Antifungal and antibiofilm activities of selected plant extracts

Aims@#Candida albicans is a regular member of the human microbiota but also one of the most frequent pathogens with a strong biofilm-forming capacity and prominent resistance to antimycotic drugs. The aim of this investigation was to evaluate the anti-C. albicans biofilm activity of ethanolic and methanolic leaf extracts of spinach, Swiss chard and garden orache.@*Methodology and results@#Antifungal activity was established by determining the minimum inhibitory (MIC) and minimum fungicidal concentrations (MFC) by the broth microdilution method. The antibiofilm activity was tested by the tissue culture plate method, followed by the determination of the biofilm inhibition. Results showed that all extracts exhibit antifungal activity, with the MIC value of 62.50 μg/mL. This is in accordance with the results of antibiofilm activity, where extracts showed the ability to decrease the biofilm-forming capacity at sub-inhibitory concentrations. Overall antibiofilm effect of spinach extracts were narrow, but biofilm inhibition activity was observed at 31.25 μg/mL of ethanolic extract. Considering the dilution range, garden orache extracts had the broadest antibiofilm activity, with a biofilm inhibition of 20.96-38.10% and 12.11-12.97% for ethanolic and methanolic extracts, respectively. Swiss chard ethanolic extract inhibited biofilm from 14.52% to 31.39% and methanolic from 37.66% to 44.70%.@*Conclusion, significance and impact of study@#Study revealed that investigated plant extracts have antifungal and antibiofilm potential against C. albicans, which could be important in light of its emerging resistance to synthetic drugs, as well as the possible toxicity of antimycotics.

Análogos sintéticos da cheferina I: interação com íons metálicos divalentes e o seu efeito na internalização celular e nas atividades anticandida e candidacida / Synthetic analogues of Shepherin I: interaction with metal divalent ions and their effect on cellular internalization and on anticandidal/candidacidal activitie

O desenvolvimento de resistência antimicrobiana e a consequente seleção de microrganismos multirresistentes consolidam-se como grandes ameaças à saúde global. Neste contexto, a busca por novas drogas antimicrobianas/microbicidas é fundamental e compostos como os peptídeos antimicrobianos (AMPs) tornaram-se alvos atraentes. Os AMPs são compostos químicos de massa molar média e grande diversidade estrutural, produzidos por todos os seres vivos e com capacidade de inibir o crescimento de e/ou matar microrganismos. O AMP Cheferina I (Chef I) foi isolado das raízes de Capsella bursa-pastoris e é resultado da proteólise de uma proteína da família das proteínas ricas em glicina, que em plantas estão relacionadas às funções de defesa e cicatrização. O nosso grupo de pesquisa foi pioneiro no desenvolvimento e estudo de análogos truncados amidados deste AMP atípico rico em glicina (67,9%) e histidina (28,6%), que se mostraram ativos frente às diferentes cepas de Candida e a S. cerevisiae pela internalização/ação celular acompanhada de manutenção da integridade da membrana plasmática; o análogo amidado (Chef Ia) e o análogo marcado com 5(6)-carboxifluoresceína/FAM (FAM-Chef Ia) tiveram as suas atividades antifúngicas potencializadas por íons Zn2+. Este trabalho deu continuidade ao estudo do efeito dos íons metálicos divalentes Zn2+, Cu2+, Ca2+ e Mg2+ nas atividades anticandida/fungistática e candidacida/fungicida a diferentes pHs e forças iônicas, estruturas e localizações intracelulares destes análogos. Os resultados na ausência de íons em pH 5,1 revelaram maior atividade do análogo fluorescente em relação à do não fluorescente. Neste mesmo pH, as atividades anticandida e candidacida de Chef Ia foram influenciadas negativamente pelos íons Ca2+ e Mg2+ (2-4 vezes) enquanto que, na presença de íons Zn2+ as atividades anticandida de ambos os análogos foram aumentadas (Chef Ia: 8-64 vezes; FAM-Chef Ia: 4-32 vezes). Os íons Cu2+ aumentaram a atividade anticandida de Chef Ia (2-4 vezes), mas não a do análogo fluorescente, mas as atividades candidacidas de ambos foram melhoradas (Chef Ia: 2-8 vezes; FAM-Chef Ia: 2 vezes). Em pH 5,1, os íons Zn2+ mantiveram a atividade anticandida de Chef Ia em alta força iônica, mas só FAM-Chef Ia exibiu atividade candidacida. Em pH 7,4 ambos análogos foram inativos em baixa e alta forças iônicas na ausência e presença de Zn2+ ou Cu2+. As maiores porcentagens de folhas-ß-antiparalelas e dobras foram observadas no espectro de DC de Chef Ia em pH 7,4, sendo que aqueles registrados em pH 5,1 e 7,4 em presença de íons Zn2 e Cu2+ indicaram a formação de quelatos estruturalmente distintos. Ambos os peptídeos são bioquelantes em potencial, sendo as proporções peptídeo: íon obtidas as seguintes: FAM-Chef Ia = 1:2 para Cu2+, 1:10 para Zn2+; Chef Ia = 1:1 para Cu2+. A análise da internalização celular de FAM-Chef Ia permitiu a suposição de dois mecanismos de internalização (translocação direta e endocitose), sendo que nas células vivas a presença de Zn2+ afetou negativamente a translocação direta (p 0,0343) e potencializou a endocitose (p 0,0002)

The development of antimicrobial resistance and the consequent selection of multiresistant microorganisms have become major threats to global health. In this context, the search for new antimicrobial/microbicidal drugs is crucial and the antimicrobial peptides (AMPs) have been seen as attractive targets. AMPs are chemical compounds of medium molecular mass and high structural diversity produced by all living beings, capable of inhibiting the growth of microorganisms and killing them. The AMP Shepherin I (Shep I) was isolated from the roots of Capsella bursa-pastoris, being a bioactive peptide encrypted in a glycine-rich protein from a family that in plants are strictly related to defense and healing functions. Our research group has pioneered the development and study of amidated truncated analogues of this atypical glycine- (67.9%) and histidine-rich (28.6%) AMP, which has shown activity against different strains of Candida and S. cerevisiae through cellular internalization with maintenance of the plasma membrane integrity. The amide analogue (Chef Ia) and its fluorescent analog labeled with 5 (6) - carboxyfluorescein / FAM (FAM-Chef Ia) had their antifungal activities potentiated by Zn2+ ions, so the present work continued examining the effect of the divalent metallic ions Zn2+, Cu2+, Ca2+ and Mg2+ on the anticandidal/fungistatic and candidacidal/fungicide activities at different pHs and ionic forces, structures and intracellular locations of these analogues. The results in the absence of those ions at pH 5.1 revealed that the fluorescently labelled analog was more potent than the nonfluorescent. At the same pH, Shep Ia anticandidal and candidacidal activities were negatively influenced by Ca2+ and Mg2+ ions (2-4 fold), whereas in the presence of Zn2+ ions the anticandidal activities of both analogues were increased (Shep Ia: 8-64 fold, FAM- Shep Ia: 4-32 fold). Cu2+ ions increased Shep Ia anticandidal activity (2-4 fold) but not that of FAM-Shep Ia, nevertheless, the candidacidal activities of both analogues were increased (Shep Ia: 2-8 fold, FAM-Shep Ia: 2 fold). Also at pH 5.1, the Zn2+ ions helped retaining the anticandidal activity of Shep Ia at high ionic strength, although only FAM-Shep Ia exhibited candidacidal activity. At pH 7.4 both analogues were inactive at low and high ionic strengths in the absence or presence of Zn2+ or Cu2+. The highest percentages of antiparallel ß-sheet and turns were observed in Shep Ia CD spectrum at pH 7.4, while those recorded at pH 5.1 and 7.4 in the presence of Zn2+ or Cu2+ ions indicated the formation of structurally different chelates. Both peptides are potential biochelates, with the following peptide:ion ratios: FAM-Shep Ia = 1: 2 for Cu2+, 1:10 for Zn2+; Shep Ia = 1: 1 for Cu2+. The analysis of the cellular internalization of FAM-Chef Ia allowed the assumption of two mechanisms of internalization (direct translocation and endocytosis) and in the living cells the presence of Zn2+ negatively affected the direct translocation (p 0.0343) and potentiated endocytosis (p 0.0002)

Ecofriendly Synthesis of Silver Nanoparticles Using Potato Steroidal Alkaloids and Their Activity Against Phytopathogenic Fungi

ABSTRACT In order to reduce the excessive reliance on the toxic chemical fungicides, the present study aimed to isolate the total potato glycoalkaloids (TPAs), and the two steroidal alkaloids α-chaconine and α-solanine from potatoes, Solanum tuberosum L. Their structures were characterized using physical and spectroscopic methods including (UV, IR, 1H, 13C--NMR, 2D 1H-1H COSY, HMBC and NOESY). Silver nanoparticles (AgNPs) were prepared from potato alkaloids through a green synthesis approach. Potato alkaloids and their nanoparticles inhibited mycelial growth of the phytopathogenic fungi Alternaria alternate, Rhizoctonia solani, Botrytis cinerea and Fusarium oxysporum f. sp. lycopersici with low minimal inhibitory and minimal fungicidal concentrations. R. solani was the most susceptible, while F. oxysporum was the most resistant. TPAs was the most fungitoxic (EC50's were 19.8, 22.5, 26.5 and 32.3 µg/ml against R. solani, A. alternate, B. cinerea and F. oxysporum respectively). A mixture of α-solanine and α-chaconine (1:1) showed a marked antifungal activity. AgNPs (size 39.5-80.3 diameter) from alkaloids showed improved fungitoxic activity (EC50's of TPAs nanoparticles ranged between 10.9 and 16.1 µg/ml). Alkaloids exhibited no or a slight phytotoxicity against wheat and radish. Results recommend the potential of using potato alkaloids and their nanoparticles as biorational alternatives to conventional fungicides.

Influência da poluição aérea gerada pelo tráfego veicular na produção do óleo essencial e das atividades antifúngica e citotóxica in vitro de Cyrtocymura scorpioides (Lam. ) H. Rob. (Asteraceae) / Air pollution influence generated by vehicular traffic in essential oil production and antifungal and cytotoxic activities in vitro Cyrtocymura scorpioides (Lam. ) H. Rob. (Asteraceae)

RESUMO Cyrtocymura scorpioides (sin. Vernonia scorpioides (Lam.) Pers.), Piracá é utilizada popularmente para tratamento de úlceras, traumatismos, candidíase, processos inflamatórios e dores musculares. Objetivou-se verificar nas plantas cultivadas na Vila Nair, Jardim São Dimas e Urbanova em São José dos Campos - SP, a influência da poluição veicular nos rendimentos da matéria seca (folhas), no óleo essencial, e no extrato bruto, bem como a ação citotóxica em células HEP-2 e L929, e identificar os componentes do óleo essencial e ação fungicida em Candida albicans. As estacas (54) foram cultivadas durante 6 meses em solo + adubo (2:1) na Universidade do Vale do Paraíba - UNIVAP, e distribuídas nas estações Dutra (E1 - tráfego intenso), Teotônio (E2 - tráfego médio) e Urbanova (E3 - tráfego baixo), onde 18 mudas foram cultivadas durante 6 meses, sendo 3 repetições de 6 plantas. O óleo essencial foi extraído por hidrodestilação e seus componentes identificados por cromatografia gasosa acoplado a espectrômetro de massas (CG-MS), através de indice de similaridade com a base de espectros Wiley L. O extrato bruto foi concentrado por rotavapor. A ação fúngica foi avaliada pelo teste de difusão em disco e a citotoxicidade pelo teste MTT. Em Urbanova (E3) verificouse maior rendimento da matéria seca, do extrato bruto e do óleo essencial. Identificou-se no óleo essencial: ß-cariofileno, α-cariofileno, germacreno D, delta-cadineno e cariofileno. O Óleo Essencial possui possui baixa ação fungicida em C. albicans, enquanto o extrato hidroalcóolico se mostrou citotóxico para L929 e HEp-2.

ABSTRACT Cyrtocymura scorpioides (syn. Vernonia scorpioides (Lam.) Pers.), known as Piracá, is popularly used for the treatment of ulcers, trauma, candidiasis, inflammatory disorders, and muscle pain. This study aimed to assess the influence of vehicular pollution on the yield of dry matter (leaves), essential oil, and crude extract, and the cytotoxic action in HEP-2 and L929 cells. This study also aimed to identify the components of the essential oil, and verify its fungicidal action against Candida albicans in plants grown in Vila Nair, Jardim São Dimas, and Urbanova, São José dos Campos - SP, Brazil. The seedlings (54) were grown in soil + fertilizer (2:1) at the Universidade do Vale do Paraiba - UNIVAP, and distributed to different stations, Dutra (E1 - heavy traffic), Teotônio (E2 - medium traffic), and Urbanova (E3 - low traffic), where 18 seedlings were cultivated for 6 months, with 3 replicates of 6 plants. The essential oil was extracted by hydrodistillation and its components were identified by by Gas chromatography - mass spectrometry (GC-MS), with a similarity index computed using the Wiley L spectra. The crude extract was concentrated in a Buchi Rotary Evaporator R-114, the fungicidal action and cytoptoxicity were evaluated using the disk diffusion method and the MTT test, respectively. In Urbanova (E3), high yields of dry matter, crude extract, and essential oil were obtained. The following components were identified in the oil: ß-caryophyllene, α -caryophyllene, germacrene D, delta-cardinene, and caryophyllene oxide. The oil was found to have low fungicidal action against C. albicans, while the hydroalcoholic extract was cytotoxic to L929 and HEP-2.

Actividad antimicrobiana in vitro del extracto foliar de zabila (Aloe vera L.) en microorganismos de interés clínico / Antimicrobial activity of leaf extract of zabila (Aloe vera L.) in growth of clinical microbes

La zabila es una planta de gran interés médico-farmacéutico, por las diversas y numerosas propiedades medicinales en enfermedades de distinto origen, corroborándose el efecto de sus extractos en estudios in vitro e in vivo. Se evaluó mediante el método de macro dilución, la actividad antimicrobiana del extracto etanólico foliar del Aloe vera L. (5% a 80%), cualitativamente por la turbidez del cultivo en medio líquido y cuantittivamente en unidades formadoras de colonia (UFC) en medio sólido, determinando la concentración mínima inhibitoria (CMI), bactericida (CMB) y fungicida (CMF) en 50 μL del inoculo de microorganismos de interés clínico, tales como: complejo Candida albicans, Escherichia coli, Pseudomonas aeruginosa y Staphylococcus aureus a 37ºC a 24 y 48 h. A las 24 horas, la CMI fue 35, 40 y 55% y la CMB fue 40, 45 y 60%, para E. coli, P. aeruginosa y S. aureus respectivamente, mientras que en C. albicans, la CMI y CMF fueron 55 y 60% respectivamente. A las 48 horas de cultivo la CMI fue 30, 35 y 25% y la CMB fue 35, 40 y 30%, para E. coli, P. aeruginosa y S. aureus, respectivamente, mientras que en C. albicans, la CMI y CMF fueron 40 y 45% respectivamente. Los resultados encontrados con la exposición a 48 h al extracto foliar de Aloe vera L. de estos microrganismos, permite concluir que los mismos son erradicados, planteándose a futuro como una eficaz y económica alternativa natural para el tratamiento de las afecciones causadas por estos organismos tan importantes clínicamente, luego que se realicen las validaciones legales in vivo, que permitan su definitivo uso comercial.

The zabila is a plant of great medical and pharmaceutical interest, for its various and numerous medicinal properties in diseases of different origin, corroborating the effect in vitro and in vivo of extracts. This study evaluated the antimicrobial activity with macro dilution method, in the ethanol extract of Aloe vera leaf (5-80%), qualitatively by the turbidity of the culture in liquid medium and quantitatively in colony forming units (CFU) on solid medium, determining the minimum inhibitory concentration (MIC), bactericidal (BIC) and fungicidal (FIC), in 50 uL of the inoculum of microorganisms of clinical interest such as Candida albicans complex, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus at 37°C for 24 and 48 h. Found that at 24 hours, MIC was 35, 40 and 55% and BIC was 40, 45 and 60% for E. coli, P. aeruginosa and S. aureus, respectively, whereas C. albicans, the MIC and MFC were 55 and 60% respectively. After 48 hours of culture MIC was 30, 35 and 25% and BIC was 35, 40 and 30% for E. coli, P. aeruginosa and S. aureus, respectively, whereas C. albicans, the MIC and MFC were 40 and 45% respectively. The 48 h exposure to the leaf extract of Aloe vera L has great potential biocide for these clinically important microorganisms, can be effectively used in the production of generic drugs of low economic value to treat conditions caused by them in the future, once the legal validation in vivo, allowing their final commercial use are made.

Phytochemical and Antimicrobial Properties of Crude n-Hexane and Methanol Extracts of Cola acuminata Nuts.

Aim: To determine the phytochemical and antimicrobial properties of crude n-hexane and methanol extracts of Cola acuminata nuts against standard and clinical strains of pathogenic bacteria and fungi species implicated in various infections. Place and Duration of study: Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Ibadan, Nigeria, between February 2009 and September 2009. Methodology: All the microorganisms were tested for their susceptibilities to the plant extracts by means of agar diffusion (Kirby-Bauer) method according to the National Committee for Clinical Laboratory Standard guidelines. Minimum Inhibitory Concentrations were determined using the broth dilution method. Results: The methanol extract showed activity on all 16 isolates tested with zones of inhibition in the range of 10 to 19 mm for fungi and 13 to 20 mm for bacteria while that of n-hexane was completely inactive. The minimum inhibitory concentration (MIC) of the methanol extract was between 62.5 and 500 μg/ml with the fungi species having higher values. Phytochemical analysis of Cola acuminata nuts reveals the presence of saponins, tannins, alkaloids and sugar. Griseofulvin, tioconazole and nystatin resistant Candida sp were sensitive to the methanolic extracts of C. acuminata. The kinetics of bactericidal and fungicidal studies show that 90% of Staphylococcus aureus and 99% of Candida albicans were killed within 3 h of contact time at a concentration of 250 μg/ml of the crude methanol extract of Cola acuminata. Conclusion: In comparison with these antibiotics, the test plant extracts fared better in their antifungal activity and are capable of being a good replacement as alternatives if processed.

Comparison of the Minimum Fungicidal Concentration of Clotrimazole, Ketoconazole, Miconazole and Terbinafine Against Clinical Isolates of Dermatophytes.

Superficial fungal infections affect millions of people worldwide. Earlier most dermatophyte strains had relatively restricted geographical distribution. But currently, dermatophytosis has become one of the most common human infectious diseases worldwide. Fungal infections are common in hot and humid climate of tropical countries like India. Topical and systemic therapies are commonly used to treat dermatophyte infections.Clotrimazole is one of the most commonly used topical antifungal drugs. This study compared the minimum fungicidal concentration (MFC) of Clotrimazole with Miconazole, Ketoconazole and Terbinafine in skin dermatophytes. The study demonstrated that Clotrimazole had lower MFCs as compared to Ketoconazole and Miconazole against Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. Clotrimazole had comparable MFCs versus Terbinafine against Trichophyton rubrum but it had lower MFCs against Trichophyton mentagrophytes and Microsporum canis. Thus, Clotrimazole is an effective antifungal agent for dermatophytosis even today.The efficacy of Clotrimazole even against strains with intermediate resistance or resistance to the older azole anti fungal drugs reiterate the current decisions of empirical treatment with topical Clotrimazole for the management of superficial dermatophyte infections.

IL-33 Priming Enhances Peritoneal Macrophage Activity in Response to Candida albicans

IL-33 is a member of the IL-1 cytokine family and plays a role in the host defense against bacteria, viruses, and fungi. In this study, we investigated the function of IL-33 and its receptor in in vitro macrophage responses to Candida albicans. Our results demonstrate that pre-sensitization of isolated peritoneal macrophages with IL-33 enhanced their pro-inflammatory cytokine production and phagocytic activity in response to C. albicans. These macrophage activities were entirely dependent on the ST2-MyD88 signaling pathway. In addition, pre-sensitization with IL-33 also increased ROS production and the subsequent killing ability of macrophages following C. albicans challenge. These results indicate that IL-33 may increase anti-fungal activity against Candida through macrophage-mediated resistance mechanisms.

Phytochemical and antimicrobial screening of crude methanolic leaf extract of Peucedanum winkleri H. Wolff.

Phytochemical investigation of the leaves of Peucedanum winkleri H. Wolff, revealed the presence of secondary metabolites. The extract from total extraction with methanol was screened for its antimicrobial activity against Staphylococcus aureus, Methicillin resistant Staphylococcus aureus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pyogenes, Proteus mirabilis, Candida albicans and Candida krusei using agar-well difussion method. The result indicated that the extract inhibited the growth of one or more test pathogens and were compareable with those of the standard drugs used. The minimun inhibitory concentration (MIC) ranges from 5-10 mg/ml and the minimun bactericidal/fungicidal concentration (MBC/MFC) ranges from 20-40 mg/ml. The result of the study shows justification for the use of the plant for the treatment of infectious diseases caused by these bacteria and fungi pathogens. It was concluded that P. winkleri H. Wolff could be a potential source of active antimicrobial agents and a detailed assessment of antimicrobial activity of the plant material in other solvents extract, isolation and characterization of active compounds from the most active extract is on-going.

Efecto biocida in vitro del extracto foliar de Azadirachta indica en Staphylococcus sp y Pseudomonas sp / Biocide effect of leaf extract of Azadirachta indica in growth of clinical microbes

El Neem es una planta de interés etnobotánico, por las potenciales propiedades medicinales, en enfermedades de distinto origen, corroborándose el efecto de sus extractos en estudios in vitro e in vivo. Se evaluó la actividad antimicrobiana del extracto etanólico foliar del Neem (5-80%), cualitativamente por la turbidez del cultivo en medio líquido y cuantitativamente en unidades formadoras de colonia (UFC) en medio sólido, determinando la concentración mínima inhibitoria (CMI), bactericida (CMB) y fungicida (CMF) en 50 μl del inoculo de microorganismos de interés clínico a 37ºC a 24 y 48 h. Las cepas evaluadas fueron el complejo Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus y Streptococcus mutans. Encontrándose a las 24 horas, una CMI de 30, 40 y 45% y una CMB de 35, 45 y 50%, para S. aureus, S. mutans y P. aeruginosa, respectivamente, mientras que en el complejo C. albicans, no se detectó efecto fungicida pero si fungistático parcial. A las 48 horas de exposición S. mutans no creció a ninguna concentración, considerándose la CMB 5%, mientras que la CMI para S. aureus fue de 25%, para el complejo C. albicans de 30% y en P. aeruginosa de 35%, en tanto que la CMB para S. aureus fue de 30%, en P. aeruginosa de 40% y la CMF para el complejo C. albicans fue a 35%. El mayor tiempo de exposición del extracto a bajas concentraciones, tiene un gran potencial biocida para estos microorganismos tan importantes clínicamente, lo que sugiere la posibilidad de utilizarlo para el tratamiento tópico de afecciones causadas por los mismos.

Neem is a plant of ethnobotanical interest, for its medicinal properties in diseases of different origin, corroborating the effect in vitro and in vivo of extracts. This study evaluated the antimicrobial activity, in the ethanol extract of neem leaf (5-80%), qualitatively by the turbidity of the culture in liquid medium and quantitatively in colony forming units (CFU) on solid medium, determining the minimum inhibitory concentration (MIC), bactericidal (BIC) and fungicidal (FIC), in 50 ul of the inoculum of microorganisms of clinical interest at 37°C at 24 and 48 h: The strains tested were complex Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus mutans. Found that at 24 hours, MIC was 30, 40 and 45% and BIC was 35, 45 and 50% for S. aureus, S. mutans and P. aeruginosa, respectively, whereas to complex Candida albicans not detected fungicidal but fungistatic partial. After 48 hours of culture S. mutans did not grow at any concentration, 5% considered bactericidal, while 25% for S. aureus, 30% in complex Candida albicans and 35% P. aeruginosa were MIC, being 30 and 40% for S. aureus and P. aeruginosa were BIC respectively and fungicidal for C. albicans was 35%. The longer exposure to low concentrations of the extract, has great potential as a biocide for these clinically important microorganisms, can be effectively used in the production of generic drugs of low economic value to treat conditions caused by them.

Combined 624-nm and 850-nm illumination at low rates leads to enhanced inhibition of Candida albicans.

Background: To determine whether combinations of red and infrared light could improve inhibition of Candida albicans and whether combining wavelengths and altering rate of energy delivery could prevent the formation of resistance to light energy. Methods: C. albicans was tested because of the common appearance in human skin and mucous membrane infections. The organism was treated in vitro with a combination of 624-nm (red) and 850-nm (infrared) light emitted from a supraluminous diode (SLD) array. Doses of 9, and 30 J/cm2 were used. Rate of energy delivery was also manipulated. Colony counts were performed and compared to untreated controls using Student t tests and one-way ANOVA with Tukey post hoc analysis. Results: The combination of 624 and 850-nm light energy at 30 J/cm2 was an effective (p ≤ 0.05) inhibitor of C. albicans across all seven stages of the experiment. The combination of 624 and 850-nm wavelengths produced a maximum kill rate [{control – treated / control} X 100] of 76.24% and an average kill rate of 54% across the seven stages of the experiment. Conclusions: A Combination of 624-nm and 850-nm light from an SLD array can inhibit the growth of C. albicans in vitro. Altering delivery rate of the energy can delay resistance formation in this organism.

Evaluation of Malawian Vernonia glabra (Steetz)Vatke leaf and Securidaca longepedunculata (Fresen) root extracts for antimicrobial activities.

The aqueous, ethanol and acetone crude extracts and dichloromethane, hexane, ethyl acetate and nbutanol fractions of the Securidaca longepedunculata roots and Vernonia glabra leaves were studied for their antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans using agar well diffusion method. The phytochemical presents, as well as the minimum inhibitory concentration (MIC) and minimum bacteriacidal/fungicidal concentration (MBC/MFC) of the extracts were also determined using standard methods. Results obtained indicated that Vernonia glabra leaves acetone extracts had excellent antimicrobial activity against E. coli, P. aeruginosa and ethanol extracts against S. aureus. The n-butanol fractions had the best activity against E. coli and S. aureus, dichloromethane fraction against P. aeruginosa and ethyl acetate fraction against C. albicans. For S. longepedunculate root ethanol extracts showed best activity against E. coli, acetone extract against P. aeruginosa and aqueous extract against C. albicans. The n-butanol fractions had best activity against E. coli, P. aeruginosa and C. albicans. These results verified the claims by traditional healers in Malawi that the plants extracts treats bacteria related ailments such as diarrhoea and could be a potential source for development of phytomedicine.

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.

Fungicidal activity of Eucalyptus tereticornis essential oil on the pathogenic fungus Fusarium oxysporum / Actividad antimicótica del aceite esencial a partir de Eucalyptus tereticornis sobre el hongo patógeno Fusarium oxysporum

The objective of present paper was to determine the antifungal activity of the Eucalyptus tereticornis (Myrtaceae) essential oil and two fractions on the Fusarium oxysporum mushroom, a pathogen with clinical and agricultural significance. The total citronelal (44.8 percent) and geraniol (9.78 percent) essential oil had a fungicidal effect at a 3 g/L concentration and a fungicidal activity at small concentrations. The A and B fractions composed most of p-mentane-3,8-diol (18.95 percent) and geraniol acetate (24.34 percent), respectively were more active than the total extract. The observations at microscopic level showed damages and changes in hyphae and chlamydospores, as well as a decrease in the number of conidia. The observed fungicidal activity and the morphologic damages were dependent on the concentration.

El objetivo de este trabajo fue determinar la actividad antifúngica del aceite esencial de Eucalyptus tereticornis (Myrtaceae) y 2 fracciones sobre el hongo Fusarium oxysporum, patógeno de importancia tanto clínica como agrícola. El aceite esencial total, compuesto principalmente por citronelal (44,8 por ciento), citronelol (9,78 por ciento) presentó un efecto fungicida a una concentración de 3 g/L y actividad fungistática a concentraciones menores. La fracciones A y B compuestas en su mayoría por p-mentano-3,8-diol (18,95 por ciento) y acetato de citronelol (24,34 por ciento) respectivamente fueron más activas que el extracto total. Las observaciones a nivel microscópico mostraron daños y cambios en hifas y clamidosporas, así como disminución en el número de conidias. La actividad fungistática observada y los daños morfológicos fueron dependientes de la concentración.

Investigations on fungicidal sensitivity of trichoderma spp and sclerotium rolfsii (collar rot pathogen) in crossandra.

Crossandra is an important flower plant of our country earning a lot of revenue and trade. Of different diseases affecting crossandra cultivation, collar rot induced by Sclerotium rolfsii Sacc. is an important soil borne disease causing devastating losses. In the present study, the sensitivity of the collar rot pathogen was investigated. Also the compatibility of fungal biocontrol agent, Trichoderma spp. with these fungicides was worked out to for further devising Integrated Management strategies for collar rot disease. Four fungicides, viz., captan 50% WP, propiconazole 25% EC, thiophanate-methyl 70% WP and thiram 75% SD were evaluated at five different concentrations against collar rot pathogen, S. rolfsii. Further, the compatibility of these fungicides and the antagonist Trichoderma isolate-1 (T1). Results indicated that the fungicides, propiconazole, thiram and captan have significantly reduced the mycelial growth of test pathogen over control. Maximum inhibition was with propiconazole (100%), followed by thiram (81%) and captan (78%). Results on compatibility of Trichoderma spp. with these fungicides revealed that the bioagent was highly compatible with thiram (32% growth inhibition), followed by captan (47.5%). However, the Trichoderma isolate is not compatible with thiophanate methyl (88% growth inhibition) and propiconazole (100% growth inhibition). The Trichoderma isolate-1 (T1) and the fungicide, thiram were selected for further studies to devise integrated management strategies against collar rot disease.

Antimicrobial activity and chemical composition of essential oil of Pelargonium odoratissimum

The chemical composition of the essential oil from the leaves of Pelargonium odoratissimum (L.) L'Hér., Geraniaceae, was determined and the antimicrobial activities against the Aspergillus flavus CML 1816, Aspergillus carbonarius CML1815 and Aspergillus parasiticus CMLA 817 fungi, as well the Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25 992 bacteria were evaluated. The essential oil was isolated by steam distillation using a modified Clevenger apparatus, and its constituents were identified and quantified by GC/MS and GC-FID analyses. In vitro bioanalytical testing was performed using a completely randomized design. The concentrations of essential oil employed ranged from 0.1 to 2 μL.mL-1 (in dimethyl sulfoxide) for the fungus species and from 1 to 500 μL.mL-1 for the bacteria. The diameters of the inhibition zones formed for bacteria and the mean diameters of mycelial growth in perpendicular directions for fungi were measured, followed by calculation of the percentage of inhibition. The essential oil from the leaves of P. odoratissimum furnished methyleugenol (96.80 percent), a phenylpropanoid. This essential oil inhibited the growth of fungi (100 percent inhibition) and exhibited a small effect on the bacteria at the concentrations tested.

Interleukin-15 augments oxidative metabolism and fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Interleukin (IL)-15 is a pleiotropic cytokine that regulates the proliferation and survival of many cell types. IL-15 is produced by monocytes and macrophages against infectious agents and plays a pivotal role in innate and adaptive immune responses. This study analyzed the effect of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with Paracoccidioides brasiliensis (Pb18), the agent of paracoccidioidomycosis. Peripheral blood monocytes were pre-incubated with IL-15 and then challenged with Pb18. Fungicidal activity was assessed by viable fungi recovery from cultures after plating on brain-heart infusion-agar. Superoxide anion (O2-), hydrogen peroxide (H2O2), tumour necrosis factor-alpha (TNF-α), IL-6, IL-15 and IL-10 production by monocytes were also determined. IL-15 enhanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by addition of anti-IL-15 monoclonal antibody. A significant stimulatory effect of IL-15 on O2- and H2O2 release suggests that fungicidal activity was dependent on the activation of oxidative metabolism. Pre-treatment of monocytes with IL-15 induced significantly higher levels of TNF-α, IL-10 and IL-15 production by cells challenged with the fungus. These results suggest a modulatory effect of IL-15 on pro and anti-inflammatory cytokine production, oxidative metabolism and fungicidal activity of monocytes during Pb18 infection.

In Vitro Antifungal Activity of Equol against Candida albicans

In this study, we demonstrate that equol has fungicidal activities against Candida albicans. The minimum inhibitory and minimum fungicidal concentrations of equol against C. albicans were 516 and 1,032 microM, respectively. Two separate viability assays found that equol changed the integrity of the C. albicans cell membrane, possibly by formation of membrane lesions. Scanning electron microscopy demonstrated ultrastructural changes.

Avaliação funcional de fagócitos em imunodeficiências com manifestações cutâneas / Functional phagocyte evaluation in immunodeficiencies with cutaneous manifestations

A pele e as mucosas constituem as primeiras barreiras na defesa contra infecções e os macrófagos são componentes essenciais do sistema imune inato, importante neste aspecto. O envolvimento destas células pode ser verificado em grande percentual das imunodeficiências primárias. Desta forma, a avaliação da função fagocitária é de extrema relevância para o reconhecimento dos distúrbios imunológicos que acometem a pele. O objetivo do presente estudo foi avaliar a metodologia laboratorial para a detecção de defeitos funcionais dos fagócitos. Para isto foram estabelecidos os seguintes testes laboratoriais: Nitro Blue Tetrazolium (NBT), Dihidrorodamina (DHR), quimiotaxia, fagocitose e a aderência de S. aureus e C. albicans por citometria de fluxo (CF), além de morte intracelular de S. aureus e C. albicans (CF). Para verificar a integridade do sistema complemento realizou-se ensaios hemolíticos para as vias clássica e alternativa (CH50 e AP50). A metodologia proposta foi aplicada em indivíduos normais para a padronização dos testes. O burst oxidativo avaliado pelo teste da dihidrorodamina (DHR) foi aplicado em 101 indivíduos saudáveis e em paralelo, 50 indivíduos sadios para o teste do NBT. Os mesmos testes foram realizados em pacientes com Candidíase mucocutânea crônica (CMC) (n=9 ), Candidíase persistente (n=5), Suspeita de distúrbios de fagócitos (SDF) (n=14), Doença Granulomatosa Crônica (DGC)(n= 7) e portadores de DGC (n=5)...

Skin and mucosa are part of the first barriers in the defense against infections, and the macrophages are essential components of the innate immune system, important when related to this aspect. The involvement of these cells can be seen in a large percentage of the primary immunodeficiencies. Therefore, the assessment of the phagocitary function is extremely important for the recognition of immunological disorders which affect the skin. The present study focus on the evaluation of the laboratorial methodology for the detection of functional defects of phagocytes. For this the following laboratorial tests were established: Nitro Blue Tetrazolium (NBT), chemotaxis, phagocytosis and adherence of S. aureus and C. albicans through flow cytometry (FC), besides the intracellular death of S. aureus and C. albicans (FC). To assess the integrity of the complement system hemolytic assays were performed for the classic and alternative pathways (CH50 and AP50). The proposed methodology was applied to normal individuals for the standardization of the assays. The oxidative burst evaluated through the dihydrorodamine essay (DHR) was applied to 101 healthy individuals and in parallel, 50 healthy individuals for the NBT assay. The same assays were performed on patients with Chronic mucocutaneous candidiasis (CMC)(n=9), persistent candidiasis (n=5), Phagocytes disorders suspicious (PDS) (n=14), Chronicle granulomatous disease (CGD)(n=7) and CGD carriers (n=5). Chemotaxis was standardized using 34 controls for neutrophils stimulated by lipopolisacharydes from e. coli (LPS) and 5 by C. albicans...

In Vitro Amphotericin B Susceptibility of Korean Bloodstream Yeast Isolates Assessed by the CLSI Broth Microdilution Method, Etest, and Minimum Fungicidal Concentration Test / 대한진단검사의학회지

BACKGROUND: Although amphotericin B (AMB) has a wide spectrum of activity that encompasses the majority of yeast isolates, there have been recent reports suggesting that some yeast isolates exhibit decreased susceptibility to AMB. However, in vitro AMB susceptibility of yeast species isolates from blood cultures in Korea has not been fully surveyed. METHODS: A total of 92 bloodstream yeast isolates from four Korean hospitals, representing 10 Candida species (69 isolates) and 4 non-Candida yeast species (23 isolates) were evaluated. AMB minimum inhibitory concentrations (MICs) were determined by two methods: the CLSI method and Etest. AMB minimum fungicidal concentrations (MFCs) were also determined. RESULTS: For all 92 yeast isolates, the CLSI method generated a restricted range of MICs (0.125 to 4 microgram/mL) with 3.3% exhibiting MICs > or =2 microgram/mL, and the corresponding MFC values ranged from 0.25 to 8 microgram/mL with 26.1% showing MFCs > or =2 microgram/mL. Etest produced the widest distribution of MICs, ranging from 0.03 to 32 microgram/mL. High AMB MICs (> or =0.38 microgram/mL) by Etest was observed in 34.8% of the isolates: Candida krusei (100%), Candida rugosa (100%), Trichosporon asashii (100%), Candida glabrata (82%), and Yarrowia lipolytica (75%). Etest disclosed that all isolates of Candida guilliermondii, Candida lusitaniae, Candida pelliculosa and Kodamaea ohmeri were highly susceptible to AMB (MIC < or =0.19 microgram/mL). CONCLUSIONS: Our study showed that Etest may be more useful to discriminate yeast isolates with reduced susceptibility to AMB, and some isolates of less common yeast species from Korea may have decreased AMB susceptibilities.