Estratégias alternativas para combater biofilmes de Candida

Dissertação de Mestrado em BiotecnologiaAs infeções fúngicas nosocomiais causadas por espécies de Candida representam as causas mais comuns de infeção do trato urinário e da corrente sanguínea. Estas infeções estão geralmente associadas a biofilmes. Nestes casos, quando ocorre infeção, os tratamentos existentes são praticamente ineficazes, existindo assim uma necessidade crescente na procura de terapias antifúngicas mais eficazes e com menores efeitos adversos colaterais. Nesta perspetiva, surgiu o mel como potencial agente antifúngico, devido às suas propriedades: antioxidantes, anti-tumorais, anti-inflamatórias e antivirais, reconhecidas por todo o mundo. Posto isto, este estudo tem como objetivo principal avaliar o efeito do mel em espécies de Candida, assim como testar o efeito da combinação de mel com diferentes agentes antifúngicos comerciais, nomeadamente, nistatina (NIS), clorohexidina (CLR) e fluconazol (FLC). Para tal, inicialmente avaliou-se o efeito do mel urze e do mel manuka em células de Candida em crescimento planctónico pelo método de difusão em agar e foram determinadas as concentrações mínimas inibitórias (CMIs). De seguida, foi avaliado o efeito antifúngico dos mesmos méis em biofilmes pelos métodos cristal violeta (CV), contagem de Unidades Formadoras de Colónias (UFC) e por último microscopia de fluorescência. Adicionalmente, numa perspetiva clínica tornou-se fulcral avaliar o efeito do mel em biofilmes polimicrobianos de C. tropicalis e P. aeruginosa, visto que na natureza os biofilmes são constituídos por mais de uma espécie microbiana, conferindo-lhes uma maior resistência. Nesta associação, foram também testadas as combinações de mel com FLC e mel com FLC e gentamicina. Os resultados demostraram que o mel consegue reduzir o crescimento de todas as espécies em estudo, quer em estado planctónico como em biofilmes, demostrando maior efeito em Candida tropicalis. Na terapia combinacional os resultados sugerem que a combinação de mel com CLR e mel com NIS são significativamente mais eficazes que o tratamento apenas com mel. Em biofilmes polimicrobianos o mel demostrou ser uma escolha promissora, visto que permitiu reduzir o biofilme para ambas as espécies. Neste caso, as terapias combinacionais não demostraram o efeito pretendido. Apesar disso, estas terapias poderão ser promissoras utilizando agentes antimicrobianos diferentes ou alterando as concentrações. Em geral, os resultados obtidos neste estudo destacaram o grande potencial do mel como estratégia alternativa ou complementar para o controle de infeções provocadas por Candida.Nosocomial fungal infections triggered by Candida species represent the most common causes of infection of the urinary tract and bloodstream. These infections are usually associated with biofilms. In these cases, when an infection occurs, the existing treatments are practically ineffective, so there is an increasing need to find more effective antifungal therapies with less adverse side effects. In this perspective, honey emerged as a potential antifungal agent, due to its antioxidant, antitumoral, anti-inflammatory and antiviral properties, recognized all over the world. Hence, the aim of this study was to evaluate the effect of honey on Candida species, as well as to test the effect of combining honey with different commercial antifungal agents, namely nystatin (NIS), chlorhexidine (CLR) and fluconazole (FLC). For this, the effect of heather honey and manuka honey on planktonic growth of Candida was initially evaluated by the agar diffusion method to determine minimum inhibitory concentrations (MICs). Then, the same types of honey were evaluated in biofilms by crystal violet (CV) staining, Colony Forming Units (CFU) counting and fluorescence microscopy. Moreover, from a clinical viewpoint, it became essential to assess the effect of honey on polymicrobial biofilms of C. tropicalis and P. aeruginosa, since biofilms are composed of more than one microbial species in nature, which gives it a higher resistance. In addition to this evaluation, the combinations of honey plus FLC and honey plus FLC and gentamicin were also tested. The results have shown that honey enables a growth reduction of all species under study, both in planktonic state and in biofilms, with a greater effect on Candida tropicalis. In combinational therapy, the results suggest that the combination of honey with CLR, and honey with NIS are significantly more effective than treatment with honey alone. In polymicrobial biofilms, honey has shown to be a promising choice, since it was able to reduce the biofilm in both species. In this case, combinational therapies did not demonstrate the intended effect. Nonetheless, these therapies could have a greater potential when using different antimicrobial agents or by altering their concentrations. In general, the results obtained in this study highlight the great potential of honey as an alternative or complimentary strategy for the control of Candida infections.Este estudo foi apoiado pela FCT no âmbito do financiamento estratégico da unidade UID/BIO/04469/2013 e COMPETE 2020 (POCI-01-0145-FEDER-006684) e operação BioTecNorte (NORTE-01-0145-FEDER-000004) financiado pelo Fundo Europeu de Desenvolvimento Regional no âmbito de Norte 2020 - Programa Operacional Regional do Norte

Simulated Vaginal Fluid: Candida resistant strains biofilm characterization and vapor phase of essential oil effect

Introduction : Vulvovaginal candidiasis is a disease that affects millions of women worldwide. Oral formulations, topical creams or ointments are the conventional dosage forms, with an increase in drug administration through vaginal via. The use of simulated biological fluids (e.g. vaginal fluid) in the evaluation of antifungal therapies may better mimic the real biological environments and therefore provide a better understanding of the behavior of the antifungal. Methods : The main objective of this work was to compare planktonic growth and biofilm formation of Candida species, on common growth medium, Sabouraud Dextrose Broth (SDB) and on vaginal simulation conditions, Simulated Vaginal Fluid (SVF), through the optical density determination, colony-forming units and scanning electron microscopy. In addition, under the same conditions this study also evaluated the ability of vapor phase of oregano and white thyme essential oils (VP-EOs), potential alternative treatment, to inhibit biofilm formation and to destroy mature biofilms of vaginal isolates, through the colony-forming units determination. Results : Candida isolates maintained the same biofilm formation capacity and morphology in both media (SVF and SDB). Furthermore, the results obtained in this work related with VP-EOs effect agree with results acquired, previously, with SDB. This means that the effect of VP-EOs is not affected by the SVF medium, and that this fluid allows the dissolution of the volatile and bioactive compounds. Conclusions : These results can predict the in vivo behaviour, suggesting a potential effective application of VP-EOs as prophylactic or therapeutic treatment for biofilm-related vulvovaginal candidiasis.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 Microelectromechanical Systems, LA/P/0029/2020 and Maria Elisa Rodrigues thanks FCT for funding through program DL 57/2016—Norma transitóriainfo: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

Candida albicans antimicrobial and antibiofilm activity of novel endodontic solvents

Background: Candida albicans is the most prevalent fungi isolated in endodontic infections. In this study, the ability of C. albicans biofilm to tolerate the novel solvent mixtures methyl ethyl ketone (MEK)/tetrachloroethylene (TCE) and MEK/orange oil (OOil) sequentially to the standard irrigation of sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic (EDTA) was evaluated. Methods: Biofilm cell cultures of C. albicans SC 5314 were treated sequentially with NaOCl and EDTA and exposed to MEK/TCE or MEK/OOil. The effect of the antimicrobial treatment was evaluated using the disk diffusion method for planktonic culture, and the enumeration of colony-forming units (CFUs) and scanning electron microscope (SEM) for biofilm cell culture. Results: C. albicans mature biofilm (24 h) was significantly reduced in biomass and cell viability after solvent mixtures exposure, compared with the previous NaOCl and EDTA treatments. MEK/OOil combination caused a total reduction of biofilm, while with MEK/TCE, there was a 3-log (CFU/cm2) reduction compared with the sequence NaOCl and EDTA, and a 4-log (CFU/cm2) reduction compared with the control. Conclusions: The additional exposure of a preformed 24 h C. albicans biofilm to novel solvent mixtures MEK/TCE and MEK/OOil caused a positive antibiofilm impact, overcoming the performance of the conventional endodontic irrigating protocol.This article was supported by National Funds through FCT-Fundação para a Ciência e a Tecnologia, I.P., within CINTESIS, R&D Unit (reference UIDB/4255/2020). This work 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; 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

Analysis of the coupling between a single-mode fiber to a multi-core fiber with long-period gratings

In this paper, the coupling of a single source injected in a single-mode fiber to all the cores of a multi-core fiber is theoretically studied. The power transfer between the core and the cladding of a fiber is promoted by long-period gratings. To promote the power transfer between the fibers, we considered cladding modes with similar effective refractive index. The results show that the coupling is possible, but the design still needs to be optimized to maximize the power transfer.publishe

Vapor-phase of essential oils as a promising solution to prevent Candida vaginal biofilms caused by antifungal resistant strains

Background: Vulvovaginal candidiasis (VVC) is a disease with high incidence, a huge impact on the quality of life and health of women, and which represents a great challenge to treat. The growing need to apply antifungal intensive therapies have contributed to an emergence of drug-resistant Candida strains. Thus, effective therapeutic options, to meet the antifungal-resistance challenge and to control high resilient biofilms, are urgently needed. This study aimed to investigate the antifungal activity of essentials oils (EOs) on drug-resistant Candida vaginal isolates. Method: Therefore, the antimicrobial effect of tea tree, niaouli, white thyme, and cajeput EOs on the planktonic growth of Candida isolates was initially evaluated by an agar disc diffusion method. Then, the vapor-phase effect of tea tree EO (VP-TTEO) on biofilm formation and on pre-formed biofilms was evaluated by crystal violet staining, XTT reduction assay, colony forming units’ enumeration, and scanning electron microscopy. Results: The results revealed high antifungal activity of EOs against drug-resistant Candida isolates. Additionally, the VP-TTEO showed a significant inhibitory effect on the biofilm formation of all tested isolates and was able to provoke an expressive reduction in mature Candida albicans biofilms. Conclusions: Overall, this study suggests that the VP-EO may be a promising solution that is able to prevent biofilm-related VVC caused by antifungal-resistant strains.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 Microelectromechanical Systems, LA/P/0029/2020 and Maria Elisa Rodrigues thanks FCT for funding through program DL 57/2016—Norma transitória.info:eu-repo/semantics/publishedVersio

Characterization of molecular features and virulence profile of Klebsiella pneumoniae and Klebsiella oxytoca isolates from companion animals in Portugal

Klebsiella spp. are important pathogens that affect both humans and animals and can cause serious life-threatening diseases. The increasing incidence of Klebsiella infections in companion animals (e.g., cats and dogs) can result in the death of animals and become a serious public health concern. The study of strains isolated from animal infections can be a means of assessing the risk of transmission to humans, including zoonotic potential. The aim of this study was to characterize the genetic and phenotypic features of Klebsiella pneumoniae and Klebsiella oxytoca previously isolated from ill companion animals by whole genome sequencing, followed by in vitro evaluation of biofilm formation. The Galleria mellonella model was also used to evaluate the in vivo pathogenicity of Klebsiella isolates. K. pneumoniae isolates tested exhibited two LPS O-types (O3B and O1/O2v2) and only one LPS O-types was detected for K. oxytoca isolates (OL104). Among the STs, ST11 and ST266 were the most frequently found. In turn, K. pneumoniae showed a high diversity of K-locus types (KL102; KL105; KL31, and KL13). Among K. pneumoniae, a specific pattern (i.e., KL105-ST11-O1/O2v2) raises concern due to its high resistance and virulence towards human hosts. Furthermore, this pattern was associated with a high inflammatory response observed in G. mellonella larvae, with approximately 80% of the larvae dead at 72 h post-infection, which is not directly related to the ability of Klebsiella spp. to form a biofilm. The present study highlights a noteworthy level of pathogenicity associated with Klebsiella spp. isolated from companion animals. Consequently, it underscores the potential for dogs and cats to serve as reservoirs of resistant Klebsiella spp. that could pose a risk of transmission to humans.This work was financially supported by: LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE), funded by national funds through FCT/MCTES (PIDDAC). In addition, this study was also funded by FCT (Portuguese Foundation for Science and Technology), under the scope of the strategic funding of UIDB/04469/2020 unit, LABELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems—LA/P/0029/2020 (CEB). J.C. also thanks FCT for the CEEC Individual (2022.06886.CEECIND).info:eu-repo/semantics/publishedVersio

Genomic and phenotypic profiling of multidrugresistant Klebsiella pneumoniae and Klebsiella variicola isolated from northern portuguese hospitals

With the alarming increase in antibiotic resistance and the global spread of resistance mechanisms, the emergence of antibiotic-resistant Klebsiella spp. has become a significant challenge in clinical settings. It is therefore important to monitor the characteristics of Klebsiella isolates in circulation to develop mitigation solutions. The aim of this study was to characterize the genetic and phenotypic characteristics of 21 Multidrug- Resistant (MDR) Klebsiella isolates, including 19 K. pneumoniae, and 2 K. variicola isolated from patients admitted to two central hospitals in northern Portugal using whole-genome sequencing (WGS) and subsequent bioinformatic analysis of several virulence factors, including capsule types (K-types), LPS O antigen serotype and sequence types (ST). Furthermore, we assessed the in vitro biofilm-forming capacity and the hypermucoviscosity of Klebsiella isolates, as well as the ability of Klebsiella spp. to infect Galleria mellonella, a larval in vivo model. Genetic results showed a high prevalence of O1/O2 serotypes (14/21; 67%) among the isolates tested, which is consistent with previous reports from Portugal. In contrast, a wide variety of K locus types was found, in our study, where ST15-KL19 (4/21; 19%) associated to serotype O1/O2v2 was the dominant. Within the O1/O2v2 serotype, a ST15-KL23 K. variicola, was the one harbouring a high number of virulence genes. We also found statistical differences in the ability to produce biofilm biomass within the strains, with a ST280-KL23 K. pneumoniae outcompeting with nine other strains. According to our results, the most prominent serotype able to cause the death of G. mellonella was the KL105-O1/O2v2. In conclusion, our results emphasize the importance of conducting continuous molecular surveillance in order to identify the key molecular features to be considered in the development of novel strategies for the treatment of Klebsiella spp.-associated infections.This work was financially supported by: LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE), funded by national funds through FCT/MCTES (PIDDAC). In addition, this study was also funded by FCT (Portuguese Foundation for Science and Technology), under the scope of the strategic funding of UIDB/04469/2020 unit, LABELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems—LA/P/0029/2020 (CEB), Inov4Agro-Associate Laboratory for Inovation, Building and Sustainability in Agri-Food Production—LA/P/0126/2020, and projects UIDB/04033/2020 and UIDP/04033/2020 (CITAB). J.C. also thanks FCT for the CEEC Individual (2022.06886.CEECIND).info:eu-repo/semantics/publishedVersio

Will e-Vaccinas be a Quality Information System?

The quality of information is essential for the survival of any institution, which is why Health Services need quality systems that effectively meet the demands of their users. The quality of the e-Vaccines was assessed through the Data Quality Assessment methodology in two districts of Alentejo, which revealed that this information system plays an important role in the quality of care delivery, particularly in vaccination at the level of efficiency and effectiveness of service to users and in reducing the risk of errors. The e-Vaccines is a quality system, since the assessment of the four dimensions proposed by the authors revealed a global result considered strong, also demonstrating that this application is an essential work tool in care delivery, facilitating communication and the information sharing, thus promoting the existence of coordinated interventions among professionals

Prognostic factors of patients admitted in a medical intermediate care unit: a prospective observational study

BackgroundMedical Intermediate Care Units (IntCU’s) are high-dependency units intended for treatment of patients who do not meet criteria for admission to intensive care units (ICU’s) but require a higher level of care than can be provided in general ward. IntCU’s operate as a transitional unit, improving patients outcome. In addition, IntCU’s permit better resource utilization, reducing the length of stay in ICU and therefore increasing availability of critical care. Regardless of admission of unstable patients in IntCU’s, studies on prognostic factors are scarce. Our purpose was to identify prognostic factors of patients admitted in IntCU.MethodsA prospective observational study was performed during 32 months in a IntCU of a central hospital. Main objective was evaluation of mortality and analysis of acute illness severity, nurse workload, comorbidity and previous functional status as prognostic factors. Assessment of these variables was performed using several standardized scores: Acute Physiology and Chronic Health Evaluation II (APACHE II), Simplified Acute Physiology Score II (SAPS II), Sequential Organ Failure Assessment (SOFA), Therapeutic Intervention Scoring System-28 (TISS-28), Nursing Activities Score (NAS), Charlson comorbidity index and Barthel index of basic activities of daily living. Bivariate and multiple logistic regression with forwards stepwise selection were used to identify prognostic factors of IntCU and in-hospital mortality.ResultsTwo hundred and eighty-eight patients were included, mean age 65,67 ± 20,38 years-old. IntCU and in-hospital mortality was 9,38 and 17,71%, respectively. All the scores applied, concerning comorbidity, functional status, acute illness severity and nurse workload were good predictors of mortality. SAPS II was the better predictor of mortality followed by NAS.ConclusionsAcute illness severity and nursing workload scores validated in ICU are useful and reliable in IntCU setting, being SAPS II and NAS the strongest predictors of mortality. Comorbidity, functional status and age were also prognostic factors. Consequently, a comprehensive assessment of patients admitted in IntCU is mandatory to reliably predict outcome. Several scores might be used to help clinical judgment, concerning admission criteria and clinical decisions. Â