Antibacterial activity of ß-sitosterol isolated from the leaves of Odontonema strictum (Acanthaceae).

The observation of a dog eating the roots of Odontonema strictum in 2008 in Lubumbashi (DR. Congo) was the starting point of this research which later led to the isolation of ß-sitosterol (BSL), a known phytosterol, isolated for the first time from the leaves of this tropical plant which has a large range of medicinal properties including anti-inflammation, anti-hypertension and antibacterial. The analysis of the 1H NMR spectrum showed that the active compound contains 60% of BSL and 40% of stigmasterol. With a melting point (m.p.) of 134-136 °C and the Rf value 0.55 in EtOAc-hexane (1:3) on silica gel TLC, the active compound was confirmed to be BSL. Here, we determined the minimal inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of BSL on Staphylococcus aureus by the broth dilution method. The MIC and MBC were found to be 1.24 mg/mL and 2.208 mg/mL, respectively. For the crude extract, the MIC and MBC were 4.33 mg/mL and the MBC was 7.66 mg/mL, respectively. The Total antibacterial activity underlined the fact that the crude extract from 1 g of plant materials could be diluted 65 times and still retains the ability to inhibit the growth of S. aureus. This is the first report of the antibacterial activity of BSL from this plant.

Antibacterial Activity against Staphylococcus Aureus of Titanium Surfaces Coated with Graphene Nanoplatelets to Prevent Peri-Implant Diseases. An In-Vitro Pilot Study.

Dental implants are one of the most commonly used ways to replace missing teeth. Nevertheless, the close contact with hard and soft oral tissues expose these devices to infectious peri-implant diseases. To prevent such infection, several surface treatments have been developed in the last few years to improve the antimicrobial properties of titanium dental implants. In this in-vitro pilot study, the antimicrobial activity of titanium surfaces coated with different types of graphene nanoplatelets are investigated. Six different colloidal suspensions of graphene nanoplatelets (GNPs) were produced from graphite intercalated compounds, setting the temperature and duration of the thermal shock and varying the number of the exfoliation cycles. Titanium disks with sand-blasted and acid-etched surfaces were sprayed with 2 mL of colloidal GNPs suspensions. The size of the GNPs and the percentage of titanium disk surfaces coated by GNPs were evaluated through a field emission-scanning electron microscope. The antibacterial activity of the specimens against Staphylococcus aureus was estimated using a crystal violet assay. The dimension of GNPs decreased progressively after each sonication cycle. The two best mean percentages of titanium disk surfaces coated by GNPs were GNPs1050°/2 and GNPs1150°/2. The reduction of biofilm development was 14.4% in GNPs1150°/2, 20.1% in GNPs1150°/3, 30.3% in GNPs1050°/3, and 39.2% in GNPs1050°/2. The results of the study suggested that the surface treatment of titanium disks with GNPs represents a promising solution to improve the antibacterial activity of titanium implants.

The Bactericidal Activity of Protein Extracts from Loranthus europaeus Berries: A Natural Resource of Bioactive Compounds.

Loranthus europaeus is a well-known and important medicinal plant, with a long history of traditional medicine use. Several studies showed that it contains many bioactive compounds with a wide range of pharmacological effects. In light of these past researches, L. europaeus were chosen to consider its potential antimicrobial action. To this aim, different protocols were performed to selectively extract protein compounds, from L. europaeus yellow fruits, and evaluate the antimicrobial activity against four phytopathogenic fungi (Aspergillus niger, Alternaria spp., Penicillium spp., Botritis cinereus) and a number of foodborne bacterial pathogens (Listeria monocytogenes, Staphylococcus aureus strains, Salmonella Typhimurium and Escherichia coli) by using serial dilutions and colony formation assays. Results evidenced no antifungal activity but a notable bactericidal efficiency of a crude protein extract against two foodborne pathogens, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values between 0.2 and 0.5 mg/mL, being S. aureus strains the most susceptible bacteria. Moreover, a strong bactericidal activity against S. aureus M7 was observed by two partially purified protein fractions of about 600 and 60 kDa molecular mass in native conditions. Therefore, these plant protein extracts could be used as natural alternative preventives to control food poisoning diseases and preserve foodstuff avoiding health hazards of chemically antimicrobial applications.

Efeito da terapia fotodinâmica antimicrobiana sobre culturas planctônicas e biofilmes de bactérias e fungos utilizando curcumina e cloro-alumínio ftalocianina veiculada por nanoemulsões / Effect of antimicrobial photodynamic therapy against planktonic and biofilm cultures of bacteria and fungi mediated by curcumin and aluminum-chloride- phthalocyanine entrapped in nanoemulsions

Esta investigação teve os seguintes objetivos: 1. avaliar in vitro o efeito da Terapia Fotodinâmica (PDT) utilizando diferentes concentrações de curcumina e doses de luz LED na inativação de suspensões planctônicas de S. aureus resistente (MRSA) e susceptível à meticilina (MSSA) e a citotoxicidade dos parâmetros antimicrobianos dessa terapia em cultura celular de fibroblastos; 2. o efeito fotodinâmico da cloro-alumínio ftalocianina (ClAlFt) veiculada em nano-emulsão catiônica e aniônica e comparar com ClAlFt diluída em solvente orgânico na inativação da C. albicans quando em suspensão planctônica e organizados em biofilmes; 3. o efeito antimicrobiano da terapia fotodinâmica utilizando a cloroalumínio ftalocianina em sistema de nanoemulsão em culturas planctônicas e biofilmes de bactérias gram-positivas (MSSA e MRSA). No primeiro estudo, suspensões de MSSA e MRSA foram tratadas com diferentes concentrações de curcumina (entre 0,1 a 20,0 µM) e expostas a diferentes fluências de LED azul (18; 25 e 37,5 J/cm2 ). Em seguida, diluições seriadas foram obtidas e alíquotas de 25 µl de cada diluição foram plaqueadas em meio de cultura Mannitol Salt Agar. Após o período de incubação de 48 horas a 37°C, as unidades formadoras de colônias foram determinadas (UFC/mL). Para as células L929, essas foram incubadas por 20 minutos com curcumina e irradiadas em seguida com LED (37,5 J/cm2 ). A viabilidade celular após a PDT foi avaliada utilizando o teste de MTT e as alterações morfológicas foram avaliadas pela microscopia eletrônica de varredura (MEV). Os resultados foram submetidos à análise descritiva, Análise de Variância (ANOVA) e post hoc de Tukey (α= 5%). As concentrações de 5, 10 e 20 µM resultaram em completa inativação do MSSA quando associadas a qualquer fluência de energia avaliada. Entretanto, somente a concentração de 20 µM associadas a dose de luz de 37,5 J/cm2 resultou em completa inativação do MRSA. Essa associação resultou em 80% de redução dos fibroblastos. Além disso, alterações na morfologia celular foram observadas, indicando que a membrana citoplasmática foi o principal alvo dessa terapia. Os controles de luz e curcumina não causaram alterações significativas tanto na contagem de colônias quanto nos valores do metabolismo celular. No segundo estudo, suspensões de C. albicans foram tratadas com a cloro-alumúnio ftalocianina (ClAlFt) na concentração de 31,8 µM encapsulada em nanoemulsões (NE) de caráter catiônico e aniônico e a ClAlFt livre por 30 minutos no escuro. Em seguida, as amostras foram centrifugadas para remoção do fármaco e 300 µl de solução salina foi adicionado previamente à irradiação com LED (660 ± 3 nm; 50 e 100 J/cm2 ). Amostras controle negativo não foram expostas a ClAlFt nem luz. Para as soluções planctônicas, as colônias foram determinadas (UFC/mL) após 48 horas de incubação. Além disso, o metabolismo celular foi avaliado por meio do teste de XTT e a técnica de citometria de fluxo foi utilizada para avaliar danos a membrana celular. Para os biofilmes, a atividade metabólica foi avaliada pelo XTT e a distribuição da ClAlFt utilizando os diferentes veículos no interior dos biofilmes foi analisada por microscopia confocal. Os dados de CFU/mL e os valores de absorbância obtidos pelo teste de XTT foram analisados pelo Kruskal-Wallis e teste de comparações múltiplas dos postos médios. Já para a citometria de fluxo foi utilizado ANOVA um critério (α= 0,05%). A viabilidade fúngica foi dependente do veículo, da carga superficial e dose de luz. A ClAlFt livre inativou completamente o fungo quando associada a maior dose de luz. Já a NE catiônica causou redução significativa tanto de CFU/mL (~ 3,1 log) quanto do metabolismo celular em cerca de 92,3% quando comparada ao controle (p<0,05). Além disso, ambas as veiculações resultaram em danos a membrana citoplasmática significativos (p<0,05). Para os biofilmes, a NE catiônica resultou em redução de 70% do metabolismo. A FS aniônica não apresentou atividade antifúngica. As imagens obtidas pela microscopia confocal demonstraram diferentes padrões de acúmulo e intensidade de fluorescência no interior dos biofilmes para os diferentes sistemas de veiculação de fármacos avaliados. No estudo 3, suspensões e biofilmes de MSSA e MRSA foram tratados com a ClAlFt livre e com os sistemas de NE catiônico e aniônico contendo esse fármaco. Após o período de pré-incubação, o fármaco foi removido e a irradiação foi realizada por um sistema de LED (660 ± 3 nm). Para as suspensões planctônicas bacterianas, foi avaliado o número de unidade formadoras de colônias (UFC/mL) e o metabolismo celular pelo teste de XTT. Já para os biofilmes, foi avaliado o metabolismo pelo XTT. Assim como para o fungo, a eficiência da PDT foi dependente do veículo, carga superficial e dose de luz. A NE catiônica e a ClAlFt livre causaram completa inativação de ambas as cepas de S. aureus. Para os biofilmes, a NE catiônica reduziu em cerca de 80 e 73% o metabolismo celular da cepa susceptível e da resistente, respectivamente. A NE aniônica, apesar de reduzir em cerca de 4 log e 1 log o número de UFC/mL para o MSSA e MRSA, respectivamente, não causou redução do metabolismo dos biofilmes do MRSA mesmo quando associada a maior dose de luz avaliada. Esses resultados sugerem que a NE catiônica representa uma veiculação viável para inativação de fungos e bactérias, incluindo cepas resistentes aos tratamentos convencionais

The aim of this investigation was to evaluate: 1. the Photodynamic Therapy (PDT) mediated by curcumin for in vitro inactivation of methicillin-resistant S. aureus (MRSA) and susceptible S. aureus (MSSA) and its citotoxic effects against L929 fibroblasts; 2. the photodynamic potential of aluminum-chloride-phthalocyanine (ClAlPc) entrapped in cationic and anionic nanoemulsions (NE) to inactivate C. albicans planktonic and biofilm cultures compared with free ClAlPc; 3. the photodynamic effect of ClAlPc encapsulated in NE on methicillin susceptible and resistant S. aureus suspensions and biofilms. In the first study, suspensions of MSSA and MRSA were treated with different concentrations of curcumin and exposed to LED. Serial dilutions were obtained from each sample, and colony counts were quantified. For fibroblasts, the cell viability subsequent to the curcumin-mediated photodynamic therapy was evaluated using the MTT assay and morphological changes were assessed by SEM analysis. Curcumin concentrations ranging from 5.0 to 20.0 µM in combination with any tested LED fluences resulted in photokilling of MSSA. However, only the 20.0 µM concentration in combination with highest fluence resulted in photokilling of MRSA. This combination also promoted an 80% reduction in fibroblast cell metabolism and morphological changes were present, indicating that cell membrane was the main target of this phototherapy. In the second study, fungal suspensions were treated with different types of delivery systems containing ClAlPc. After 30 minutes, the drug was washed-out and samples were illuminated with LED source (660 ± 3 nm). Negative control samples were not exposed to ClAlPc or light. For planktonic suspensions, colonies were counted (CFU/ml) and cell metabolism was evaluated by XTT assay. (Kruskal-Wallis and Multiple Comparison test; α= 0.05%). Flow cytometry was used to evaluate damage to cell membrane. (One-way ANOVA; α= 0.05%). For biofilms, the metabolic activity was evaluated by means of XTT reduction assay and ClAlPc distribution using the different delivery systems through three-dimensional architecture of the biofilms were analyzed by confocal laser scanning microscopy (CLSM). Fungal viability was dependent on the delivery system, superficial charge and light dose. Free ClAlPc caused photokilling of the yeast when combined with a higher light dose. Cationic NE-ClAlPc reduced significantly both CFU/mL (~3.1 log10) and cell metabolism (92.3%) compared with the negative control (p<0.05). In addition, cationic NE-ClAlPc and free-ClAlPc caused significant damage to the cell membrane (p<0.05). For the biofilms, cationic NE-ClAlPc reduced cell metabolism by 70%. Anionic NE-ClAlPc did not present antifungal activity. CLSM showed different accumulation and fluorescence intensities emissions on biofilm structure for the evaluated delivery systems. In the third study, suspensions and biofilms of MRSA and MSSA were treated with different delivery systems containing ClAlPc. After the preincubation period, the drug was washed-out and irradiation was performed with LED source (660 ± 3 nm). Negative control samples were not exposed to ClAlPc or light. For the suspensions, colonies were counted (CFU/mL). The metabolism of S. aureus suspensions and biofilms were evaluated by the XTT assay. The efficiency was dependent on the delivery system, superficial load and light dose. Cationic NEClAlPc and free-ClAlPc caused photokilling of the both strains of S.aureus. For biofilms, cationic NE-ClAlPc reduced cell metabolism by 80 and 73% of susceptible and resistant strains, respectively. Although anionic NE-ClAlPc caused a significant CFU/mL reduction for MSSA and MRSA, it was not capable of reducing MRSA biofilm metabolism. ClAlPc entrapped in cationic NE or in its free form combined with LED caused photokilling of both S. aureus strains evaluated and reduction of the metabolic activity of biofilms. This therapy may represent an alternative treatment for eradicating resistant strains

Avaliação da influência da infecção bacteriana secundária na evolução da leishmaniose cutânea em Corte de Pedra, Bahia / Evaluation of secondary bacterial infection's influence on the course of cutaneous leishmaniasis in Corte de Pedra, Bahia

In order to study the prevalence of secondary bacterial infection in ulcerated lesions and its relationship to the healing process, 84 leishmaniotic patients were evaluated. Diagnosis of the secondary infection was made by bacterial aerobic culture of peripheral tissue specimen of the ulcer. All patients received antimonial therapy during 20 days and washed their ulcers with common soap. Cases were composed mainly of adolescent and adult farmer patients with single lesions. The evaluated ulcers were encountered on legs and feet in 47.6%. Secondary bacterial infection was found in 45/83 (54.2%), and was more frequent in lesions located below the knee. Staphylococcus aureus predominated (89%). The ulcers' healing process, evaluated in 79 patients one month after finishing treatment, was not influenced by the secondary bacterial infection.

Foram avaliados 84 pacientes leishmanióticos com o objetivo de verificar a prevalência de infecção bacteriana secundária das úlceras cutâneas e de estudar sua relação com a cicatrização das lesões. A infecção secundária foi diagnosticada mediante cultura bacteriana aeróbica de amostra de tecido da lesão. Todos os pacientes receberam tratamento antimonial durante 20 dias e fizeram lavagem da úlcera com água e sabão comum. A casuística foi composta principalmente de adolescentes e de adultos dedicados à lavoura, apresentando lesão única. Em 47,6%, as úlceras estudadas estavam localizadas nas pernas e nos pés. Verificou-se infecção secundária em 45/83 (54,2%), sendo mais freqüente nas lesões localizadas abaixo dos joelhos. O Staphylococcus aureus predominou (88,9%). A reepitelização completa das úlceras, avaliada em 79 pacientes um mês após o fim do tratamento, não foi influenciada pela infecção secundária.