared to those present in the literature (higher yields and larger scope). The antifungal activities of most of these glycolipids were satisfactory. In particular, sucrose palmitoleate (URB1537) showed good activity against Candida albicans ATCC 10231, Fusarium spp., and Aspergillus fumigatus IDRAH01 (MIC value: 16, 32, 64 µg/mL, respectively), and was further characterized through radical scavenging, anti-inflammatory, and biocompatibility tests. URB1537 has been shown to control the inflammatory response and to have a safe profile.

Sugar-Based Monoester Surfactants: Synthetic Methodologies, Properties, and Biological Activities.

Glycolipids are biocompatible and biodegradable amphiphilic compounds characterized by a great scientific interest for their potential applications in various technological areas, including pharmaceuticals, cosmetics, agriculture, and food production. This report summarizes the available synthetic methodologies, physicochemical properties, and biological activity of sugar fatty acid ester surfactants, with a particular focus on 6-O-glucose, 6-O-mannose, 6-O-sucrose, and 6'-O-lactose ones. In detail, the synthetic approaches to this class of compounds, such as enzymatic lipase-catalyzed and traditional chemical (e.g., acyl chloride, Steglich, Mitsunobu) esterifications, are reported. Moreover, aspects related to the surface activity of these amphiphiles, such as their ability to decrease surface tension, critical micelle concentration, and emulsifying and foaming ability, are described. Biological applications with a focus on the permeability-enhancing effect across the skin or mucosa, antimicrobial and antifungal activities, as well as antibiofilm properties, are also presented. The information reported here on sugar-based ester surfactants is helpful to broaden the interest and the possible innovative applications of this class of amphiphiles in different technological fields in the future.

Disinfection efficacy of chlorine and peracetic acid alone or in combination against Aspergillus spp. and Candida albicans in drinking water.

The aim of the present study was to evaluate the fungicidal activity of chlorine and peracetic acid in drinking water against various pathogenic Aspergillus spp. and Candida albicans strains. A. nidulans exhibited the greatest resistance, requiring 10 ppm of chlorine for 30 min contact time for a complete inactivation. Under the same experimental conditions, peracetic acid was even less fungicidal. In this case, A. niger proved to be the most resistant species (50 ppm for 60 min for complete inactivation). All Aspergillus spp. were insensitive to 10 ppm even with extended exposure (>5 h). The combination of chlorine and peracetic acid against Aspergillus spp. did not show synergistic effects except in the case of A. flavus. Complete growth inhibition of C. albicans was observed after about 3 h contact time with 0.2 ppm. C. albicans was less sensitive to peracetic acid. Hence the concentrations of chlorine that are usually present in drinking water distribution systems are ineffective against several Aspergillus spp. and peracetic acid cannot be considered an alternative to chlorine for disinfecting drinking water. The combination of the two biocides is not very effective in eliminating filamentous fungi at the concentrations permitted for drinking water disinfection.

Role of melanin in the black yeast fungi Aureobasidium pullulans and Zalaria obscura in promoting tolerance to environmental stresses and to antimicrobial compounds.

The role of melanin in Aureobasidium pullulans ATCC 15233 and Zalaria obscura LS31012019, under simulated osmotic, oxidative, and high temperature stress conditions, on the susceptibility to essential oils (EOs) or antifungals and on the resistance to UV-C radiation was investigated. 93.6% of melanized A. pullulans and 92% of Z. obscura survived to 40 °C for 1 h compared to 77% and 76% of the non-melanized ones, while both yeasts tolerated a high concentration of NaCl (up to 30%) and H2O2 (up to 400 mM) regardless of melanin production. Higher EOs antifungal efficacy was observed in non-melanized cells (growth inhibition zone >30 mm) compared to the melanized ones (25 mm). Similarly, the lowest Minimum Inhibitory Concentrations (MIC) and Minimum Fungicidal Concentration (MFC) values were evidenced for Fluconazole, Clotrimazole, Bifonazole and Amphotericin in the non-melanized fungi. Increasing UV-C intensity (up to 2004.5 J/m2) caused total death in the non-melanized strains compared to about 30% growth reduction in the melanized ones. The results of this investigation, the first focused on the biological role of melanin in "black-fungi", are novel and encourage a better understanding of the biochemical features of melanin in the environmental adaptive ability of the new species Z. obscura.

Synthesis and Biological Characterization of the New Glycolipid Lactose Undecylenate (URB1418).

As a follow-up to our previous studies on glycolipid surfactants, a new molecule, that is lactose 6'-O-undecylenate (URB1418), was investigated. To this end, a practical synthesis and studies aimed at exploring its specific properties were carried out. URB1418 showed antifungal activities against Trichophyton rubrum F2 and Candida albicans ATCC 10231 (MIC 512 µg/mL) and no significant antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. At the same time, it presented anti-inflammatory properties, as documented by the dose-dependent reduction in LPS-induced NO release in RAW 264.7 cells, while a low antioxidant capacity in the range of concentrations tested (EC50 > 200 µM) was also observed. Moreover, URB1418 offers the advantage of being more stable than the reference polyunsaturated lactose esters and of being synthesized using a "green" procedure, involving an enzymatic method, high yield and low manufacturing cost. For all these reasons and the absence of toxicity (HaCaT cells), the new glycolipid presented herein could be considered an interesting compound for applications in various fields.

A combination of sugar esters and chitosan to promote in vivo wound care.

In recent years, researchers are exploring innovative green materials fabricated from renewable natural substances to meet formulation needs. Among them, biopolymers like chitosans and biosurfactants such as sugar fatty acid esters are of potential interest due to their biocompatibility, biodegradability, functionality, and cost-effectiveness. Both classes of biocompounds possess the ability to be efficiently employed in wound dressing to help physiological wound healing, which is a bioprocess involving uncontrolled oxidative damage and inflammation, with an associated high risk of infection. In this work, we synthesized two different sugar esters (i.e., lactose linoleate and lactose linolenate) that, in combination with chitosan and sucrose laurate, were evaluated in vitro for their cytocompatibility, anti-inflammatory, antioxidant, and antibacterial activities and in vivo as wound care agents. Emphasis on Wnt/ß-catenin associated machineries was also set. The newly designed lactose esters, sucrose ester, and chitosan possessed sole biological attributes, entailing considerable blending for convenient formulation of wound care products. In particular, the mixture composed of sucrose laurate (200 µM), lactose linoleate (100 µM), and chitosan (1%) assured its superiority in terms of efficient wound healing prospects in vivo together with the restoring of the Wnt/ß-catenin signaling pathway, compared with the marketed wound healing product (Healosol®), and single components as well. This innovative combination of biomaterials applied as wound dressing could effectively break new ground in skin wound care.

Microbiological validation of a robot for the sterile compounding of injectable non-hazardous medications in a hospital environment.

Objectives: To design and execute a comprehensive microbiological validation protocol to assess a brand-new sterile compounding robot in a hospital pharmacy environment, according to ISO and EU GMP standards. Methods: Qualification of the Class-A inner environment of the robot was performed through microbial air and surface quality assessment utilising contact plates, swabs and particulate matter monitoring. To evaluate the effectiveness of the microbial decontamination process (UV rays) challenge test against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis spores and Candida albicans was used. The challenge Media Fill test was used to validate the aseptic processing. Results: After 3 hours, no microorganisms retained viability. Monitoring inside the equipment evidenced complete absence of microorganisms. The Media Fill test was always negative. Conclusions: According to our results, the APOTECAunit meets the requirements for advanced aseptic processing in the hospital pharmacies and the pharmaceutical industry in general, providing advantages in terms of safety for patients compared with conventional procedures of parenteral preparation production. The protocol has demonstrated to be a comprehensive and valuable tool in validating, from a microbial point of view, a sterile-compounding technology. This study might represent an important benchmark in developing a contamination control strategy, as required, for example, in the Performance Qualification of the GMP in the case of drug manufacturing.

Evaluation of fungal community involved in the bioderioration process of wooden artworks and canvases in Montefeltro area (Marche, Italy).

Microbiological monitoring represents one of the most useful methods to assess potential risks related to the integrity of cultural heritage. The objective of this work was to evaluate the fungal community prevalent in 64 different artworks of Montefeltro area (Marche, central Italy). A total of 293 swabs were collected and, among these, 2.3% resulted negative to cultural method, while 87.7% were positive for the presence of filamentous fungi, yeasts and Actinomycetes. Totally, 11 genera and 12 different species were recovered from Sabouraud Dextrose Agar (SDA), Czapek Dox Agar (CDA), Malt Extract Agar (MEA), including 73 strains of Mycelia sterilia. Penicillium spp. was isolated mostly from canvases (28.8%), while Aspergillus spp. was most present in wooden artworks (25.3%). SDA was the best performing medium with 57.1% of isolations, followed by CDA and MEA with percentages of 24.8 and 18.1% respectively. This study could be useful to better understand the microorganism-related phenomena in cultural heritage of Marche region, identifying the potential risks and defining preventive protecting such as climate control, frequent cleaning and environmental monitoring.

Disinfection of Mycobacterium avium subspecies hominissuis in drinking tap water using ultraviolet germicidal irradiation.

Nontuberculous mycobacteria are resistant to conventional water treatments, and are opportunistic human pathogen, particularly in hospitalized patients. The aim of this investigation was to assess the effectiveness of an ultraviolet UV-C lamp treatment against Mycobacterium avium subspecies hominissuis in drinking tap water. Ultraviolet treatments (0-192 mJ/cm2) were performed using UV lamp immerged onto cylindrical glass tubes containing artificially contaminated water. The results showed that susceptibility to UV varied considerably according to the strains and the diameter of the tube. With a dose of 32 mJ/cm2, a significant inactivation (p < .05) of 3 log (99.9%) or more was obtained in only 5 of the 14 strains. To obtain a complete inactivation of all strains an irradiation of 192 mJ/cm2 was needed, a dose that is much higher than the limits recommended by the international standards for UV disinfection of drinking water. In conclusion, it may be difficult to standardize a UV dose for the elimination of waterborne mycobacteria.

Activity of Brassica oleracea leaf juice on foodborne pathogenic bacteria.

Many vegetables of the Cruciferae family have been found to possess antimicrobial properties against several microorganisms of clinical importance. In this study, we reported the antibacterial effect of Brassica oleracea juice on several food-borne pathogens. The juice was found to be effective in inhibiting the growth of Salmonella Enteritidis, verotoxigenic Escherichia coli O157:H7, E. coli HB producing thermolabile toxin, nontoxigenic E. coli, and Listeria monocytogenes, but not Enterococcus faecalis. All cauliflower cultivars tested suppressed bacterial growth in a dose-dependent manner after 5 h of treatments, and the reduction in the number of viable cells ranged from 1 log with a 10% juice concentration to more than 3 log with a 20% juice concentration. The foodborne bacteria tested were also markedly reduced by isothiocyanates, natural components abundant in the genus Brassica, indicating that glucosinolate-derived isothiocyanates can play a major role in the antimicrobial activity of cauliflower. The antimicrobial effect of juice was reduced in presence of cysteine, suggesting that one mechanism of action of the juice involves blocking bacterial sulfhydryl groups.

Isolation of Escherichia coli O157 from human and bovine faeces in the Urbino area, Italy.

We examined 476 faecal samples from subjects aged from 0 to >60 years, 283 with diarrhoea and 193 with illnesses involving other sites or clinically healthy, and 154 samples of faeces of healthy cattle, in order to define the diffusion of E. coli O157 in the Urbino area. The samples were seeded by both direct streaking onto cefixime tellurite sorbitol Mac Conkey agar (CT-SMAC) and previous enrichment in cefixime tellurite tryptone soya broth for human specimens and in cefixime vancomicin tryptone soya broth for bovine samples. The strains of E. coli O157 were characterized by verocytotoxin and adhesin eae genes detection. We isolated one strain of E. coli O157 (0.2%) from a man 68 year old who had bloody diarrhoea, and one strain (0.64%) from a weaned calf. Both isolates carried the adhesin eae gene, but only the bovine strain was VT2+. The study shows a low diffusion of E. coli O157 in the Urbino area, confirming the epidemiological data on the national territory.

Antifungal properties of the human Metschnikowia strain IHEM 25107.

Metschnikowiaceae may show natural, strain-dependent antifungal properties, so they are employed in agriculture as natural, safe alternatives to pesticides. With this paper, we documented the ability of the recently described Metschnikowia IHEM 25107 to inhibit growth of several molds, including diverse Aspergillus species and dermatophytes. This biocontrol activity enables pulcherrimin-producing strains to naturally antagonize competing microorganisms.

Swimming pools and fungi: an environmental epidemiology survey in Italian indoor swimming facilities.

A growing number of people attend swimming facilities for recreational activities, rehabilitative treatments, or sport. Filamentous fungi and yeast can be isolated from contaminated air, water and surfaces and may represent a biological risk for employees and users. Here we investigated the occurrence of mycotic species, in a sample of Italian swimming pools (n = 10). Detection and identification of isolated species were achieved by cultural and morphological methods. Results revealed moderate mycotic titres and a high biodiversity. Penicillium spp., Aspergillus spp., Cladosporium spp. and Alternaria sp., were constantly detected in air and surfaces sampled by the swimming area, while pathogenic yeast Candida albicans was never detected. Fusarium spp. was the most common taxon isolated from surfaces. For one facility, we typed the genotypic profiles and studied, by genetic typing, the spatial and temporal distribution of isolates. Phylogenetic relationships between species were analysed by alignment of small ribosomal subunit RNA sequences.