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

Portrait of gene expression in C. glabrata with stress induced by drugs

Eurobiofilms 2017 - 5th European Congress on Microbial BiofilmsCandidiasis have globally increased over the last years, being a major cause of morbidity and mortality, especially in immunosuppressed and hospitalized patients. Candida albicans remains to be the most common species responsible in candidiasis, but Candida glabrata has appeared as second most common Candida in the USA and the third in Europe. Biofilms of this species are extremely difficult to eradicate and are a source of many recalcitrant infections. The goal of this study was to evaluate how C. glabrata cells try to adjust their biofilm composition in response to an antifungal drug treatment based on a series of profiles of eight genes expression - BGL2, FKS1, FKS2, GAS2, KHN1, UPG1, XOG1 and MNN2 known to be related to the production of -1,3, -1,6-glucans and mannans.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.info:eu-repo/semantics/publishedVersio

Combination of Posaconazole and Amphotericin B in the treatment of Candida glabrata biofilms

Candidemia cases have been increasing, especially among immunosuppressed patients. Candida glabrata is one of the most resistant Candida species, especially to the azole drugs, resulting in a high demand for therapeutic alternatives. The minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and minimum biofilm eradication concentration (MBEC) were determined for posaconazole (Pcz) and amphotericin B (AmB). The drug combinations of both drugs were evaluated on pre-formed biofilms of C. glabrata ATCC 2001, through XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay, colony forming units (CFU), crystal violet, and the fractional inhibitory concentration index (FICI). C. glabrata revealed higher susceptibility and biofilm reduction in the presence of AmB alone, but both drugs revealed a good capacity in the biomass elimination. In the majority of the tested combinations, the interactions were defined as indifferent (FICI 4). The combination of the two drugs does not seem to bring a clear advantage in the treatment of biofilms of C. glabrata.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) PhD grant.info: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

Mice infection with Candida glabrata biofilm cells: inflammatory cell recruitment and antifungal treatment efficacy

Background: Candida glabrata is one of the most widespread Candida spp. associated to systemic candidiasis. This species is particularly critical in hospitalized, catheterized and immunosuppressed patients, due to a high drug resistance, specially to the azoles, but also to the ability to rapidly develop echinocandin resistance. Objectives: The goal of this work was to simulate a systemic infection exclusively derived from C. glabrata biofilm cells and to evaluate the effectiveness of two echinocandins caspofungin (Csf) and micafungin (Mcf) - in its treatment. The host-pathogen response was also studied, by analyzing the inflammatory cell recruitment. Methods: CD1 mice were infected exclusively with 48 h-biofilm cells of C. glabrata and then treated with Csf or Mcf. After 72h, the efficacy of each drug was evaluated assessing organ fungal burden through CFU counting. Moreover, the immune cell recruitment into target organs was evaluated by flow cytometry and histopathology analysis. Results: Fungal burden was higher in the liver than in the kidneys. Nevertheless, none of the drugs was effective in eradicating completely the infection. At the evaluated time point, flow cytometry analysis, showed a predominant mononuclear response in the spleen, which was also evident in liver and kidneys of the infected mice, as observed by the histopathology analysis. Together, these observations confirmed C. glabrata as a low inflammatory species and indicated that two-dose treatment with Csf and Mcf do not have a significant impact on liver and kidney fungal burden, or recruited inflammatory infiltrate, when mice are i.v. infected with C. glabrata biofilm-grown cells.info:eu-repo/semantics/publishedVersio

Educar para a Liberdade : análise da UL 3, A Liberdade, do Programa de EMRC do 8º ano do 3º ciclo do Ensino Básico e Secundário

O trabalho e investigação espelhados neste relatório convergem para uma afirmação da inegável pedagogia da cultura religiosa, passível de ser ensinada e aprendida na disciplina de EMRC por abordar valores e saberes fundamentais à formação do indivíduo e a uma nova inteligibilidade do mundo. Especificamente, no âmbito da PES, trabalhámos, de forma analítica e crítica, a unidade letiva 3 – A Liberdade – referente ao 8º ano do terceiro ciclo do Ensino Básico e Secundário, com intenção de, por um lado, sustentar para este público-alvo, sedento de ferramentas de ajuda à compreensão da realidade, a importância da reflexão sobre o conceito universal de liberdade, que está no núcleo originário da dignidade humana, por outro lado, sustentar a identidade da disciplina e a distinção clara, embora complementar, que deve existir entre ela e a Catequese, nem sempre salvaguardada pelo Programa de EMRC, pela abordagem dos livros adotados e pela ação imparcial do professor.The work and research mirrored in this report converge towards an affirmation of the undeniable pedagogy of religious culture, which can be taught and learned in the discipline of EMRC by addressing values and knowledge fundamental to the formation of the individual and to a new intelligibility of the world. Specifically, in the scope of PES, we worked, in an analytical and critical way, the UL3 - Freedom - referring to the 8th year of Basic and High School, with the intention of, on the one hand, sustaining for this target audience, thirsty for tools to help them understand reality, the importance of reflection on the universal concept of freedom, which is at the original core of human dignity, on the other hand, to sustain the identity of the discipline and the clear, though complementary, distinction that must exist between it and Catechesis, not always safeguarded by the EMRC Program, the approach of the books adopted and the impartial action of the teacher

Third-generation cephalosporin-resistant klebsiella pneumoniae: Does the origin matter?

info:eu-repo/semantics/publishedVersio

Effect of azoles in Candida glabrata biofilms and its relation with ERG genes expression

The occurrence of fungal infections has been significantly increasing, thus contributing to higher morbidity and mortality. The use of broad-spectrum antibiotics, catheters, immunosuppression diseases, chemo and radiotherapy are predisposing factors for invasive fungal infection development. Candida albicans is the predominant species in both health and disease conditions, yet, in the last two decades the number of infections due to non-Candida albicans Candida species has increased significantly. Once believed as non-pathogenic, Candida glabrata rapidly was perceived to be responsible for many human diseases. Despite lacking a number of virulence factors allied to the majority of Candida pathogenicity, C. glabrata possesses high ability to colonize medical devices and human epithelium, resulting generally in biofilms formation ability. Its intrinsically low susceptibility to azoles, such as triazoles (e.g. fluconazole (Flu), voriconazole (Vcz)) and its biofilms tolerance is another problem. The aim of this study was to evaluate the effects of Flu (largely used) and Vcz (hospitalenvironment exclusive) in the control of C. glabrata biofilms and its relation with the expression of genes encoding for Ergosterol: ERG3, ERG6 and ERG11. Three isolates of C. glabrata (vaginal, urine and reference strain) were used. The Minimum Inhibitory Concentration was determined for planktonic cells and biofilms were formed during 24h and treated (for 24h) with different concentrations of both antifungal agents. The effects of Flu and Vcz were analyzed by Colony Forming Units determination and by total biomass quantification using Crystal Violet staining. Biofilms were also analyzed by scanning electron microscopy. Total proteins and carbohydrates were quantified from biofilms’ matrices. Moreover, ergosterol present in the matrices was also quantified by HPLC. To end, qRT-PCR was used to study the gene expression of selected ERG genes. Our results show that, unlike Flu, Vcz had a very good Candida biofilm eradication capacity. No fluctuations between the two azoles were noticed in terms of proteins and carbohydrates, both presenting a blocked production in the first and an overmetabolism in the second. A new finding was the detection of ergosterol in the matrices. The gene expression study showed overexpression of ERG genes, in the presence of the both drug compounds. This work reveals the extraordinary capacity of C. glabrata to change with the purpose of overcome the adversities of the environment. The increase in ergosterol present in matrices and the overexpression of Erg genes could be an explanation for higher C. glabrata biofilms tolerance. This aptitude makes hampered the action of drugs against the cells, and when passing to the progeny, is, undoubtedly, a great advantage to the development of resistance to antifungals, in Candida species

Novel strategies to fight Candida infections: natural honey

Poster apresentado no "CEB Annual Meeting", em Braga, Portugal, 2017The incidence of Candida infections (Candidosis) has increased remarkably in the last years, being attributed to the rise in the elderly population, the increasing number of immunocompromised patients, and the widespread use of indwelling medical devices. Candida albicans remains as the most prevalent species of these infections, but a clear rise in the proportion of non-Candida albicans Candida (NCAC) species has been noted. These species have an inherent level of resistance to certain antifungal agents higher than C. albicans though their virulence factors are much less understood. A major virulence factor is the ability to adhere and to form biofilms in medical devices and host tissues, because of a higher tolerance to antifungal therapy. Consequently, there is an urgent need to develop new strategies to fight these infections. Natural compounds are attracting increased interest in this field, among which honey. AIM: To evaluate honey as a novel strategy to fight C. tropicalis infections.This study was supported by 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.info:eu-repo/semantics/publishedVersio