A Multidisciplinary Approach in Examining the Susceptibility to Microbial Attack of Polyacrylic and Polyurethane Resins Used in Art Restoration.

The synthetic polymers used to protect artworks from deterioration process can be colonized by the fungi and bacteria responsible for the biodeterioration process. In this study, the susceptibility of synthetic polyacrylics and polyurethane resins to microorganisms (Aspergillus niger ATCC 9642, Aureobasidium pullulans ATCC 15233, Chaetomium globosum ATCC 6205, Cladosporium cladosporioides ATCC 16022, Alternaria alternata BC01, Penicillium citrinum LS1 and Pseudomonas aeruginosa ATCC 9027) was investigated. The microbial attack was simulated alone and with a biocide and the related growth was observed up to 21 days for bacteria and 28 days for fungi. The polyacrylic and polyurethane resins were subjected to microbial attack, regardless of the biocide treatment, with a fungal growth from 60% to the complete coverage of the plate surface. Penicillium citrinum showed the greatest adaptation ability and was found in all the examined resins. P. aeruginosa was visible in all the different resins, regardless of the presence of biocide. An environmental scanning electron microscope (ESEM) revealed the presence of fungal conidia and hyphae in the inoculated resins and the Fourier transform IR spectroscopy (FTIR-ATR) indicated chemical transformations in the IR spectra, particularly the hydrolysis of esters, with some differences between the polyacrylic and polyurethane resins, which were probably due to their different chemical features. Overall, our data stress that the chemical, physical and biological deterioration caused by microorganisms capable of degrading synthetic polymers is still a problem in art restoration and that new strategies must be considered to counteract this phenomenon.

Il Silenzio: The First Renaissance Oil Painting on Canvas from the Uffizi Museum Restored with a Safe, Green Antimicrobial Emulsion Based on Citrus aurantium var. amara Hydrolate and Cinnamomum zeylanicum Essential Oil.

Preserving artworks from the attacks of biodeteriogens is a primary duty of humanity. Nowadays, restorers use chemicals potentially dangerous for both artworks and human health. The purpose of this work was to find a green and safe formulation based on natural substances with fungicidal activity to restore ancient oil paintings, particularly "Il Silenzio" (by Jacopo Zucchi) preserved at the Uffizi Museum in Florence, Italy. The study was divided into two phases. First phase (in vitro study): three essential oils (EOs) and four hydrolates (Hys) were analysed by GC-mass spectrometry and in vitro tested against six ATCC strains of molds. An emulsion based on the more active natural compounds was tested on aged and unaged canvases samples to evaluate both their fungicidal activity and the impact on chemical-physical parameters. Finally, an in vivo toxicity test performed on the Galleria mellonella model assessed the safety for health. Second phase (in situ application): the emulsion was sprayed on the back of the painting and left to act for 24 h. Biodeteriogens present on the "Il Silenzio" painting were microbiologically identified before and after the treatment. The emulsion formulated with C. zeylanicum EO and C. aurantium var. amara Hy showed the best antifungal activity both in vitro and in situ without altering the chemical-physical characteristics of paintings. Furthermore, no in vivo toxicity was shown. For the first time, a green antimicrobial emulsion based on Hy and EO, safe for operators, was used to decontaminate an artwork colonised by fungi before the restoration practices.

Anti-Mold Effectiveness of a Green Emulsion Based on Citrus aurantium Hydrolate and Cinnamomum zeylanicum Essential Oil for the Modern Paintings Restoration.

A modern painting is characterized by multi-material bases extremely exposed to biodeteriogenic attacks. The aim of this work was to test the antifungal effectiveness of a natural, eco-friendly, and safe emulsion based on Citrus aurantium L. var. amara hydrolate and Cinnamomum zeylanicum Blume (from bark) essential oil, named "Zeylantium green emulsion" (Zege), on modern paintings. Colored unaged and aged canvas samples, performed with modern techniques (acrylic, vinylic and alkyd), were used to test in vitro both the antifungal effectiveness of Zege and its impact on the chemical-physical characteristics. Microbiological tests were performed according to the EUCAST international guidelines. pH measurements and colorimetric analysis were performed on unaged and aged canvases before and after Zege spray treatment. Finally, in situ tests were performed using the spray emulsion on canvas samples obtained from Ilaria Margutti's modern artwork, which had been colonized by molds. Microbiological tests on canvas prototypes showed a time- and dose-dependent effectiveness of the Zege spray. None of the techniques underwent relevant changes in pH. Only the acrylic colors were unaffected in the colorimetric analysis, among all colored unaged or aged canvases. Tests made with modern artwork samples confirmed the in situ antifungal effectiveness. The Zege spray showed encouraging results in regard to the use of this formulation in the restoration of modern paintings.

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.

Isolation and molecular identification of a strain belonging to the new species Zalaria obscura from a deteriorated wooden artwork.

We report the case of an outdoor deteriorated wooden sculpture of Madonna, completely blackened in the face, and thus suspected of fungal attack. A multi-disciplinary approach, including microbiological analysis, molecular biology, and Fourier transform infrared (FT-IR) spectroscopy, was applied to understand the real nature of the observed alteration. FT-IR showed that the blackening was due to the application of a natural terpene resin subjected to alteration over time. The microbiological assay allowed to isolate a particular black fungus that has been recovered in the vegetative phase, growing as the only species adapted to the examined substrate. Basic Local Alignment Search Tool (BLAST) analysis of the ITS (internal transcribed spacer) region sequence identified the fungus (LS31012019) as Zalaria obscura, a black yeast belonging to the new genus Zalaria, family Dothideales. Overall, this study evidenced the importance of a multi-disciplinary approach to understand the real causes of observed deterioration of artworks. More interestingly, the recovery of a strain identified as Z. obscura from this type of substrate is never reported in the literature and this finding could offer the possibility to investigate the role of this microorganism in the deterioration process of cultural heritage.

Moulds on cementitious building materials-problems, prevention and future perspectives.

Materials rich in organic and inorganic compounds, such as building materials or paints, represent an excellent substrate for the development of moulds. Several conditions affect mould's growth on cementitious materials, such as nutrient and water availability, temperature, pH and moisture. Microorganisms, and especially moulds, attack these surfaces and contribute to their erosion, thereby reducing the life of the structure itself and negatively affecting human health through inhalation, ingestion and dermal contact with spores. Interventions are based on The European Communities Council Directive 89/106/EEC, that obliges the use of materials, products and building elements that are resistant to fungi and other forms of degradation, and that do not constitute a health risk for users and the environment. This mini-review summarises the current state of problems related to mould growth on cementitious building materials, emphasising new innovative approaches for limiting or contrasting their growth. In particular, the use of nanoparticles and the related nanomaterials as well as the potential use of new "biocides" from natural sources is discussed.

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.

Bioeffects of Prunus spinosa L. fruit ethanol extract on reproduction and phenotypic plasticity of Trichoplax adhaerens Schulze, 1883 (Placozoa).

The aim of this work was to test and analyse the bioeffects of Prunus spinosa L. (Rosacaee) fruit ethanol extract on Trichoplax adhaerens Schulze, 1883 (Placozoa) laboratory cultures which-for the first time-were employed as in vivo biological model to assess the bioactivity of a natural extract. The ethanol extract of P. spinosa was administrated during a 46 day experimental period; ultrastructural (by optical, confocal, TEM and SEM microscopy) and morphometric analyses indicated that treated Trichoplax adhaerens showed significant differences in viability, reproductive modalities, body shape and colour with respect to the control group. Finally, P. spinosa bioactive compounds seem to exert profound protective effects on T. adhaerens reproduction and phenotype. Our results may support additional investigations related to other bioactive compounds properties useful for nutraceutical preparations to be used as food supplements.

[Risk assessment related to galenic products]. / Ipotesi e valutazione dei rischi correlati alle preparazioni galeniche in farmacia.

The risks associated with the preparation of galenic products prepared at a local pharmacy have been hypothesized and evaluated, also carrying out a microbiological environmental monitoring of the used surfaces. Three possible situations risk were evaluated: the first one related to the physical separation of the production phases (medium risk of occurrence), the second to the failure to restore hygienic conditions in the transition from different formulations (high risk of occurrence), the third to contamination caused by the operator itself (unacceptable risk of occurrence). This last analysis was supported by the microbiological data of environmental sampling that showed procedural errors of the operator during the cleaning phases. From our assessments it is advisable to apply a simplified system of self-control based on risk assessment and validation of critical phases including cleaning procedures for the galenic preparations.

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.

Dendrimers and Polyamino-Phenolic Ligands: Activity of New Molecules Against Legionella pneumophila Biofilms.

Legionnaires' disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems in order to minimize legionellosis outbreaks.

Evaluation of ultraviolet irradiation efficacy in an automated system for the aseptic compounding using challenge test.

OBJECTIVE: Ultraviolet (UV) irradiation efficacy in the intravenous compounding robot APOTECAchemo was evaluated to define the best operative conditions in terms of sterility and time optimization. DESIGN: The challenge test was used against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Bacillus subtilis spores and Candida albicans. Inoculated plates were placed inside the robot and irradiated for different times. Microbial air and surface quality inside the equipment were monitored utilizing settle and contact plates, swabs. RESULTS: After 4 h, no microorganisms were viable with killing rates ranging from 5- to 7-log for different microorganisms after 1 h of exposition. In confirmation of the efficacy of the UV irradiation program adopted, the microbial monitoring inside the equipment always gave negative results. CONCLUSIONS: This is the first exhaustive investigation of UV irradiation efficacy in the aseptic pharmaceutical production. We demonstrated that UV irradiation plays an essential role in maintaining the sterility condition of the workplace inside the APOTECAchemo and assuring the standards for aseptic manufacturing of medicinal drugs, as required for Class A clean areas. A 4-h UV irradiation also ensures sterility in the case of very resistant microorganisms and in the presence of high microbial charge (10(8) CFU/ml), but a killing rate of 5 or more is already recorded after the first hour of exposition. The results provide useful information for the best operative conditions in terms of both sterility and time optimization, not only for the automated compounding, but also for the traditional aseptic manufacturing processes.

Evaluation of Escherichia coli viability by flow cytometry: A method for determining bacterial responses to antibiotic exposure.

BACKGROUND: In this study, we check for the presence of specific resistance genes by polymerase chain reaction (PCR) and then we used flow cytometry (FCM) to evaluate antibiotic-induced effects in different strains of Escherichia coli (E. coli). METHODS: The presence of resistance genes was investigated by PCR in 10 strains of E. coli isolated from Foglia River. Bacterial responses to different antibiotics were also tested with FCM techniques by evaluating both the degree of decrease in viability and the light scatter changes in all of the strains. RESULTS: PCR revealed that only one strain exhibits the presence of one resistance gene. Despite this, analyses of strains using FCM evidenced the presence of viable subpopulations after antibiotic treatment. Furthermore, analyses of scatter signals revealed profound changes in the Forward Scatter and Side Scatter of the bacterial populations as a consequence of antibiotic exposure, confirming the viability and membrane potential data. The riverine strains were in general less sensitive to antibiotics than the reference strain (ATCC 25922). CONCLUSIONS: Antibiotic resistance is a widespread phenomena. The multiparametric approach based on FCM used in this study, providing results about different aspects (cell viability, membrane potential, light scatter changes), may overcome the limitation of PCR and could represent an adequate method for the evaluation of bacteria responses to antibiotic exposure.

Effect of starvation on survival and virulence expression of Aeromonas hydrophila from different sources.

Aeromonas hydrophila is an aquatic bacterium responsible for several human illnesses. The aim of this work was to investigate the survival ability and virulence expression of two strains from different sources (fish, strain 87 and surface water, strain LS) maintained in a seawater microcosm. The strains were analyzed for the total and viable bacterial counts, adhesion ability to Hep-2 cells and aerA gene expression by qPCR throughout the experiment (35 days). Both strains reached a putative VBNC state and lost adhesive properties but exhibited a different behavior in the expression of aerA. This could be due to the different origin of the two strains; the former adapted to a habitat rich of nutrient and the latter already used to survive in a more hostile environment. Moreover, our results indicate that the quantitative determination of aerA mRNA can be a useful indicator of virulence expression under stress conditions.

Role of biofilm in protection of the replicative form of Legionella pneumophila.

The dual nature of Legionella pneumophila enables its survival in free and intracellular environments and underpins its infection and spread mechanisms. Experiments using bacterial cultures and improved RTqPCR protocols were devised to gain fresh insights into the role of biofilm in protecting the replicative form of L. pneumophila. mip gene expression was used as a marker of virulence in sessile (biofilm-bound) and planktonic (free-floating) cells of L. pneumophila serotype 1 ATCC 33152. The ratio of mip gene expression to transcriptionally active Legionella cells increased both in sessile and free-floating cells demonstrating an up-regulation of mip gene under nutrient depletion. However, a different trend was observed between the two forms, in planktonic cells the mip gene expression/transcriptionally active Legionella cells increased until the end of the experiment, while in the biofilm such increase was observed at the end of the experiment. These findings suggest a possible association between the switch to the transmissive phase of Legionella and a mip up-regulation and a role for biofilm in preserving Legionella cells in replicative form. Moreover, it has been shown that improved RTqPCR protocols are valuable tools to explore bacterial virulence.

Evaluation of Escherichia coli viability by flow cytometry; a method for determining bacterial responses to antibiotic exposure.

BACKGROUND: In this study, we check for the presence of specific resistance genes by PCR and then we used flow cytometry to evaluate antibiotic-induced effects in different strains of Escherichia coli. METHODS: The presence of resistance genes was investigated by PCR in 10 strains of Escherichia coli isolated from Foglia River. Bacterial responses to different antibiotics were also tested with flow cytometry techniques by evaluating both the degree of decrease in viability and the light scatter changes in all of the strains. RESULTS: PCR revealed that only one strain exhibits the presence of one resistance gene. Despite this, analyses of strains using flow cytometry evidenced the presence of viable subpopulations after antibiotic treatment. Furthermore, analyses of scatter signals revealed profound changes in the Forward Scatter (FSC) and Side Scatter (SSC) of the bacterial populations as a consequence of antibiotic exposure, confirming the viability and membrane potential data. The riverine strains were in general less sensitive to antibiotics than the reference strain (ATCC 25922). CONCLUSIONS: Antibiotic resistance is a widespread phenomena. The multiparametric approach based on flow cytometry used in this study, providing results about different aspects (cell viability, membrane potential, light scatter changes), may overcome the limitation of PCR and could represent an adequate method for the evaluation of bacteria responses to antibiotic exposure. This article is protected by copyright. All rights reserved.

Chemical and microbiological monitoring of air in two waste incineration plants.

The study evaluated emissions from two incinerator plants and occupational exposure of workers during a six-year monitoring period (2004-2009). Chemical and microbiological analyses were performed by collecting environmental and staff individual air samples. Inspirable and respirable particulate and metals were measured in both environmental and staff samples; concentrations of Volatile Organic Compounds and the presence of microorganisms were investigated only in environmental samples. The concentrations of all organic and inorganic compounds investigated were very low and always below the Italian legal limits and the threshold limits recommended by the American Conference of Governmental Industrial Hygienists. In addition, microbial contamination was generally low in all the working areas considered. Study results revealed a good quality of air in the incineration plants and no apparent risk situation for the workers.

Isolation of a strain of Aspergillus fumigatus able to grow in minimal medium added with an industrial cyanide waste.

The present note refers the results about the isolation of an Aspergillus fumigatus strain able to grow on an industrial cyanide waste as nitrogen source. The fungus was selected from an alkaline unpolluted soil in enrichment cultures in 50 ml of Minimal Medium added with 20 mmol glucose and supplemented initially with 0.1 mmol KCN and then with 70 µl of a waste solution from a jewelry industry containing free cyanide and cyanide complexes of heavy metal ions including copper, silver, nickel, and others. The cyanide content of the waste was 1,500 ppm. The fungal growth was monitored determining dry weight, protein content and glucose consumption. The fungus efficiently utilized the cyanide as evidenced by the decrease in the inoculated medium of the compound under detection limits within 24 h and the concomitant growth within 15 days during which periodical additions of the waste to the cultures were made. The amount of the cyanide in the biomass of the fungus grown in presence of the waste was very scarce and comparable to that in absence of the pollutant. Furthermore the fungus was able to sequestrate metals such Ag, Cu, and Ni as a resistance mechanism against heavy metals. In conclusion our results are of interest for biodegradation plans of electroplating industrial wastes containing cyanide based pollutants.

Putative virulence properties of Aeromonas strains isolated from food, environmental and clinical sources in Italy: a comparative study.

The distribution of virulence properties in 142 strains of Aeromonas isolated from diarrhoeic patients, food and surface water in Italy and identified by biochemical and molecular methods was investigated. The virulence properties studied were the presence of genes for the aerolysin (aerA), heat-stable cytotonic enterotoxin (ast), heat-labile cytotonic enterotoxin (alt), cytotoxic enterotoxin (act); and cytotoxicity for Vero cells and adhesion on Hep-2 cells. A. hydrophila and A. caviae were the species most commonly isolated from clinical and environmental samples (9/30; 30.0% and 5/27; 18.5%, respectively) while mesophilic A. salmonicida was most common in food samples (19/80; 23.7%). Out of 142 strains, 86 (60.6%) were positive for at least one of the virulence properties. All the toxin genes were present in 4/18 (22.3%) of clinical strains. Most of the food isolates (54/55; 98.2%) were cytotoxic and most of the environmental strains (12/13; 92.3%) were adhesive. The aerA gene was present in most toxigenic strains (72/86; 83.7%), irrespective of their origin. The growth temperature affected the expression of cytotoxicity and adhesivity. Aeromonas strains from food and surface water frequently had toxin gene patterns similar to those of clinical strains and expressed virulence properties at human body temperature. These findings indicate that aeromonads have the potential to cause human illness and confirm the role of food and water as vehicles for Aeromonas diseases.

Determination of viability of Aeromonas hydrophila in increasing concentrations of sodium chloride at different temperatures by flow cytometry and plate count technique.

Aeromonads in waters and foods can represent a risk to human health. Factors such as sodium chloride concentration and temperature can affect growth and viability of several food and water-borne pathogens. The behaviour of an Aeromonas hydrophila strain in the presence of 1.7%, 3.4% and 6% NaCl concentrations at 24 degrees C and 4 degrees C was studied over a 188 day period. Viability and membrane potential were assessed by flow cytometry; growth was evaluated by plate count technique. Flow cytometry evidenced that A. hydrophila retained viability over the period although varying according to temperature and salt concentrations. Colony Forming Units were generally lower in number than viable cells especially in the presence of 6% NaCl, indicating the occurrence of stressed cells which maintain metabolic activity yet are not able to grow on agar plates. In conclusion, A. hydrophila showed a long-term halotolerance even at elevated (6%) NaCl concentrations and a lesser sensitivity to salt at low temperature; therefore, low temperature and salt, which are two important factors limiting bacterial growth, do not assure safety in the case of high initial contamination. Finally, cytometry appears a valid tool for the rapid detection of the viability of pathogenic bacteria in food and environmental matrices to control and prevent health risks.