Influence of the agrochemical benomyl on Cryptococcus gattii-plant interaction in vitro and in vivo.

Cryptococcus gattii, an environmental fungus, is one of the agents of cryptococcosis. The influence of agrochemicals on fungal resistance to antifungals is widely discussed. However, the effects of benomyl (BEN) on fungal interaction with different hosts is still to be understood. Here we studied the influence of adaptation to BEN in the interaction with a plant model, phagocytes and with Tenebrio molitor. First, the strain C. gattii L24/01 non-adapted (NA), adapted (A) to BEN, and adapted with further culture on drug-free media (10p) interact with Nicotiana benthamiana, with a peak in the yeast burden on the 7th day post-inoculation. C. gattii L24/01 A and 10p provided lower fungal burden, but these strains increased cell diameter and capsular thickness after the interaction, together with decreased fungal growth. The strains NA and A showed reduced ergosterol levels, while 10p exhibited increased activity of laccase and urease. L24/01 A recovered from N. benthamiana was less engulfed by murine macrophages, with lower production of reactive oxygen species. This phenotype was accompanied by increased ability of this strain to grow inside macrophages. Otherwise, L24/01 A showed reduced virulence in the T. molitor larvae model. Here, we demonstrate that the exposure to BEN, and interaction with plants interfere in the morphophysiology and virulence of the C. gattii.

Does sorghum phenolic extract have antifungal effect?

This study aimed to evaluate the antifungal effect of SC319 sorghum phenolic extract (SPE) on the Aspergillus, Fusarium, Penicillium, Stenocarpella, Colletotrichum, and Macrophomina genera. SPE was extracted by 20% ethanol and used in four assays: (1) against Fusarium verticillioides in solid (PDA) and liquid (PD) potato dextrose media; (2) Minimum Inhibitory Concentration (MIC) assay with 16 fungi isolates; (3) Conidial Germination Rate (CGR) with 14 fungi isolates and (4) Growth Curve (GC) with 11 fungi isolates. There was no reduction in the mycelial growth (colony diameter and dry weight) and in the number of Fusarium verticillioides spores in assay 1 (PDA and PD). The colony's dry weight was almost six times higher in the presence than in the absence of SPE. All SPE samples presented MIC (assay 1) above the maximum concentration tested (5000 µg.mL-1) for the 16 isolates. Also, there was no inhibitory effect of SPE on conidia germination rate (CGR). Oppositely, in GC assay, the control had a higher CFU count than the samples with SPE in 24 h. This result suggests that SPE can delay the fungal growth in the first hours of incubation, which is an important finding that may help reduce the severity of fungal diseases in plants. However, further studies are needed to confirm these results, including sorghum genotypes with different profiles of phenolic compounds. Although the SC319 SPE was not effective as an antifungal agent, it may have potential as a growth promoter of beneficial fungi in the food and pharmaceutical industries.

Detection of Cladosporium spinulosum on an engraving by Rembrandt and susceptibility profile to eco-friendly antifungal treatments.

Interdisciplinary studies on cultural heritage artworks provide efficient solutions to control fungal growth and the negative effects of biodeterioration. In this study, we aimed to identify the population of filamentous fungi colonizing an engraving by the Dutch painter Rembrandt, whose conservation status was compromised and showed visible stains of biodeterioration. Microbiological techniques, such as cultivation-dependent approaches and molecular biology, have been used to identify fungal populations. In addition, the anaerobic atmosphere technique and eco-friendly antifungal agents, such as essential oils (EOs) of Curcuma longa, Thymus vulgaris, and Melaleuca alternifolia, were tested against the metabolically active fungal population Cladoposporium spinulosum. Furthermore, in vitro assays revealed that the interaction between the fungal strains and EO was positive, inhibiting the growth of these fungi, and the EOs from T. vulgaris and M. alternifolia showed low minimum inhibitory concentration values. Exposure to anaerobic conditions for 35 days was effective in the total elimination of isolated fungal strains. In conclusion, this study demonstrated the effectiveness of a nondestructive technique for artwork on engraving colonized by fungal strains and using EO as an alternative to toxic antifungals used in conventional treatments in artworks. Thus, this interdisciplinary study involving applied microbiology and botanical and preventive conservation presents a tool to control microbial growth while maintaining artwork integrity.

Identification and evaluation of thermotolerance of yeasts from milk in natura exposed to high temperature and slow and fast pasteurization.

Milk is considered one of the basic raw materials of animal origin; it must present hygienic quality and physical-chemical properties suitable for processing and human consumption. Thus, the ingestion of milk in natura when not properly treated can be characterized as an opportunistic route of transmission of possible microbial pathogens, which can offer risks to public health. The present study aimed the yeast identification, to analyze the thermo-resistance of yeasts isolated from fresh milk, and to trace the susceptibility profile of the isolates to antifungal agents. For this, 23 samples of fresh milk type B, collected by manual or mechanical milking, were stored in collective refrigeration tanks of farms located in the Metropolitan Region of Natal and nearby, State of Rio Grande do Norte (RN), Brazil. Twenty samples of fresh milk commercially traded in the city of Ceará-Mirim RN were also analyzed. The yeasts were quantified by count of colony-forming units (CFU). All isolated species were treated by slow pasteurization (62-64 °C for 30 min) and fast (72-75 °C for 20 s), as well as by boiling (100 °C). Fifty yeast strains were obtained, and the species were identified as Candida tropicalis (28%), Candida parapsilosis (14%), Candida albicans (12%), Candida glabrata (10%), Candida krusei (10%), Kluyveromyces marxianus (10%), Candida guilliermondii (8%), Candida rugosa (2%), Candida orthopsilosis (2%), Pichia manshurica (2%), and Kodamaea ohmeri (2%). Five isolates showed resistance to the antifungal agents tested. Among all the isolates submitted to heat treatment, 80% were resistant to fast pasteurization and 60% to boiling, but none of them resisted the slow pasteurization. The milk collected through mechanical milking and stored in collective cooling tanks, presented higher rates of yeast contamination, compared to milk samples collected by manual milking and kept under the same storage conditions.

Antibiofilm and Anti-Candidal Activities of the Extract of the Marine Sponge Agelas dispar.

This study aimed to determine the antifungal and antibiofilm activities of Agelas dispar on biofilm-producing Candida species. The methanolic extract of A. dispar was obtained and the fraction Ag2 showed inhibitory activity for all 13 Candida strains tested, in concentrations ranging from 2.5 to 0.15625 mg/mL. Antifungal activity of fungicidal nature was seen between 5.0 and 0.3125 mg/mL of extract against the strains. All the strains were classified as biofilm producers. The methanolic extract Ag2 was tested at concentrations of 2.5 and 1.25 mg/mL for antibiofilm activity against the biofilm formation and maturation in all the strains of the genus Candida. Treated and untreated biofilm samples were selected for visualization using scanning electron microscopy (SEM). SEM allowed the visualization of the quantitative decrease in the microbial community, alterations of structural morphology, and destruction of both the formation and maturation of biofilms, at the cellular level. The mechanism of action of this fraction is suggested to be at the plasma membrane and/or cell wall alteration level. Therefore, the use of the methanolic extract of A. dispar may be a promising antifungal and antibiofilm therapeutic strategy against different species of the genus Candida.

Evaluation of microbiological air parameters and the fungal community involved in the potential risks of biodeterioration in a cultural heritage of humanity, Ouro Preto, Brazil.

The research of the microbiological air quality of a building considered a human historical and cultural heritage site by the United Nations Educational, Scientific, and Cultural Organization is fundamental for preventive conservation action, mainly because it identifies cultivable fungal species around the collections and suggests the appropriate treatment choice. This study investigated the air microbiological parameters inside the Nossa Senhora da Conceição Church and identified the population of airborne fungi. Sixty filamentous fungal isolates were detected with ten distinct taxa. The counts of colony forming units (CFUs) performed at 10 different points were in accordance with Brazilian legislation. In addition, the presence of two fungal species was detected colonizing artworks covered with gold leaves: Cladosporium cladosporioides and Aspergillus versicolor. Air quality monitoring inside the church was in accordance with the required Brazilian legislation standards. The composition of the filamentous fungal community included the presence of human fungal pathogens; for this reason, the use of personal protective equipment was recommended during the restoration work. Thus, characterization of the air microbiological parameters helps to preserve not only the building's collection, but also the health of the faithful, conservators-restorers, tourists, and researchers.

Application of near-infrared hyperspectral (NIR) images combined with multivariate image analysis in the differentiation of two mycotoxicogenic Fusarium species associated with maize.

Maize (Zea mays L.) is one of the most versatile crops worldwide with high socioeconomic relevance. However, mycotoxins produced by pathogenic fungi are of constant concern in maize production, as they pose serious risks to human and animal health. Thus, the search for rapid detection and/or identification methods for mycotoxins and mycotoxin-producing fungi for application in food safety remain important. In this work, we implemented use of near infrared hyperspectral images (HSI-NIR) combined with pattern recognition analysis, partial-least-squares discriminant analysis (PLS-DA) of images, to develop a rapid method for identification of Fusarium verticillioides and F. graminearum. Validation of the HSI-NIR method and subsequent analysis was realized using 15 Fusarium spp. isolates. The method was efficient as a rapid, non-invasive, and non-destructive assessment was achieved with 100% accuracy, sensitivity, and specificity for both fungi.

Occurrence of Aspergillus niger strains on a polychrome cotton painting and their elimination by anoxic treatment.

This study aimed to isolate and identify the population of filamentous fungi colonizing a cotton painting, whose conservation status was compromised and showed signs of biodeterioration due to dirt accumulation and microbial metabolism. In addition, microbiological techniques such as cultivation-dependent approach and molecular biology were used to identify microbial populations and to eliminate their metabolic action. For this, the nondestructive anoxic atmosphere technique was used, in which the microbial metabolism was affected by the absence of oxygen. Prior to exposure to an anoxic atmosphere, only one fungal species, Aspergillus niger, was identified at 12 points sampled in the obverse and reverse of the artwork; no fungal species persisted as a result of anoxic treatment. These results showed that exposure to anoxic conditions was effective for the total elimination of isolated fungal strains as well as their spores. In conclusion, this study proved the unprecedented effectiveness of a nondestructive technique for artwork on textile colonized by black fungi species. Thus, this interdisciplinary work involving conservation, microbiology, and chemistry presents a tool to eliminate microorganisms, while maintaining the integrity of artwork and safety of the restorer, that can be applied prior to artwork restoration.

N-acetylcysteine reduces amphotericin B deoxycholate nephrotoxicity and improves the outcome of murine cryptococcosis.

Cryptococcosis is a life-threatening fungal infection, and its current treatment is toxic and subject to resistance. Drug repurposing represents an interesting approach to find drugs to reduce the toxicity of antifungals. In this study, we evaluated the combination of N-acetylcysteine (NAC) with amphotericin B (AMB) for the treatment of cryptococcosis. We examined the effects of NAC on fungal morphophysiology and on the macrophage fungicidal activity 3 and 24 hours post inoculation. The therapeutic effects of NAC combination with AMB were investigated in a murine model with daily treatments regimens. NAC alone reduced the oxidative burst generated by AMB in yeast cells, but did not inhibit fungal growth. The combination NAC + AMB decreased capsule size, zeta potential, superoxide dismutase activity and lipid peroxidation. In macrophage assays, NAC + AMB did not influence the phagocytosis, but induced fungal killing with different levels of oxidative bursts when compared to AMB alone: there was an increased reactive oxygen species (ROS) after 3 hours and reduced levels after 24 hours. By contrast, ROS remained elevated when AMB was tested alone, demonstrating that NAC reduced AMB oxidative effects without influencing its antifungal activity. Uninfected mice treated with NAC + AMB had lower concentrations of serum creatinine and glutamate-pyruvate transaminase in comparison to AMB. The combination of NAC + AMB was far better than AMB alone in increasing survival and reducing morbidity in murine-induced cryptococcosis, leading to reduced fungal burden in lungs and brain and also lower concentrations of pro-inflammatory cytokines in the lungs. In conclusion, NAC + AMB may represent an alternative adjuvant for the treatment of cryptococcosis.

Investigating cross-contamination by yeast strains from dental solid waste to waste-handling workers by DNA sequencing.

Trying to widen the discussion on the risks associated with dental waste, this study proposed to investigate and genetically compare yeast isolates recovered from dental solid waste and waste workers. Three samples were collected from workers' hands, nasal mucosa, and professional clothing (days 0, 30, and 180), and two from dental waste (days 0 and 180). Slide culture, microscopy, antifungal drug susceptibility, intersimple sequence repeat analysis, and amplification and sequencing of internal transcribed spacer regions were performed. Yeast strains were recovered from all waste workers' sites, including professional clothes, and from waste. Antifungal susceptibility testing demonstrated that some yeast recovered from employees and waste exhibited nonsusceptible profiles. The dendrogram demonstrated the presence of three major clusters based on similarity matrix and UPGMA grouping method. Two branches displayed 100% similarity: three strains of Candida guilliermondii isolated from different employees, working in opposite work shifts, and from diverse sites grouped in one part of branch 1 and cluster 3 that included two samples of Candida albicans recovered from waste and the hand of one waste worker. The results suggested the possibility of cross-contamination from dental waste to waste workers and reinforce the need of training programs focused on better waste management routines.

Atorvastatin as a promising anticryptococcal agent.

Cryptococcosis caused by Cryptococcus gattii leads to pneumonia and meningoencephalitis, and has a high mortality rate worldwide due to the inadequacy of available therapy and increasing drug resistance. There is a need to develop effective treatments, and drug repositioning is an interesting alternative to achieve new strategies to treat cryptococcosis. Atorvastatin (ATO), a statin currently used to treat hypercholesterolaemia, was tested in this study as an adjuvant to control infections caused by C. gattii. Several aspects of the effect of ATO on the host and the yeast were evaluated, with particular focus on the association of ATO with fluconazole (FLC), which (i) reduced ergosterol content in the cell membrane and altered properties of the polysaccharide capsule of C. gattii; (ii) increased the production of reactive oxygen species by macrophages; and (iii) reduced yeast phagocytosis and the intracellular proliferation rate. In an animal model, infected mice treated with ATO + FLC showed increased survival, improved clinical condition, and reduced fungal burden in the lungs and brain. This study is the first to perform in vivo tests with ATO + FLC for the treatment of cryptococcosis. The results suggest that ATO may be an important adjuvant for the treatment of cryptococcosis.

Analysis of microbial load on surgical instruments after clinical use and following manual and automated cleaning.

BACKGROUND: We aimed to monitor the microbial load and identify the microorganisms recovered from surgical instruments after clinical use and following manual and automated cleaning. METHODS: This experimental study was carried out in the Laboratory of Oral Microbiology and Anaerobes at the Federal University of Minas Gerais in Brazil. Microbial samples were taken from 125 surgical instruments used in 25 types of gastrointestinal surgeries. RESULTS: The average microbial load was 93.1 CFU/100 mL after clinical use and 41 CFU/100 mL and 8.24 CFU/100 mL on instruments following 2 sequential steps of manual cleaning, respectively, and 75 CFU/100 mL and 16.1 CFU/100 mL on instruments after automated cleaning. Surgical wound classification significantly affected the microbial load recovered on instruments. Coagulase-negative Staphylococcus, Escherichia coli, Pseudomonas spp, Stenotrophomonas maltophilia, and Acinetobacter baumannii complex were recovered. CONCLUSIONS: The average microbial load observed after the cleaning steps decreased, and the decrease in microbial load was more pronounced using the manual method compared with that observed using the automated method.

2-(benzylideneamino)phenol: a promising hydroxyaldimine with potent activity against dermatophytoses.

Infections caused by dermatophytes, mainly Trichophyton rubrum,are often vulnerable to relapses upon cessation of antifungal therapy, reinforcing the need of new antifungals. Aldimines have potential biological activities, but there are few reports on their antifungal profile. The aim of this study was to evaluate the antifungal activity of 2-(benzylideneamino)phenol (3A3) and 4-(benzylideneamino)phenol (3A4) against dermatophytes. We determined the minimum inhibitory concentration, minimum fungicidal concentration, time-kill curves and fractional inhibitory concentration of the combination of 3A3, 3A4 and itraconazole against a set of isolates of T. rubrum and T. interdigitale. 3A3 was tested in a murine model of dermatophytoses caused by T. rubrum, and the effect on phagocytosis was assessed. The MIC values ranged from 8 to 32 µg/mL for 3A3 and from 64 to 256 µg/mL for 3A4. The interaction between 3A3 and 3A4 with itraconazole proved to be synergistic and indifferent, respectively. 3A3 was as efficient as itraconazole in reducing the fungal burden on the skin of mice, being this effect associated with the influx of neutrophil and macrophage. Also, 3A3 was able to increase the internalization of conidia by macrophages. Altogether, our data encourage future clinical studies with 3A3 to treat dermatophytoses.

Effectiveness of flexible gastrointestinal endoscope reprocessing.

The practice of reprocessing endoscopes and its effectiveness was evaluated in 37 services. Contamination of at least 1 endoscope could be identified in 34 (91.6%) of 37 services. Bacteria, fungi, and/or mycobacteria were isolated from 84.6% (33/39) of the colonoscopes (110-32,000 colony-forming units [CFUs]/mL) and from 80.6% (50/62) of the gastroscopes (100-33,000 CFUs/mL). Not all services followed recommended guidelines. Therefore, patients who underwent gastrointestinal endoscopies were exposed to diverse pathogens.

Prevalence of dermatomycosis in a Brazilian tertiary care hospital.

A total of 233 specimens obtained from suspected cases of dermatomycosis from 189 patients were examined for causative fungi from December 2009 to May 2010 in a tertiary care hospital in the city of Belo Horizonte, state of Minas Gerais, southeastern Brazil. Yeast and fungal isolates obtained from specimens were regarded as conclusive diagnosis of mycoses in 82 cases (35.19 %), with the exception of two patients with pityriasis versicolor (2.4 %), in which the diagnosis was made only by direct examination plus clinical diagnostics of individuals. Forty-four subjects (23.28 %) were infected in more than one anatomical site. There was a higher occurrence on female patients (146, 77.2 %) than male (43, 22.8 %). Most of the infected patients were aged between 41 and 70 years (68.29 %). There were no statistically significant differences between occurrence of fungal infection and gender, presence of secondary disease and contact with animals. The largest number of examined material occurred in samples from toenails, which resulted in 50 % of positive cultures. Candida species were the most frequent group causing dermatomycosis in many anatomical sites, mainly in toenails and fingernails. Candida parapsilosis was the most representative (40.24 %) among all agents causing dermatomycosis of toenails and fingernails, followed by Candida tropicalis (20.73 %) and Trichophyton rubrum (10.98 %). Among the dermatophytes, Trichophyton genus represented over 80 % of the isolates, with T. rubrum representing 64.29 %, T. interdigitale (T. mentagrophytes) (21.43 %) and Microsporum gypseum (14.29 %).