Liposomal and Deoxycholate Amphotericin B Formulations: Effectiveness against Biofilm Infections of Candida spp.

Background: candidiasis is the primary fungal infection encountered in patients undergoing prolonged hospitalization, and the fourth leading cause of nosocomial bloodstream infections. One of the most important Candida spp. virulence factors is the ability to form biofilms, which are extremely refractory to antimicrobial therapy and very difficult to treat with the traditional antifungal therapies. It is known that the prophylaxis or treatment of a systemic candidiasis are recurrently taken without considering the possibility of a Candida spp. biofilm-related infections. Therefore, it is important to assess the effectiveness of the available drugs and which formulations have the best performance in these specific infections. Methods: 24-h-biofilms of four Candida spp. and their response to two amphotericin B (AmB) pharmaceutical formulations (liposomal and deoxycholate) were evaluated. Results: generally, Candida glabrata was the less susceptible yeast species to both AmBs. MBECs revealed that it is therapeutically more appealing to use AmB-L than AmB-Deox for all Candida spp. biofilms, since none of the determined concentrations of AmB-L reached 10% of the maximum daily dose, but both formulations showed a very good capacity in the biomass reduction. Conclusions: the liposomal formulation presents better performance in the eradication of the biofilm cells for all the species in comparison with the deoxycholate formulation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and Célia F. Rodrigues’ [SFRH/BD/93078/2013] Ph.D. grant. The authors thank the Project “BioHealth—Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2—O Novo Norte), QREN, FEDER.info:eu-repo/semantics/publishedVersio

Portrait of matrix gene expression in Candida glabrata biofilms with stress induced by different drugs

(1) Background: Candida glabrata is one of the most significant Candida species associated with severe cases of candidiasis. Biofilm formation is an important feature, closely associated with antifungal resistance, involving alterations of gene expression or mutations, which can result in the failure of antifungal treatments. Hence, the main goal of this work was to evaluate the role of a set of genes, associated with matrix production, in the resistance of C. glabrata biofilms to antifungal drugs. (2) Methods: the determination of the expression of BGL2, XOG1, FKS1, FKS2, GAS2, KNH1, UGP1, and MNN2 genes in 48-h biofilms cells of three C. glabrata strains was performed through quantitative real-time PCR (RT-qPCR), after contact with Fluconazole (Flu), Amphotericin B (AmB), Caspofungin (Csf), or Micafungin (Mcf). (3) Results: Mcf induced a general overexpression of the selected genes. It was verified that the genes related to the production of -1,3-glucans (BGL2, XOG1, GAS2) had the highest expressions. (4) Conclusion: though -1,6-glucans and mannans are an essential part of the cell and biofilm matrix, C. glabrata biofilm cells seem to contribute more to the replacement of -1,3-glucans. Thus, these biopolymers seem to have a greater impact on the biofilm matrix composition and, consequently, a role in the biofilm resistance to antifungal drugs.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and Célia F. Rodrigues’ [SFRH/BD/93078/2013] Ph.D. grant. We also would like to acknowledge MSD® and Astellas® for the kind donation of Caspofungin and Micafungin, respectivelyinfo:eu-repo/semantics/publishedVersio

Inflammatory cell recruitment in Candida glabrata biofilm cell-infected mice receiving antifungal chemotherapy

(1) Background: Due to a high rate of antifungal resistance, Candida glabrata is one of the most prevalent Candida spp. linked to systemic candidiasis, which is particularly critical in catheterized patients. The goal of this work was to simulate a systemic infection exclusively derived from C. glabrata biofilm cells and to evaluate the effectiveness of the treatment of two echinocandinscaspofungin (Csf) and micafungin (Mcf). (2) Methods: CD1 mice were infected with 48 h-biofilm cells of C. glabrata and then treated with Csf or Mcf. After 72 h, the efficacy of each drug was evaluated to assess the organ fungal burden through colony forming units (CFU) counting. The immune cell recruitment into target organs was evaluated by flow cytometry or histopathology analysis. (3) Results: Fungal burden was found to be higher in the liver than in the kidneys. However, none of the drugs was effective in completely eradicating C. glabrata biofilm cells. At the evaluated time point, flow cytometry analysis showed a predominant mononuclear response in the spleen, which was also evident in the liver and kidneys of the infected mice, as observed by histopathology analysis. (4) Conclusions: Echinocandins do not have a significant impact on liver and kidney fungal burden, or recruited inflammatory infiltrate, when mice are intravenously (i.v.) infected with C. glabrata biofilm-grown cells.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of the Norte 2020 - Programa Operacional Regional do Norte, financially supported by project UID/EQU/00511/2019 — Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE) funded by national funds through FCT/MCTES (PIDDAC), Célia F. Rodrigues’ (SFRH/BD/93078/20130) PhD grant and M. Elisa Rodrigues (SFRH/BPD/95401/2013) post-doc grant.info:eu-repo/semantics/publishedVersio

Cost-effective bioethanol production at low content of nitrogen source from carob syrup

Ethanol, as biofuel, has received great interest in the latest decades due to its potential as an alternative transport fuel. Nowadays, ethanol can be produced through fermentative processes, using sugar rich agricultural raw material and it may have a significant role in reducing environmental impact of fossil fuels

The effectiveness of voriconazole in therapy of Candida glabratas biofilms oral infections and its influence on the matrix composition and gene expression

Candida glabrata is one of most prevalente yeast in fungal infections, especially in immunocompromised patients. Its azole resistance results in a low therapeutic response, particularly when associated with biofilms. The main goal of this work was to study the effectiveness of voriconazole (Vcz) against C. glabrata biofilms oral pathologies, as esophageal or oropharyngeal candidiasis. Antifungal susceptibilities were determined in pre-formed 24-h-biofilms and ERG genes expression was determined by qRT-PCR. Protein quantification was performed using BCA Kit, carbohydrate was estimated according to the Dubois assay and b-1,3 glucans concentration were determined using Glucatell kit. Finally, ergosterol, Vcz, and fluconazole (Flu) concentrations within the bio-film matrices were determined by RP-HPLC. Results showed that C. glabrata biofilms were more susceptible to Vcz than to Flu and that ERG genes expression evidenced an overexpression of the three ERG genes in the presence of both azoles. The matrix content presented a remarked decrease in proteins and an increase in carbohydrates, namely b-1,3 glucans. Ergosterol was successfully detected and quantified in the biofilm matrices, with no differences in all the considered conditions. Vcz demonstrated better diffusion through the biofilms and better cell penetration capacities, than Flu, indicating that the structure of the drug molecule fully influences its dissemination through the biofilm matrices. This work showed that Vcz is notably more effective than Flu for the treatment of resistant C. glabrata oral biofilms, which demonstrates a clinical relevance in its future use for the treatment of oropharyngeal/esophageal candidiasis caused by this species.This work was supported by the Programa Operacional, Fatores de competitividade— COMPETE and by national funds through FCT—Fundação para a Ciência e a Tecnologia on the scope of the projects FCT [PTDC/SAU-MIC/119069/2010], [RECI/EBB-EBI/0179/2012], [PEst-OE/EQB/LA0023/2013] and Célia F. Rodrigues’ [SFRH/ BD/93078/2013] PhD Grant. The authors thank the Project ‘‘BioHealth—Biotechnology and Bioengineering approaches to improve health quality’’, Ref. NORTE-07-0124-FEDER000027, co-funded by the Programa Operacional Regional do Norte (ON.2—O Novo Norte), QREN, FEDER. The authors would like to acknowledge Pfizer, S.A. for the kindly donation of Voriconazole and Fluconazole.info:eu-repo/semantics/publishedVersio

Design of an antifungal surface embedding liposomal Amphotericin b through a mussel adhesive-inspired coating strategy

Microbial colonisation of urinary catheters remains a serious problem for medicine as it often leads to biofilm formation and infection. Among the approaches reported to deal with this problem, surfaces functionalization to render them with antimicrobial characteristics, comprises the most promising one. Most of these strategies, however, are designed to target bacterial biofilms, while fungal biofilms are much less taken into account. In real-life settings, fungi will be inevitably found in consortium with bacteria, especially in the field of biomaterials. The development of antifungal coating strategies to be combined with antibacterial approaches will be pivotal for the fight of biomaterial-associated infections. The main goal of the present study was, therefore, to engineer an effective strategy for the immobilization of liposomal amphotericin B (LAmB) on polydimethylsiloxane (PDMS) surfaces to prevent Candida albicans colonization. Immobilization was performed using a two-step mussel-inspired coating strategy, in which PDMS are first immersed in dopamine solution. Its self-polymerization leads to the deposition of a thin adherent film, called polydopamine (pDA), which allowed the incorporation of LAmB, afterwards. Different concentrations of LAmB were screened in order to obtain a contact-killing surface with no release of LAmB. Surface characterization confirmed the polymerization of dopamine and further functionalization with LAmB yielded surfaces with less roughness and more hydrophilic features. The proposed coating strategy rendered the surfaces of PDMS with the ability to prevent the attachment of Candida albicans and kill the adherent cells, without toxicity towards mammalian cells. Overall results showed that LAmB immobilization on a surface retained its antifungal activity and reduced toxicity, holding therefore a great potential to be applied for the design of urinary catheters. Since the sessile communities commonly found associated to these devices exhibit a polymicrobial nature, the next challenge will be to co-immobilize LAmB with antibacterial agents to prevent the establishment of catheter urinary-associated infections.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors also acknowledge the support, through the Programa Operacional Competitividade e Internacionalizacao (COMPETE2020) and by national funds, through the Portuguese Foundation for Science and Technology (FCT), of the POLY-PrevEnTT project (PTDC/BTM-SAL/29841/2017-POCI-01-0145-FEDER-029841).info:eu-repo/semantics/publishedVersio

Candida auris: a quick review on identification, current treatments, and challenges

Candida auris is a novel and major fungal pathogen that has triggered several outbreaks in the last decade. The few drugs available to treat fungal diseases, the fact that this yeast has a high rate of multidrug resistance and the occurrence of misleading identifications, and the ability of forming biofilms (naturally more resistant to drugs) has made treatments of C. auris infections highly difficult. This review intends to quickly illustrate the main issues in C. auris identification, available treatments and the associated mechanisms of resistance, and the novel and alternative treatment and drugs (natural and synthetic) that have been recently reported. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.L.C. thanks the Slovak Research and Development Agency under contract No. ˇ APVV-15-0347 and the grant of VEGA 1/0537/19 from the Ministry of Education, Science, Research, and the Sport of the Slovak Republic. C.F.R. would like to acknowledge the UID/EQU/00511/2020 Project—Laboratory of Process Engineering, Environment, Biotechnology and Energy (LEPABE)— financed by national funds through FCT/MCTES (PIDDAC).info:eu-repo/semantics/publishedVersio

Modulating an antimicrobial release approach by dopamine chemistryto fight infections associated to orthopedic implants

Alongside with orthopedic implants contribution for modern healthcare improvements, theres the risk associated to their microbial colonization and biofilm formation, compromising the performance of the implant itself and representing niches for infection. This study aimed to engineer an antimicrobial release coating for stainless steel surfaces (SS) to empower them with the ability to prevent Staphylococci colonization. Surface modification was based on dopamine chemistry, which self-polymerization results in the deposition of a thin, adhered film called polydopamine (pDA). Chlorohexidine (CHX) was chosen to confer the antimicrobial features. Its immobilization was performed through a 2-step approach, including pDA formation and immersion in CHX solution, and 1-step strategy, in which dopamine and CHX were dissolved together and SS coupons were immersed in this solution. An additional layer of pDA was also performed for both strategies. SEM and AFM confirmed pDA coating by the presence of self-polymerized pDA particles without altering the roughness of SS surfaces. Immobilization of CHX using a 1-step approach yielded surfaces with a more homogenous coating than the 2-step approach. Different pDA-based strategies yielded different CHX release profiles: the amount of CHX released was higher for the 2-step approach and the addition of another pDA layer reduced the amount of CHX released. The antimicrobial performance of the modified surfaces was evaluated against S. aureus and S. epidermidis and the results showed that all the strategies caused a significant reduction (more than 3 LOG) in the number of cells adhered to the surfaces and in suspension, after 24 h. The 2-step approach was able to impart SS surfaces with antimicrobial activity even after 10 days of exposure. In conclusion, dopamine chemistry can modulate CHX release from the surfaces to obtain an antimicrobial coating strategy with great potential to fight infections associated with orthopedic implants.info:eu-repo/semantics/publishedVersio

Big data warehouse framework for smart revenue management

Revenue Management’s most cited definitions is probably “to sell the right accommodation to the right customer, at the right time and the right price, with optimal satisfaction for customers and hoteliers”. Smart Revenue Management (SRM) is a project, which aims the development of smart automatic techniques for an efficient optimization of occupancy and rates of hotel accommodations, commonly referred to, as revenue management. One of the objectives of this project is to demonstrate that the collection of Big Data, followed by an appropriate assembly of functionalities, will make possible to generate a Data Warehouse necessary to produce high quality business intelligence and analytics. This will be achieved through the collection of data extracted from a variety of sources, including from the web. This paper proposes a three stage framework to develop the Big Data Warehouse for the SRM. Namely, the compilation of all available information, in the present case, it was focus only the extraction of information from the web by a web crawler – raw data. The storing of that raw data in a primary NoSQL database, and from that data the conception of a set of functionalities, rules, principles and semantics to select, combine and store in a secondary relational database the meaningful information for the Revenue Management (Big Data Warehouse). The last stage will be the principal focus of the paper. In this context, clues will also be giving how to compile information for Business Intelligence. All these functionalities contribute to a holistic framework that, in the future, will make it possible to anticipate customers and competitor’s behavior, fundamental elements to fulfill the Revenue Managemen

Susceptibility of Candida glabrata biofilms to echinocandins: alterations in the matrix composition

Candidiases are the most recurrent fungal infections, especially among immunosuppressed patients. Although Candida albicans is still the most widespread isolated species, non-Candida albicans Candida species have been increasing. The goal of this work was to determine the susceptibility of C. glabrata biofilms to echinocandins and to evaluate their effect on the biofilm matrix composition, comparing the results with other Candida species. Drug susceptibilities were assessed through the determination of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and minimum biofilm eradication concentration (MBEC) of caspofungin (Csf) and micafugin (Mcf). The -1,3 glucans content of the matrices was assessed after contact with the drugs. The data suggest that, generally, after contact with echinocandins, the concentration of -1,3 glucans increased. These adjustments in the matrix composition of C. glabrata biofilms and the chemical differences between Csf and Mcf, seem responsible and may determine the effectivity of the drug responses.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 [POCI-01–0145-FEDER-006684] and BioTecNorte operation [NORTE-01–0145-FEDER-000004] funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, Célia F. Rodrigues’ [SFRH/BD/93078/2013] PhD grant and M. Elisa Rodrigues [SFRH/BPD/95401/2013] post-doctoral grant.info:eu-repo/semantics/publishedVersio