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

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

Natural honey to fight Candida infections

Eurobiofilms 2017 - 5th European Congress on Microbial BiofilmsThe 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

Mnn2 gene affects drug resistance in Candida glabratas biofilms

Eurobiofilms 2017 - 5th European Congress on Microbial BiofilmsInfections caused by Candida species have increased worldwide substantially over the latest decades, and are a significant cause of morbidity and mortality, mostly among critically ill patients. Candida glabrata is the second most common Candida responsible for these infections in the USA and the third in Europe, and is characterized by a high antifungal resistance. The goal of this study was to understand the role of mannans in C. glabrata biofilms and in biofilm cells resistance to antifungal drugs (fluconazole - Flu, amphotericin B - AmB, caspofungin - Csf and micafungin - Mcf)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 Norteinfo:eu-repo/semantics/publishedVersio

Honey as a strategy to fight Candida tropicalis in mixed-biofilms with Pseudomonas aeruginosa

Fungal contaminations with Candida species are commonly responsible for several infections, especially when associated to bacteria. The therapeutic approach commonly used is being compromised due to microbial resistances of these microorganisms to antimicrobial agents, especially in biofilm. The use of honey as an antimicrobial agent has been emerging as a valuable solution and proving its potential in planktonic and in biofilm cells. This work aims to assess the effect of different honeys on biofilms of Candida tropicalis and Pseudomonas aeruginosa. The effect of Portuguese heather (PH) and manuka honeys on planktonic growth of Candida was initially evaluated by determination of the minimum inhibitory concentrations (MIC). Then, the same effect was evaluated in mixed biofilms, by colony-forming units numeration and fluorescence microscopy. The combinations of honey plus fluconazole and gentamicin were also tested. The results showed that the honeys tested enabled a great reduction of C. tropicalis, both in planktonic (12.5% and 25% of MIC for PH and manuka) and in biofilm. In polymicrobial biofilms, the use of PH and manuka honeys was revealed to be a promising choice and an alternative treatment, since they were able to reduce cells from both species. No synergistic effect was observed in antimicrobial combinations assays against polymicrobial biofilms.This work 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 ofNorte2020—Programa Operacional Regional do Norte and by PTDC/CVT-EPI/4008/2014 project.info:eu-repo/semantics/publishedVersio

Essential oils as a good weapon against drug-resistant Candida auris

Candida auris is a recently found Candida species, mainly associated with nosocomial outbreaks in intensive care hospital settings, and unlike other Candida species, it can be transmitted through person-to-person or by contact with surfaces. C. auris is described as resistant to first-line antifungals and, consequently, associated with high mortality. Nowadays, essential oils (EOs) are known to be effective against fungal and bacterial infections. This work aimed to evaluate the effect of four EOs (tea tree, niaouli, white thyme and cajeput) against C. auris. The EO’s effect on C. auris planktonic growth was evaluated by the minimum inhibitory concentration determination and by the agar disc diffusion method. Then, the same effect was evaluated on biofilm by colony-forming units’ enumeration. The results showed that EOs were able to inhibit the C. auris planktonic growth, with an MIC50 between 0.78 and 1.56% and halos of 20–21 mm for white thyme and tea tree and 13–14 mm for cajeput and niaouli. In addition, the EOs were also able to completely inhibit biofilm formation. Moreover, white thyme and cajeput completely eradicate pre-formed biofilms, while tea tree and niaouli significantly reduce it. Thus, this work demonstrates that EOs are a possible therapeutic alternative and a future perspective for the hard fight against C. auris.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and grant ref 2020.05720.BD for Liliana Fernandes. Also, this study was supported by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020 and M. Elisa Rodrigues thanks FCT for funding through program DL 57/2016—Norma transitória.info:eu-repo/semantics/publishedVersio

The chemical structure of the biofilm matrices of Candida glabrata induce resistance to antifungal drugs

Candida infections are often associated to biofilms and consequently to high resistance to the most common drugs. These resistance mechanisms are not only associated with the biofilm yeast physiology, but also with the presence of a barrier imposed by the biofilm matrix. However, the biochemical role of the biofilm components remains very unclear. Therefore, this work intends to further enlighten the effect of antifungal agents on C. glabrata biofilm resistance.As a good biofilm former, Candida glabrata ATCC 2001 was selected to this study. Several antifungal drugs, belonging to different groups, were used in this work, namely fluconazole, voriconazole, amphotericin B, caspofungin and micafungin and their effect on biofilm matrix was assessed. Biofilm matrix chemical composition and structure was evaluated by analytical methods, specifically by HPLC and mass spectroscopy.As expected, C. glabrata biofilms were resistant to the antifungals used in an agent-dependent manner. The results showed significant differences in polysaccharides and proteins contents, in the matrix of biofilms formed in the absence and in the presence of the drugs. Moreover, the diffusion through the matrix was evaluated demonstrating that different agents, even belonging to the same group, present very different diffusion profiles, explaining the different tolerance registered. So, with this study we confirmed that C. glabrata biofilms resistance to antifungal drugs is a very complex mechanism, where the matrix plays a major role

Pseudomonas aeruginosa-Candida albicans polymicrobial biofilms in ventilator-associated infections (VAP): evaluating the post-antimicrobial effect of amphotericin B/polymyxin B combined activity

Introduction: Mixed bacterial-fungal colonization of the endotracheal tubes is now evident, with microbial interplay withstanding common antimicrobial therapy and paying for persistent and severe VAP infections. While alternative therapeutic strategies effectively targeting inter-kingdom biofilms are required, the role of each microorganism need to be appraised to deliver effective treatments. Hypothesis and aims: We earlier reported the combination therapy involving polymyxin B (PMB) and amphotericin B (AMB) as holding an attractive therapeutic option to treat dual-species biofilms. This study aimed to determine the post-antimicrobial phenomenon of PMB/AMB combined action in P. aeruginosa (PA) +Candida albicans (CA) biofilms, and to ascertain the events underlying biofilm growth restoration. Methodology: Post-antimicrobial effect of PMB combined with AMB was assessed in 24-h dual-species biofilms. Cell culturability and viability were evaluated by CFU and Live/Dead staining, respectively. The gene expression profile was assessed by qPCR. Results: Results showed that PA+CA biofilms lost their culturability straightaway being exposed to PMB/AMB combined solution. However, 24h was enough to both species recover their growth onto agar medium, with microbial counts approximating those observed for pre-treated biofilms. Following the subsequent treatment cycle, CFU estimation was only slightly disturbed. L/D results revealed that PA and CA populations displayed a compromised status at the end of the first PMB/AMB treatment cycle. Finishing the 24-h-regrowth cycle, most biofilm-encased species exhibited viability, which endured after the second treatment period. Transcriptional analysis of dual-species biofilms exposed to PMB/AMB combined action showed a high expression level in all PA resistance-encoded genes anrB, galU, mexA and algD and in ERG3 and ALS2 CA genes. Conclusion: Our finsings showed that PA+CA biofilms were able to escape to the combined action of PMB/AMB, and both species had a preeminent role while retaining adaptive resistance mechanisms that likely contributed for their recovery and adaptation on the ensuing treatments.info:eu-repo/semantics/publishedVersio

L. crispatus protects Hela cells against G. vaginalis cytotoxicity

Eurobiofilms 2017 - 5th European Congress on Microbial BiofilmsBacterial vaginosis (BV) is the most common lower genital tract disorder among women of reproductive age and is characterized by a shift in the vaginal flora from the dominant Lactobacillus to a polymicrobial flora, being Gardnerella vaginalis the predominant species of the biofilm mass. However, G. vaginalis vaginal colonization does not always result in BV. Accurate in vitro model systems mimicking in vivo conditions are required to better understand the complex host-microbe and microbe-microbe interactions. In effort to analyse the adaptation and interaction of the commensal vaginal microbiota and pathogens in the vaginal environment, we used a genital tract simulant medium (mGTS) to evaluate the growth of resident vaginal Lactobacillus crispatus and G. vaginalis in the adopted growth medium. Also, to understand the differences between G. vaginalis strains isolated from women with and without BV, we performed in vitro assays to compare the virulence properties of G. vaginalis strains. In this sense, G. vaginalis strains were characterised for their cytotoxicity activity after adhesion on a monolayer of epithelial cells pre-adhered with L. crispatus, mimicking the healthy vagina environment. Furthermore, transcript levels of vaginolysin and sialidase genes were also evaluated. These assays revealed that a BV isolate of G. vaginalis was significantly more cytotoxic than a non-BV isolate after 3 hours in the contact with a monolayer of HeLa cells. However, when L. crispatus was pre-adhered on a monolayer of HeLa cells, the cytotoxicity effect of both strains observed was drastically reduced. Significant differences in the transcript levels of both genes were also observed in the presence of L. crispatus. Thus, this work highlights not only the discrepant virulence potential of two distinct variants of G. vaginalis but also the beneficial role of vaginal lactobacilli in protecting the vaginal epithelium from G. vaginalis infection.info:eu-repo/semantics/publishedVersio

Insights into Pseudomonas aeruginosa and Candida albicans consortia challenged by antimicrobials

Ventilator associated pneumonia (VAP), an usual nosocomial infection in the intensive care units and the most common in mechanically ventilated patients, is a serious problem due to high mortality and morbidity rates associated. The presence of the endotracheal tube is the principal risk factor for developing VAP because its surface is prone to microbial adhesion and the formation of biofilms, deserving thus high attention in clinical settings. Cell-to-cell communication is an important mechanism of interaction between VAP microorganisms, being involved in the process known as quorum-sensing (QS) that regulate the expression of virulence. To evaluate bacteria fungi cross-talk in co-infection, the biofilm-forming ability of Pseudomonas aeruginosa and Candida albicans, individually or jointly, before and after antibiotic and antifungal co-treatment was tested. Biofilms were characterized in terms of total mass and cell viability. Results showed that no antimicrobial combination was successful in the binary biofilms eradication. In some cases, the tolerance of the polymicrobial consortia was higher than that of single biofilms, highlighting that P. aeruginosa and C. albicans established synergistic relationships. To gain knowledge helping to explain those interactions, a quantitative real-time PCR approach was followed to inspect the expression profiles of some cell-cell communication genes involved in biofilm resistance. To overcome the tolerance issues, new antimicrobial combinatorial approaches using QS-inhibitors are being tested. Some combinations involving chlorogenic acid and ciprofloxacin displayed promising anti-biofilm potential