Lipid-based nanostructures for the delivery of natural antimicrobials

Encapsulation can be a suitable strategy to protect natural antimicrobial substances against some harsh conditions of processing and storage and to provide efficient formulations for antimicrobial delivery. Lipid-based nanostructures, including liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid nanocarriers (NLCs), are valuable systems for the delivery and controlled release of natural antimicrobial substances. These nanostructures have been used as carriers for bacteriocins and other antimicrobial peptides, antimicrobial enzymes, essential oils, and antimicrobial phytochemicals. Most studies are conducted with liposomes, although the potential of SLNs and NLCs as antimicrobial nanocarriers is not yet fully established. Some studies reveal that lipid-based formulations can be used for co-encapsulation of natural antimicrobials, improving their potential to control microbial pathogens

Desenvolvimento de lipossomas nanométricos para armazenamento e liberação controlada de peptídeos antimicrobianos

Os compostos antimicrobianos naturais são um tema de grande interesse devido ao aumento da demanda por alimentos seguros e de alta qualidade. A utilização de lipossomas é uma alternativa interessante para proteger antimicrobianos nos alimentos, além de fornecer compostos naturais de liberação controlada. Os lipossomas revestidos com polissacarídeos apresentam melhor estabilidade, representando uma alternativa aos lipossomas convencionais. Inicialmente, os nanolipossomas que encapsulam a nisina foram preparados com fosfatidilcolina de soja (PC) e pectina ou ácido poligalacturônico. Os lipossomas desenvolvidos apresentaram alta eficiência de encapsulação, baixo índice de polidispersão e foram estáveis durante 21 dias a 7 °C e 25 °C. A atividade antimicrobiana foi observada contra cinco cepas diferentes de Listeria em placas de ágar de leite, com uma melhor eficiência contra L. innocua 6a. Em um segundo momento, as características estruturais dos lipossomas foram estudadas por dispersão de raios-X de pequeno ângulo (SAXS) e as amostras foram submetidas a ciclos de temperatura (20-60 °C). Para isso, os lipossomas foram desenvolvidos contendo pectina ou ácido poligalacturônico pelos métodos de hidratação de filme e evaporação em fase reversa, para encapsular nisina. A análise de SAXS confirmou a presença de estruturas lamelares em todas as amostras. Além disso, parte da estrutura multilamelar tornou-se cúbica, provavelmente devido à presença de nisina nos lipossomas. A adição de polissacarídeos mostrou diferenças entre as fases cúbicas formadas. Em última análise, a mistura de lisozima e nisina foi encapsulada em lipossomas contendo polissacarídeos. O diâmetro médio dos lipossomas foi de 85,6 e variou para 77,3 e 79,9 nm com a incorporação de pectina ou ácido poligalacturônico, respectivamente. O potencial zeta dos lipossomas com polissacarídeos foi de cerca de -30 mV, mostrando alta eficiência de encapsulação. A atividade antimicrobiana foi avaliada a 37 °C, mostrando que a PC-pectina reduziu a população de L. monocytogenes em 2 log UFC/mL e 5 log UFC/mL em leite integral e desnatado, respectivamente. Em refrigeração, a PC-pectina reduziu a população de L. monocytogenes para quase zero por até 25 dias em leite desnatado. Portanto, pode dizer-se que os lipossomas que contêm polissacarídeos podem ser uma tecnologia promissora para o encapsulamento da lisozima e nisina. Além disso, a existência de estrutura cúbica nos lipossomas pode proporcionar liberação controlada de antimicrobianos.Natural antimicrobial compounds are a topic of utmost interest due to the increased demand for safe and high-quality foods. The use of liposomes is an interesting alternative to protect antimicrobials in food, also providing controlled release natural compounds. Polysaccharides coated liposomes present better stability, representing an alternative to conventional liposomes. Initially, nanoliposomes encapsulating nisin were prepared with soy phosphatidylcholine (PC) and pectin or polygalacturonic acid. The liposomes developed presented high encapsulation efficiency, low polydispersity index, and were stable for 21 days at 7°C and 25°C. The antimicrobial activity was observed against five different strains of Listeria in milk-agar plates, with a better efficiency against L. innocua 6a. In a second moment, structural characteristics of liposomes were studied by small angle X-ray scattering (SAXS) and the samples were submitted to temperature cycles (20-60°C). For this, liposomes were developed containing pectin or polygalacturonic acid by the thin-film hydration method and reverse phase evaporation method for nisin encapsulation. The analysis of SAXS confirmed the presence of lamellar structures in all the samples. In addition, part of the multilamellar structure became cubic, probably due to the presence of nisin in the liposomes. The addition of polysaccharides showed differences between the cubic phases formed. Ultimately, the mixture of lysozyme and nisin were encapsulated in liposomes containing polysaccharides. The mean diameter of the liposomes was 85.6 and varied to 77.3 and 79.9 nm with the incorporation of pectin or polygalacturonic acid, respectively. The zeta potential of liposomes with polysaccharides were around -30 mV, showing high encapsulation efficiency. The antimicrobial activity was assessed at 37 °C, showing that PC-pectin reduced the population of L. monocytogenes to 2 log CFU/mL and 5 log CFU/mL in whole and skim milk, respectively. At under refrigeration, PC-pectin reduced the population of L. monocytogenes to almost zero for up to 25 days in skim milk. Therefore, it can say that the liposomes containing polysaccharides can be a promising technology for the encapsulation of lysozyme and nisin. In addition, the existence of cubic structure in the liposomes can provide controlled release of antimicrobials

S. epidermidis isolates from a tertiary care Portuguese hospital show very high antibiotic non-susceptible rates and significant ability to form biofilms

Healthcare-associated infections (HAIs) have been increasing during recent decades, leading to long hospital stays and high morbidity and mortality rates. The usage of antibiotics therapy against these infections is enhancing the emergence of more multiple-drug resistant strains, in particular in Staphylococcus epidermidis. Hence, this study focused on the resistance pattern of S. epidermidis isolates from clinical settings and its association with phenotypic and molecular traits. Our results showed that HAIs were more prevalent among infants and older adults, and the most frequent type of HAI was central line-associated bloodstream infection. Half of the patients received antibiotic therapy before laboratory diagnosis. Preceding microbiological diagnosis, the number of patients receiving antibiotic therapy increased by 29.1%. Eighty-six per cent of the clinical isolates presented a multidrug resistance (MDR) profile, and a quarter were strong biofilm producers. Furthermore, polysaccharide intercellular adhesin (PIA)-dependent biofilms presented higher biomass production (p=0.0041) and a higher rate of antibiotic non-susceptibility than PIA-independent biofilms, emphasizing the role of icaABDC operon in infection severity. Therefore, this study suggests that a thorough understanding of the phenotypic and molecular traits of the bacterial cause of the HAIs may lead to a more suitable selection of antibiotic therapy, improving guidance and outcome assessment.Fundação para a Ciência e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009 (FCOMP-01-0124-FEDER-014309). The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2—O Novo Norte), QREN, FEDER and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462)info:eu-repo/semantics/publishedVersio

Augmented Reality Musical System for Rehabilitation of Patients with Duchenne Muscular Dystrophy

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Isolation of clinical strains of Staphylococcus epidermidis from a Portuguese hospital and assessment of their relationship between biofilm formation capacity and antimicrobial resistance

Staphylococcus epidermidis has been documented as an emergent pathogen responsible for many healthcareassociated infections (HAIs). These infections are an increasing cause of major concern not only due to the high distribution of methicillin resistance, but also due to their ability to form biofilm, which increases their persistence, impairs patient’s quality of life and leads to failed treatment and extra costs. Portugal has one of highest incidence rates of HAIs in Europe. However, bacteriological information that may shed light on the clinical significance of S. epidermidis Portuguese isolates and provide data for control as well as epidemiological measures is missing. In order to fill this gap, the aim of this study was to isolate and determinate the antibiotic resistance profile of clinical strains of S. epidermidis and ensure its association with phenotypic and genotypic biofilm-associated determinants. Of the 89 studied patients, 52 (58.4%) were men and the mean age was 45 years old. Bloodstream infections (69.7%) were the most frequently reported infections during the study period and almost a third of all infections were catheter-related. The majority (85.4%) of the clinical isolates were mecA-positive and among those, 92.1% were also resistant to 3 or more of the antimicrobial agent groups tested and hence considered multidrug-resistant (MDR). Resistance also reaches higher levels among -lactam antibiotics (96.4%) and erythromycin (79.8%). Notwithstanding, positive associations were found between MDR and MRSE strains, between MDR strains and prescription of at least one antimicrobial agent and between patients under antibiotic therapeutic and MRSE strains. Regarding the phenotypic and molecular features, the majority (64%) of the clinical isolates were considered biofilm producers and all strong producers were carriers of the icaA gene, although equal distributed among MRSE and MSSE strains. The genetic combination most frequently observed was icaA+aap+ (41.6%) followed by icaA+aap+bhp+ (21.3%). Additionally, strains with the genetic combination icaA+aap+bhp+ were positively associated with both MRSE and MDR phenotype. Our results confirmed the impact of S. epidermidis on hospital-acquired infections and highlight the burden of antimicrobial resistance, mainly multidrug resistance that reached alarming levels in this tertiary-care hospital. Moreover, this data raised concerns regarding antimicrobial strategies previously adopted. In addition, an association between the carriage of some virulent-associated genes and biofilm phenotype was clear, mainly regarding the carriage of icaA gene that demostrated to be essential in the biofilm process of S. epidermidis clinical strains

Virulence factors in coagulase-negative staphylococci

Coagulase-negative staphylococci (CoNS) have emerged as major pathogens in healthcare-associated facilities, being S. epidermidis, S. haemolyticus and, more recently, S. lugdunensis, the most clinically relevant species. Despite being less virulent than the well-studied pathogen S. aureus, the number of CoNS strains sequenced is constantly increasing and, with that, the number of virulence factors identified in those strains. In this regard, biofilm formation is considered the most important. Besides virulence factors, the presence of several antibiotic-resistance genes identified in CoNS is worrisome and makes treatment very challenging. In this review, we analyzed the different aspects involved in CoNS virulence and their impact on health and food.: V.G. and N.L. acknowledge the FCT fellowship, respectively, SFRH/BD/13145/2017 and SFRH/BD/136998/2018.info:eu-repo/semantics/publishedVersio

"História do cerco de Lisboa" - uma história diferente

Using José Saramago's (1922-2010) novel "The history of the siege of Lisbon" (1989) as a model and example, this article attempts to demonstrate the function of multiperspective narrative structures in metahistoriographical novels, making reference to Vera and Ansgar Nünning. It discusses the problem of supposed objectivity in history studies and the invisibility of marginalised groups, who should no longer be exclusively the focus of the description, but finally also the describers. It also takes a brief digression on Hayden White's argument on the relationship between literature and historiography. By analysing the structure of perspective and by looking at the means of irony in the novel, the article aims to show how far it critically addresses these problems. It concludes that it is necessary to replace the collective singular of history (according to Reinhart Koselleck) with a more pluralistic understanding of history(-ies).Usando como modelo e exemplo o romance "História do cerco de Lisboa" de José Saramago, este artigo tenta demonstrar a função de estruturas narrativas em torno da multiperspetiva em romances metahistoriográficos. Discute o problema da suposta objetividade nos estudos de história e a invisibilidade dos grupos colocados à margem, que já não deveriam ser exclusivamente o foco da descrição, mas, afinal, também os que descrevem, exercendo sua voz. Ao analisar a estrutura da perspetiva e ao olhar para os meios de ironia do romance, o artigo pretende mostrar até que ponto a obra de Saramago aborda de forma crítica estes problemas. Conclui-se, enfim, que é necessário substituir o singular coletivo da história por uma compreensão mais pluralista da história(s)

Quantification of biofilm-associated genes in Staphylococcus epidermidis biofilms: its impact in biofilm formation and 3D structure

Staphylococcus epidermidisis a common commensal coloniser of the human skin and is currently the most frequent cause of biomaterial associated infections. Several studies have attempted to identify the determinants that distinguish invasive from commensals S. epidermidisstrains. Its pathogenesis is directly related to its ability to establish multi-layered and highly structured biofilms, resistant to antimicrobial agents. This bacterium expresses several factors that are responsible for the development of the biofilm, including the contribution of specific factors (icaA, aap and bhp genes) in the accumulation phase. Recently, several research groups have been trying to understand the contribution of the genes involved in biofilm formation. Thus, the main goal was to analyse the gene expression of icaA, aap and bhp and compare with the formation of the biofilm structure. Two S. epidermidis strains, a clinical and a commensal were characterized at the level of biofilm formation, at different incubation times. According to our results both strains showed an increase of biomass production overtime, revealing the importance to use screening assays with more than 24 h of incubation. A biofilm structure analysis was also performed to detect the presence of poly-N-acetylglucosamine (PNAG), the major component of S. epidermidis biofilm matrix. The results revealed a higher production of PNAG only after 48 h for SECOMO034.A1. Finally, the gene expression at two different incubation times was determined, confirming the importance of the icaA gene in the accumulation stage, explaining the high production of biomass and PNAG. On the other hand, the aap and bhp expression levels raised some questions about their role in the biofilm process

Color difference between the vita classical shade guide and composite veneers using the dual-layer technique

The purpose of this in vitro study was to evaluate the color difference between the Vita Classical Shade Guide and composite veneers using the dual-layer technique. Thirty samples were fabricated using a custom-made mold (Easy Layering Shade Guide Kit

Modelling of bulk density as related to aggregate size distribution in clayey Ferralsols

Among microaggregated soils, there are Ferralsols that show little or no distinct horizonation. Their macrostructure is weak to moderate and they have typically a strong microstructure. In most Brazilian clayey Ferralsols, physical properties are closely related to the development of microstructure that consists of subrounded microaggregates 50 to 500 µm in diameter