The transcription factor Ndt80 is a repressor of candida parapsilosis virulence attributes

Candida parapsilosis is an emergent opportunistic yeast among hospital settings that affects mainly neonates and immunocompromised patients. Its most remarkable virulence traits are the ability to adhere to prosthetic materials, as well as the formation of biofilm on abiotic surfaces. The Ndt80 transcription factor was identified as one of the regulators of biofilm formation by C. parapsilosis; however, its function in this process was not yet clarified. By knocking out NDT80 (CPAR2-213640) gene, or even just one single copy of the gene, we observed substantial alterations of virulence attributes, including morphogenetic changes, adhesion and biofilm growth profiles. Both ndt80Δ and ndt80ΔΔ mutants changed colony and cell morphologies from smooth, yeast-shaped to crepe and pseudohyphal elongated forms, exhibiting promoted adherence to polystyrene microspheres and notably, forming a higher amount of biofilm compared to wild-type strain. Interestingly, we identified transcription factors Ume6, Cph2, Cwh41, Ace2, Bcr1, protein kinase Mkc1 and adhesin Als7 to be under Ndt80 negative regulation, partially explaining the phenotypes displayed by the ndt80ΔΔ mutant. Furthermore, ndt80ΔΔ pseudohyphae adhered more rapidly and were more resistant to murine macrophage attack, becoming deleterious to such cells after phagocytosis. Unexpectedly, our findings provide the first evidence for a direct role of Ndt80 as a repressor of C. parapsilosis virulence attributes. This finding shows that C. parapsilosis Ndt80 functionally diverges from its homolog in the close related fungal pathogen C. albicans.info:eu-repo/semantics/publishedVersio

Antibacterial action mechanisms of honey: physiological effects of avocado, chestnut, and polyfloral honey upon Staphylococcus aureus and Escherichia coli

Numerous studies have explored the antibacterial properties of di erent types of honey from all around the world. However, the data available describing how honey acts against bacteria are few. The aim of this study was to apply a flow cytometry (FC) protocol to examine and characterize the primary e ects of three varieties of honey (avocado, chestnut and polyfloral) upon physiological status of Staphylococcus aureus and Escherichia coli cells to reveal their antibacterial action mechanisms. The e ects of honey samples on membrane potential, membrane integrity, and metabolic activity were assessed using di erent fluorochromes, in a 180 min time course assay. Time-kill experiments were also carried out under similar conditions. Exposure of S. aureus and E. coli to the distinct honey samples resulted in physiological changes related to membrane polarization and membrane integrity. Moreover, honey induced a remarkable metabolic disruption as primary physiological e ect upon S. aureus. The di erent honey samples induced quite similar e ects on both bacteria. However, the depth of bacteria response throughout the treatment varied depending on the concentration tested and among honey varieties, probably due to compositional di erences in the honey.This work was supported by the Consejería de Sanidad of Junta de Castilla y León; under Grant GRS 551/A/10. Patricia Combarros-Fuertes was funded by Consejería de Educación of Junta de Castilla y León and European Social Fund.info:eu-repo/semantics/publishedVersio

Evaluation of physiological effects induced by manuka honey upon staphylococcus aureus and escherichia coli

Several studies have explored the antimicrobial properties of manuka honey (MkH). However, the data available regarding antibacterial action mechanisms are scarcer. The aim of this study was to scrutinize and characterize primary e ects of manuka honey (MkH) upon the physiological status of Staphylococcus aureus and Escherichia coli (as Gram-positive and Gram-negative bacteria models, respectively), using flow cytometry (FC) to reveal its antibacterial action mechanisms. E ects of MkH on membrane potential, membrane integrity and metabolic activity were assessed using di erent fluorochromes in a 180 min time course assay. Time-kill experiments were carried out under the same conditions. Additionally, MkH e ect on e ux pumps was also studied in an E. coli strain with an over-expression of several e ux pumps. Exposure of bacteria to MkH resulted in physiological changes related to membrane potential and membrane integrity; these e ects displayed slight di erences among bacteria. MkH induced a remarkable metabolic disruption as primary physiological effect upon S. aureus and was able to block effux pump activity in a dose-dependent fashion in the E. coli strain.This work was supported by the Consejería de Sanidad of Junta de Castilla y León; under grant GRS 551/A/10. Patricia Combarros-Fuertes was funded by Consejería de Educación of Junta de Castilla y León and European Social Fund.info:eu-repo/semantics/publishedVersio

Antibacterial Action Mechanisms of Honey: Physiological Effects of Avocado, Chestnut, and Polyfloral Honey upon Staphylococcus aureus and Escherichia coli

[EN] Numerous studies have explored the antibacterial properties of different types of honey from all around the world. However, the data available describing how honey acts against bacteria are few. The aim of this study was to apply a flow cytometry (FC) protocol to examine and characterize the primary effects of three varieties of honey (avocado, chestnut and polyfloral) upon physiological status of Staphylococcus aureus and Escherichia coli cells to reveal their antibacterial action mechanisms. The effects of honey samples on membrane potential, membrane integrity, and metabolic activity were assessed using different fluorochromes, in a 180 min time course assay. Time-kill experiments were also carried out under similar conditions. Exposure of S. aureus and E. coli to the distinct honey samples resulted in physiological changes related to membrane polarization and membrane integrity. Moreover, honey induced a remarkable metabolic disruption as primary physiological effect upon S. aureus. The different honey samples induced quite similar effects on both bacteria. However, the depth of bacteria response throughout the treatment varied depending on the concentration tested and among honey varieties, probably due to compositional differences in the honeySIThis work was supported by the Consejería de Sanidad of Junta de Castilla y León; under Grant GRS 551/A/10. Patricia Combarros-Fuertes was funded by Consejería de Educación of Junta de Castilla y León and European Social Fun

Development of cross-resistance by Aspergillus fumigatus to clinical azoles following exposure to prochloraz, an agricultural azole

Background: The purpose of this study was to unveil whether azole antifungals used in agriculture, similar to the clinical azoles used in humans, can evoke resistance among relevant human pathogens like Aspergillus fumigatus, an ubiquitous agent in nature. Additionally, cross-resistance with clinical azoles was investigated. Antifungal susceptibility testing of environmental and clinical isolates of A. fumigatus was performed according to the CLSI M38-A2 protocol. In vitro induction assays were conducted involving daily incubation of susceptible A. fumigatus isolates, at 35°C and 180 rpm, in fresh GYEP broth medium supplemented with Prochloraz (PCZ), a potent agricultural antifungal, for a period of 30 days. Minimal inhibitory concentrations (MIC) of PCZ and clinical azoles were monitored every ten days. In order to assess the stability of the developed MIC, the strains were afterwards sub-cultured for an additional 30 days in the absence of antifungal. Along the in vitro induction process, microscopic and macroscopic cultural observations were registered. Results: MIC of PCZ increased 256 times after the initial exposure; cross-resistance to all tested clinical azoles was observed. The new MIC value of agricultural and of clinical azoles maintained stable in the absence of the selective PCZ pressure. PCZ exposure was also associated to morphological colony changes: macroscopically the colonies became mostly white, losing the typical pigmentation; microscopic examination revealed the absence of conidiation. Conclusions: PCZ exposure induced Aspergillus fumigatus morphological changes and an evident increase of MIC value to PCZ as well as the development of cross-resistance with posaconazole, itraconazole and voriconazole.IFR and IMM are supported by FCT (Fundação Ciência e Tecnologia). IFR is supported by FCT PhD grant (SFRH/BD/91155/2012). I.MM is supported by FCT, Ciência 2008 and co-financed by the European Social Fund. The authors would like to thank Isabel Santos for the excellent technical assistance. Part of the results was presented at the 52nd Interscience Conference on Antimicrobial Agents and Chemotherapy, ICAAC, held San Francisco, USA, September 2012.info:eu-repo/semantics/publishedVersio

Environmental azole fungicide, prochloraz, can induce cross-resistance to medical triazoles in Candida glabrata

Acquisition of azole resistance by clinically relevant yeasts in nature may result in a significant, yet undetermined, impact in human health. The main goal of this study was to assess the development of cross-resistance between agricultural and clinical azoles by Candida spp. An in vitro induction assay was performed, for a period of 90 days, with prochloraz (PCZ) - an agricultural antifungal. Afterward, the induced molecular resistance mechanisms were unveiled. MIC value of PCZ increased significantly in all Candida spp. isolates. However, only C. glabrata developed cross-resistance to fluconazole and posaconazole. The increased MIC values were stable. Candida glabrata azole resistance acquisition triggered by PCZ exposure involved the upregulation of the ATP binding cassette multidrug transporter genes and the transcription factor, PDR1. Single mutation previously implicated in azole resistance was found in PDR1 while ERG11 showed several synonymous single nucleotide polymorphisms. These results might explain why C. glabrata is so commonly less susceptible to clinical azoles, suggesting that its exposure to agricultural azole antifungals may be associated to the emergence of cross-resistance. Such studies forward potential explanations for the worldwide increasing clinical prevalence of C. glabrata and the associated worse prognosis of an infection by this species.info:eu-repo/semantics/publishedVersio

An alternative respiratory pathway on Candida krusei : implications on susceptibility profile and oxidative stress

Our aim was to detect the presence of an alternative oxidase (AOX) in Candida krusei clinical strains and its influence on fluconazole susceptibility and in reactive oxygen species (ROS) production. Candida krusei clinical isolates were tested to evaluate the presence of AOX. Debaromyces hansenii 2968 (AOX positive) and Saccharomyces cerevisiae BY4742 (AOX negative) were used as control strains. Measurements of oxygen consumption were performed in the presence of 1 mM KCN, an inhibitor of the classical respiratory chain, and 5 mM salicylhydroxamic acid (SHAM). AOX expression was monitored by Western blotting using an AOX monoclonal antibody. Interactions between fluconazole and SHAM were performed using checkerboard assay. ROS production was evaluated in the presence of SHAM plus fluconazole, H2O2, menadione, or plumbagin. AOX was present in all C. krusei tested. The combination of fluconazole with SHAM resulted in an indifferent effect. In the presence of SHAM, the treatment with ROS inductors or fluconazole increased ROS production, except in the AOX-negative strain. An alternative respiratory pathway resistant to cyanide is described for the first time as a characteristic of C. krusei species. This AOX is unrelated to fluconazole resistance; however, it protects C. krusei from oxidative stress.Fundação para a Ciência e a Tecnologia - SFRH/BD/27662/200

<i>Candida parapsilosis</i> Virulence and Antifungal Resistance Mechanisms: A Comprehensive Review of Key Determinants

Candida parapsilosis is the second most common Candida species isolated in Asia, Southern Europe, and Latin America and is often involved in invasive infections that seriously impact human health. This pathogen is part of the psilosis complex, which also includes Candida orthopsilosis and Candida metapsilosis. C. parapsilosis infections are particularly prevalent among neonates with low birth weights, individuals who are immunocompromised, and patients who require prolonged use of a central venous catheter or other indwelling devices, whose surfaces C. parapsilosis exhibits an enhanced capacity to adhere to and form biofilms. Despite this well-acknowledged prevalence, the biology of C. parapsilosis has not been as extensively explored as that of Candida albicans. In this paper, we describe the molecular mechanistic pathways of virulence in C. parapsilosis and show how they differ from those of C. albicans. We also describe the mode of action of antifungal drugs used for the treatment of Candida infections, namely, polyenes, echinocandins, and azoles, as well as the resistance mechanisms developed by C. parapsilosis to overcome them. Finally, we stress the importance of the ongoing search for species-specific features that may aid the development of effective control strategies and thus reduce the burden on patients and healthcare costs

Comparison of Two Probes for Testing Susceptibilities of Pathogenic Yeasts to Voriconazole, Itraconazole, and Caspofungin by Flow Cytometry

A cytometric approach to determine the susceptibilities of Candida spp. and Cryptococcus neoformans to voriconazole, itraconazole, and caspofungin is described. A total of 63 clinical isolates with different susceptibility patterns were exposed for 1, 2, 4, and 6 h to serial concentrations of each antifungal agent, followed by staining with two fluorescent probes: propidium iodide (PI) and FUN-1. FUN-1 was able to identify the susceptibility patterns of the assayed strains to the three agents after 1 h. PI penetrated a maximum of 50% of the cells treated with PI, at the highest concentration of caspofungin, 16 μg/ml, after 6 h of incubation (this percentage varied with the strain and was drug concentration and time of incubation dependent) and did not stain cells treated with high concentrations of either azole after 6 h. The use of FUN-1 appears to be an excellent fast and reliable alternative to the classical dilution method for determining the susceptibility of Candida spp. and C. neoformans to these three antifungal agents