Molecular characterization and virulence profile of Klebsiella pneumoniae and Klebsiella oxytoca isolated from ill cats and dogs in Portugal.

Klebsiella spp. are important pathogens of humans and companion animals such as cats and dogs, capable of causing severe life-threatening diseases. The aim of this study was to characterize the molecular and phenotypic properties of Klebsiella pneumoniae and Klebsiella oxytoca isolated from ill companion animals by whole genome sequencing, followed by in vitro assessment of biofilm formation and in vivo pathogenicity using the Galleria mellonella model. Two LPS O-types were identified for all the K. pneumoniae isolates tested (O3B and O1/O2v2) and only one for K. oxytoca isolates (OL104), and the most common STs found were ST11 and ST266. Furthermore, a high diversity of K-locus types was found for K. pneumoniae (KL102; KL105; KL31, and KL13). Within K. pneumoniae, one specific O/K/ST-types combination (i.e., KL105-ST11-O1/O2v2) showed results that were of concern, as it exhibited a high inflammatory response at 12 h post-infection in G. mellonella with 80% of the larvae dead at 72 h post-infection. This virulence potential, on the other hand, did not appear to be directly related to the biofilm-forming capacity. Also, virulence and resistance scores obtained for this set of strains did surpass score 1. The present study demonstrated that Klebsiella spp. isolated from companion animals belonging to STs that can cause human infections and present virulence on an invertebrate model. Thus, this study underscores the role of dogs and cats as reservoirs of resistant Klebsiella spp. that could potentially be transmitted to humans.

Multifunctional Magnetoelectric Sensing and Bending Actuator Response of Polymer-Based Hybrid Materials with Magnetic Ionic Liquids.

With the evolution of the digital society, the demand for miniaturized multifunctional devices has been increasing, particularly for sensors and actuators. These technological translators allow successful interaction between the physical and digital worlds. In particular, the development of smart materials with magnetoelectric (ME) properties, capable of wirelessly generating electrical signals in response to external magnetic fields, represents a suitable approach for the development of magnetic field sensors and actuators due to their ME coupling, flexibility, robustness and easy fabrication, compatible with additive manufacturing technologies. This work demonstrates the suitability of magnetoelectric (ME) responsive materials based on the magnetic ionic liquid (MIL) 1-butyl-3-methylimidazolium tetrachloroferrate ([Bmim][FeCl4]) and the polymer poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE) for magnetic sensing and actuation device development. The developed sensor works in the AC magnetic field and has frequency-dependent sensitivity. The materials show voltage responses in the mV range, suitable for the development of magnetic field sensors with a highest sensitivity (s) of 76 mV·Oe-1. The high ME response (maximum ME voltage coefficient of 15 V·cm-1·Oe-1) and magnetic bending actuation (2.1 mm) capability are explained by the magnetoionic (MI) interaction and the morphology of the composites.

Effect of Vapor-Phase Oregano Essential Oil on Resistant Candida Species Biofilms: Mechanisms of Action.

Vulvovaginal candidiasis (VVC) is one of the most prevalent vaginal infectious diseases. The increasing incidence of drug-resistant Candida strains and the limited therapeutic options make the discovery of effective alternative therapies fundamental. Essential oils (EOs) have been suggested as a promising alternative, and interestingly, vapor-phase essential oils (VP-EOs) present more advantages than their direct application. Thus, this study aims to evaluate the effect of oregano VP-EO (VP-OEO) on biofilms of antifungal-resistant vaginal isolates of Candida species (Candida albicans and Candida glabrata) and determine its mode of action. CFU, membrane integrity, and metabolic activity were evaluated. Furthermore, a reconstituted vaginal epithelium was used to mimic vaginal conditions and evaluate the effect of VP-OEO on Candida species infection, analyzed by DNA quantification, microscopy, and lactate dehydrogenase activity. The results revealed high VP-OEO antifungal activity. There was a significant reduction (>4 log CFU) in Candida species biofilms. Furthermore, the results show that the mechanisms of action of VP-OEO are related to membrane integrity and metabolic activity. The epithelium model confirms the effectiveness of VP-OEO. This study suggests that VP-EO can be considered a first approach for the development of an alternative form of VVC treatment. IMPORTANCE This work presents a new approach to the application of essential oils, exposure to the vapor phase, which can be considered a first approach for the development of a complementary or alternative form of vulvovaginal candidiasis (VVC) treatment. VVC is a significant infection caused by Candida species and remains a common disease that affects millions of women every year. The great difficulty in treating VVC and the extremely limited effective therapeutic options make the development of alternative treatments crucial. In this scope, this study aims to contribute to the development of effective, inexpensive, and nontoxic strategies for the prevention and treatment of this infectious disease, based on natural products. Moreover, this new approach has several advantages for women, such as lower costs, easy access, an easier mode of application, avoidance of skin contact, and, therefore, fewer negative impacts on women's health.

The epidemiology of the COVID-19 pandemic in the small, low-resource country of Timor-Leste, January 2020 - June 2022.

Abstract: Timor-Leste, a small, mountainous half-island nation which shares a land border with Indonesia and which is 550 km from Australia, has a population of 1.3 million and achieved independence for the second time in 2002. It is one of the poorest nations in Asia. In response to the global coronavirus disease 2019 (COVID-19) pandemic, the Timor-Leste Ministry of Health undertook surveillance and contact tracing activities on all notified COVID-19 cases. Between 1 January 2020 and 30 June 2022, there were 22,957 cases of COVID-19 notified which occurred in three waves, the first which was delayed until April 2021 (community transmission of B.1.466.2 variant following major flooding), followed by waves in August 2021 (B.1.617.2 Delta variant transmission) and February 2022 (B.1.1.529 Omicron variant transmission). There were 753 people hospitalised due to COVID-19 and 133 deaths. Of the 133 deaths, 122 (92%) were considered not fully vaccinated (< 2 COVID-19 vaccines) and none had received boosters. Timor-Leste implemented measures to control COVID-19, including: rapid closure of international borders; isolation of cases; quarantining of international arrivals and close contacts; restrictions on internal travel; social and physical distancing; and, finally, a country-wide vaccination program. The health system's capacity was never exceeded.

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 behavior, suggesting a potential effective application of VP-EOs as prophylactic or therapeutic treatment for biofilm-related vulvovaginal candidiasis.

Candida auris, a singular emergent pathogenic yeast: its resistance and new therapeutic alternatives.

Nowadays, fungal infections affect millions of people across the world. Candida auris, a new emergent yeast, is a worrisome pathogen because it associates with a high rate of incidence and prevalence, including in the nosocomial environment. The hard identification, the phenotypic plasticity, and the easy adaptation to stressful conditions are some of the C. auris traits that render this latest yeast singular challenging. C. auris infections have already been reported from more than 30 countries and are associated with high mortality rates. This is the result from rapid transmission and the difficulty of prevention, control, and eradication. There are several factors related to the high virulence of C. auris, such as the multidrug resistance, biofilm development, and the ability to escape the response of the innate immune system. So, C. auris infections are a serious and alarming problem, not only because of the high pathogenicity of the fungal agent but also because of the low effectiveness of the treatments available. Although new formulations have been developed against C. auris strains, a better understanding is essential to efficiently treat, prevent, and control C. auris infections.

Electrospun Magnetic Ionic Liquid Based Electroactive Materials for Tissue Engineering Applications.

Functional electrospun fibers incorporating ionic liquids (ILs) present a novel approach in the development of active microenviroments due to their ability to respond to external magnetic fields without the addition of magnetic particles. In this context, this work reports on the development of magnetically responsive magneto-ionic fibers based on the electroactive polymer poly(vinylidene fluoride) and the magnetic IL (MIL), bis(1-butyl-3-methylimidazolium) tetrathiocyanatocobaltate ([Bmim]2[(SCN)4Co]). The PVDF/MIL electrospun fibers were prepared incorporating 5, 10 and 15 wt.% of the MIL, showing that the inclusion of the MIL increases the polar ß-phase content of the polymer from 79% to 94% and decreases the crystallinity of the fibers from 47% to 36%. Furthermore, the thermal stability of the fibers decreases with the incorporation of the MIL. The magnetization of the PVDF/MIL composite fibers is proportional to the MIL content and decreases with temperature. Finally, cytotoxicity assays show a decrease in cell viability with increasing the MIL content.

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.

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.

The Risk of Salt Reduction in Dry-Cured Sausage Assessed by the Influence on Water Activity and the Survival of Salmonella.

Water activity (aw) is the main hurdle for microbial control in dry-cured sausages. The aw can be influenced by drying or adding electrolytes or humectants. Dry-cured meat products are partially dried, which, together with added salt, results in safe aw values. Currently, there is a trend to reduce salt in meat products, which can compromise the preservation process. The present work aims to evaluate the influences of added salt levels (1% or 3%) and the use or omission of phosphates and wine on the aw of a dry-cured sausage, and to evaluate the possibility of estimating the aw from the moisture loss and the behavior of Salmonella during dry-cured sausage (chouriço) processing. There was a strong relationship between moisture and aw, regardless of the salt level and the presence of phosphates or wine. Predicting aw from moisture loss is possible using the Boltzmann sigmoid function. The salt level strongly influences Salmonella behavior, mainly through aw reduction. An increase in aw by 0.01 units reduced the odds of achieving a 5-log reduction in Salmonella counts to half. Increasing added salt from 1% to 3% increased the odds of achieving a 5-log Salmonella reduction 7.5-fold. The current trend to reduce salt in foods must be carefully approached if applied to cured meat products, as it has substantial consequences on aw evolution and Salmonella survival.

Ionic Liquid-Based Materials for Biomedical Applications.

Ionic liquids (ILs) have been extensively explored and implemented in different areas, ranging from sensors and actuators to the biomedical field. The increasing attention devoted to ILs centers on their unique properties and possible combination of different cations and anions, allowing the development of materials with specific functionalities and requirements for applications. Particularly for biomedical applications, ILs have been used for biomaterials preparation, improving dissolution and processability, and have been combined with natural and synthetic polymer matrixes to develop IL-polymer hybrid materials to be employed in different fields of the biomedical area. This review focus on recent advances concerning the role of ILs in the development of biomaterials and their combination with natural and synthetic polymers for different biomedical areas, including drug delivery, cancer therapy, tissue engineering, antimicrobial and antifungal agents, and biosensing.

Influence of cellulose nanocrystal surface functionalization on the bending response of cellulose nanocrystal/ionic liquid soft actuators.

This work reports the development of renewable cellulose nanocrystal (CNC) and ionic liquid (IL) hybrid materials for bending actuator applications. For this purpose, cellulose nanocrystals with different surface charges (neutral, positive and negative) were prepared and increasing amounts of the IL 2-hydroxy-ethyl-trimethylammonium dihydrogen phosphate ([Ch][DHP]) (10 and 25 wt%) were incorporated into the CNC hosting matrix. The morphology of the samples was evaluated, proving that both surface charge and IL incorporation do not affect the characteristic layered structure of the CNC. Atomic force microscopy results reveal a sea-island morphology in the hybrid films, where CNC bundles are surrounded by [Ch][DHP]-rich regions. An increase in the electrical conductivity is observed upon IL incorporation into the CNC matrix, regardless of the CNC surface charge. The highest electrical conductivity values are observed for IL/CNC (+) 25 wt% with an electrical conductivity of 3.18 × 10-5± 2.75 × 10-7 S cm-1 and IL/CNC (-) 10 wt% (1.26 × 10-5± 5.92 × 10-6 S cm-1). The highest bending displacement of 2.1 mm for an applied voltage of 4.0 Vpp at a frequency of 100 mHz was obtained for the IL/CNC (+) 25 wt% composite, demonstrating the suitability of cellulose to develop soft actuators.

Portuguese honeys as antimicrobial agents against Candida species.

BACKGROUND AND AIM: Honey has been recognized worldwide for its antioxidant, anti-tumor, anti-inflammatory and antimicrobial properties. Among them, the antifungal properties associated to honey make it an attractive alternative treatment for Candida-associated infections, particularly for topical application to the mucous membranes and skin. In this sense, the main purpose of this work was to evaluate physicochemical properties of five Portuguese honeys and Manuka honey (an Australian honey with well recognized medical proprieties, used as control) and to evaluate the antifungal activity in Candida species planktonic and biofilm assays. EXPERIMENTAL PROCEDURE: Pollen analysis, pH determination, color, concentration of protein and methylglyoxal, conductivity, total phenolics and flavonoids, hydrogen peroxide concentration, and characterization by differential scanning calorimetry in honey samples were determined. Additionally, the effect of honeys on planktonic growth of Candida was initially evaluated by determination of the minimum inhibitory concentrations. Then, the same effect of those honeys was evaluated in biofilms, by Colony Forming Units enumeration. RESULTS AND CONCLUSION: It has been shown that Portuguese heather (Erica cinereal) honey presented the most similar physicochemical properties to manuka honey (specially phenolic and flavonoids contents). The five Portuguese honeys under study, presented in general a potent activity against planktonic multi-resistant yeast pathogens (several clinical isolates and reference strains of Candida species) and S. aureus and P. aeruginosa bacteria cultures. Additionally, it was also concluded that Portuguese heather honey (50% and 75% (w/v)) can also act as a good Candida species biofilm reducer, namely for C. tropicalis.

Erratum: Development of Poly(l-Lactic Acid)-Based Bending Actuators. Polymers 2020, 12, 1187.

The authors wish to make a change to their published paper [...].

Design of Ionic-Liquid-Based Hybrid Polymer Materials with a Magnetoactive and Electroactive Multifunctional Response.

Multifunctional materials with sensor and actuator capabilities play an increasing role in modern technology. In this scope, hybrid materials with magnetic sensing and an electromechanical actuator response based on magnetic ionic liquids (MILs) and the polymer poly(vinylidene fluoride) (PVDF) have been developed. MILs comprising different cation alkyl chain lengths [Cnmim]+ and sharing the same anion [FeCl4]- were incorporated at 20 wt % into the PVDF matrix and the morphological, physical, chemical, and functional properties of the materials were evaluated. An increasing IL alkyl chain length leads to the formation of a porous structure, together with an increase in the electroactive PVDF ß-phase content of the polymer and a decrease in the crystallinity degree and thermal stability. The magnetic susceptibility of the [Cnmim][FeCl4]/PVDF films reveals a paramagnetic behavior. The multifunctional response is characterized by a magnetoionic response that decreases with increasing IL alkyl chain length, the highest magnetoionic coefficient (1.06 ± 0.015 V cm-1 Oe-1) being observed for [C2mim][FeCl4]/PVDF. The electromechanical actuator response is characterized by a highest displacement of 1.1 mm for the [C4mim][FeCl4]/PVDF film by applying a voltage of 4 V at a frequency of 100 mHz. Further, their solution processing makes these multiresponsive materials compatible with additive manufacturing technologies.

Environmental pH modulates biofilm formation and matrix composition in Candida albicans and Candida glabrata.

Candida species are fungal opportunistic pathogens capable of colonizing and infecting various human anatomical sites, where they have to adapt to distinct niche-specific pH conditions. The aim of this study was to analyse the features of Candida albicans and Candida glabrata biofilms developed under neutral and vaginal acidic (pH 4) conditions. C. albicans produced thicker and more filamentous biofilms under neutral than under acidic conditions. On the other hand, the formation of biofilms by C. glabrata was potentiated by the acidic conditions suggesting the high adaptability of this species to the vaginal environment. In general, both species developed biofilms containing higher amounts of matrix components (protein and carbohydrate) under neutral than acidic conditions, although the opposite result was found for one C. glabrata strain. Overall, this study contributes to a better understanding of the modulation of C. albicans and C. glabrata virulence by specific pH conditions.

Development of Poly(l-Lactic Acid)-Based Bending Actuators.

This work reports on the development of bending actuators based on poly(l-lactic acid) (PLLA)/ionic liquid (IL) blends, through the incorporation of 40% wt. of the 1-ethyl-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Emim][TFSI]) IL. The films, obtained by solvent casting at room temperature and 50 °C, were subjected to several post-thermal treatments at 70, 90, 120 and 140 °C, in order to modify the crystallinity of the films. The influence of the drying temperature and of [Emim][TFSI] blending on the morphological, structural, mechanical and electrical properties of the composite materials were studied. The IL induced the formation of a porous surface independently of the processing conditions. Moreover, the [Emim][TFSI] dopant and the post-thermal treatments at 70 °C promoted an increase of the degree of crystallinity of the samples. No significant changes were observed in the degree of crystallinity and Young Modulus for samples with thermal treatment between 70 and 140 °C. The viability of the developed high ionic conductive blends for applications as soft actuators was evaluated. A maximum displacement of 1.7 mm was achieved with the PLLA/[Emim][TFSI] composite prepared at 50 °C and thermally treated at 140 °C, for an applied voltage of 10 Vpp, at a frequency of 100 mHz. This work highlights interesting avenues for the use of PLLA in the field of actuators.

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.

Ionic-Liquid-Based Printable Materials for Thermochromic and Thermoresistive Applications.

Smart materials exhibiting thermochromic and themoresistive properties based on the electroactive polymer poly(vinylidene fluoride) (PVDF) and the ionic liquid (IL) bis(1-butyl-3-methylimidazolium) tetrachloronickelate ([Bmim]2[NiCl4]) have been developed with different contents of [Bmim]2[NiCl4] (10, 20, and 40 wt %) within the polymer matrix. The morphology of the composites is studied, and the thermochromic and thermoresistive properties are evaluated. Independently of the IL content, the PVDF/[Bmim]2[NiCl4] composites present a porous morphology and thermochromic response, revealed by the color change of the composites from transparent to dark blue, attributed to the tetrahedral complex NiCl42- formed after a dehydration process. Further, the electrical conductivity increases with increasing IL content and decreases with increasing temperature. It is also shown that the incorporation of the IL into the PVDF matrix leads to an increase in the electroactive ß phase and a decrease in the degree of crystallinity and thermal stability with increasing [Bmim]2[NiCl4] content. The printability and applicability of the developed materials as sensors are also demonstrated.

Study of TiAlN PVD Coating on Stamping Dies Used in Tinplate Food Package Production.

The food industry is increasingly demanding in terms of the quality and appearance of its product packages. The present study focuses on identifying the main wear mechanisms developed during the stamping process of these packages. During the stamping process, the presence of a tin layer on the surface of the sheet used creates difficulties in the stamping due to the transfer of material from the sheet to the tool, addition of the coefficient of friction, and premature wear of some surfaces of the tool where the contact is more pronounced. In order to understand and avoid these phenomena, a TiAlN coating deposited by a physical vapor deposition (PVD) process was used, which was studied in the laboratory to analyze the evolution of the friction force on the contact and to verify the reaction of the coating on contact with tinplate. Afterwards, the tool was coated and practical tests were performed on the stamping. The obtained results allow confirmation of a significant improvement of the wear behavior of the tool when provided with the coating and also verify that this coating presented better wear resistance than others previously tested in the same working conditions.