Cassia alata and the preclinical search for therapeutic agents for the treatment of opportunistic infections in AIDS patients.

In our search for therapeutic agents from natural sources with potential for the treatment of opportunistic infections in patients afflicted with acquired immunodeficiency syndrome (AIDS), we investigated antibacterial and antifungal activities of water extracts of Cassia alata (C. alata). The extracts are traditionally used in Ivory Coast, West Africa to treat bacterial infections caused by Escherichia coli (E. coli), and fungal infections caused by Candida albicans (C. albicans) and dermatophytes. Our working hypothesis was that the extract contains active ingredient(s) which can be isolated, identified and developed into useful antibacterial/antifungal agents for the treatment of opportunistic infections in patients with AIDS. We used the broth dilution and agar dilution methods. Specifically, we focused on E. coli and C. albicans and the effectiveness of the extracts was evaluated relative to those of standard antibacterial agent chloramphenicol and antifungal agent amphotericin B. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the water extract of C. alata against E. coli were 1.6 mg/ml and 60 mg/ml, respectively; corresponding data for chloramphenicol were 2 micrograms/ml and 10 micrograms/ml. Similarly, the MIC and minimum fungicidal concentration (MFC) for the extract against C. albicans were 0.39 mg/ml and 60 mg/ml in contrast to 0.58 micrograms/ml and 0.98 micrograms/ml for amphotericin B. From the dose-response curve plots, the extract had an IC50 of 31 mg/ml for E. coli and 28 mg/ml for C. albicans. The data suggest that C. alata extracts contain agent(s) which have therapeutic potential and might be useful if isolated and developed for the treatment of opportunistic infections of AIDS patients.     

Effect of Mexican propolis extracts from Apis mellifera on Candida albicans in vitro growth

Propolis is a resinous substance collected by bees (Apis mellifera) from different trees and bushes. Due to its antifungal, antibacterial, antiviral and antiparasitic properties, it has continued to be very popular throughout the time showing variable activity depending on its geographical origin. In Mexico, information about this product is very limited. The aim of this work was to evaluate the antifungal activity of four propolis ethanolic extracts from three different Mexican states, and four commercial extracts on Candida albicans growth. A reference strain (ATCC 10231) and 36 clinical isolates of C. albicans were used. The Minimal Inhibitory Concentration (MIC) was determined by the dilution on agar method. Growth curves on Sabouraud Dextrose broth with and without different propolis ethanolic extracts concentrations were performed. In addition, whether the effect was fungistatic or fungicide was determined. The propolis ethanolic extract obtained from Cuautitlán Izcalli, State of Mexico, showed the best biological activity, inhibiting 94.4% from the clinical isolates at 0.8 mg/ml; the reference strain was inhibited at 0.6 mg/ml. The propolis effect was fungistatic in low concentrations and fungicide in concentrations higher to MIC. The Mexican propolis ethanolic extract could be further investigated for its alternative use for the treatment of some C. albicans infections.     

中药提取物对酵母菌抗真菌活性研究

目的探讨6味中药2种方法提取成分对酵母菌的抑菌和杀菌作用。方法采用药基琼脂稀释法,测定6味中药水提和醇提成分对白念珠菌和糠秕马拉色菌的MIC和MFC。结果对白念珠菌：水提黄连、醇提黄柏、醇提土槿皮MIC范围分别为0．625—1．25mg／mL、0．625～1．25mg/mL、0．313—0．625mg／mL;均值均为0．625mg／mL;对糠秕马拉色菌：水提和醇提黄连MIC范围分别为0．625～1．25mg／mL和1．25mg／mL,均值均为1．25mg／mL。对白念珠菌：醇提土槿皮MFC范围0．625～2．5mg/mL,均值0．625rag／mL。结论水提黄连、醇提黄柏和土槿皮对白念珠菌有较强抑菌作用,其中醇提土槿皮有较强杀菌作用。水提和醇提黄连对糠秕马拉色菌有较强抑菌作用。     

Increased in vitro sensitivity of Candida albicans to amphotericin B when grown in mixed culture with Escherichia coli.

The growth of Candida albicans was inhibited by some Escherichia coli strains both in conventional batch cultures and also in a chemostat under conditions of constant addition of fresh medium. Concentrations of 0.2 microgram amphotericin B per millilitre and of 2 microgram nystatin per millilitre, which caused a slight inhibition of C. albicans in pure culture, exerted a strong fungicidal effect when the yeast was placed in mixed cultures with certain strains of E. coli. Candida albicans cells, inhibited by either E. coli or in mixed culture with polyene antibiotics, appeared larger and less uniformly stained by acridine orange than control cells from pure cultures. Addition of chloramphenicol to the mixed cultures, in quantities sufficient to kill the E. coli cells, abolished the increased sensitivity of C. albicans to amphotericin B or nystatin. In preliminary in vivo tests, E. coli did not sensitize C. albicans to the polyene antibiotics.     

Fungicidal effect of prenylated flavonol, papyriflavonol A, isolated from Broussonetia papyrifera (L.) vent. against Candida albicans

Papyriflavonol A (PapA), a prenylated flavonoid (5,7,3',4'-tetrahydroxy-6,5'-di-(r,r-dimethylallyl)-flavonol), was isolated from the root barks of Broussonetia papyriferra. Our previous study showed that PapA has a broad-spectrum antimicrobial activity against pathogenic bacteria and fungi. In this study, the mode of action of PapA against Candida albicans was investigated to evaluate PapA as antifungal agent. The minimal inhibitory concentration (MIC) values were 10~25 microgram/ml for C. albicans and Saccharomyces cerevisiae, gram-negative bacteria (Escherichia coli and Salmonella typhimurium) and gram-positive bacteria (Staphylococcus epidermidis and Staphylococcus aureus). The kinetics of cell growth inhibition, scanning electron microscopy, and measurement of plasma membrane florescence anisotrophy revealed that the antifungal activity of PapA against C. albicans and S. cerevisiae is mediated by its ability to disrupt the cell membrane integrity. Compared with amphotericin B, a cell membrane disrupting polyene antibiotic, the hemolytic toxicity of PapA was negligible. At 10~25 microgram/ml of MIC levels for the tested strains, the hemolysis ratio of human erythrocytes was less than 5%. Our results suggest that PapA could be a therapeutic fungicidal agent having a broad spectrum antimicrobial agent.     

In vitro activity of amphotericin B and anidulafungin against Candida spp. biofilms

Invasive infections caused by Candida spp. are increasing worldwide and are becoming an important cause of morbidity and mortality in immunocompromised patients. A large number of manifestations of candidiasis are associated with the formation of biofilms on inert or biological surfaces. Candida spp. biofilms are recalcitrant to treatment with conventional antifungal therapies. The aim of this study was dual 1) to determine the prevalence of biofilm producers among clinical isolates from catheter (16 C. albicans ) and blood culture (2 C. albicans and 30 C. tropicalis), and 2) to determine the activity of amphotericin B and anidulafungin against C. albicans and C. tropicalis biofilms of 24 and 48 hours of maturation. Biofilms were developed using a 96-well microtitre plate model and production and activity of antifungal agents against biofilms were determined by the tetrazolium (XTT) reduction assay. Of catheter and blood isolates, 62.5 and 56.25%, respectively, produced biofilms. By species, 68.42% of C. albicans and 53.33% of C. tropicalis were biofilm producers. C. albicans biofilms showed more resistance to amphotericin B and anidulafungin than their planktonic counterparts. Complete killing of biofilms was never achieved, even at the highest concentrations of the drugs tested. Anidulafungin displayed more activity than amphotericin B against C. albicans biofilms of 24 hours of maturation (GM MIC 0.354 vs. 0.686 microg/ml), but against C. tropicalis biofilms amphotericin B was more active (GM MIC 11.285 vs. 0.476 microg/ml). In contrast, against biofilms with 48 hours maturation, amphotericin B was more active against both species.     

Antimicrobial effects of Piper guineense 'Uziza' and Phyllantus amarus 'Ebe-benizo' on Candida albicans and Streptococcus faecalis

Two pathogens were employed, Streptococcus faecalis and Candida albicans for the study of the antimicrobial effects of Piper guineense and Phyllantus amarus using agar-well diffusion and disc-diffusion methods. Phytochemical screening of ethanol, cold and hot water extracts detected the presence of tannins, saponins, flavonoids, terpenoids and phlobatannins. Antimicrobial effect of the plant extracts showed that the organic solvent and aqueous solvents of P. amarus were inhibitory to S. faecalis while the extracts of Phyllantus amarus were not inhibitory to Candida albicans. Agar-well determined Minimum Inhibitory Concentration (MIC) values ranged between 3.125 mg/ml and 6.25 mg/ml while the disc diffusion determined MIC values ranged between 6.25 mg/ml and 25.0 mg/ml. The agar-well determined MIC values for the ethanolic P. amarus extracts (3.12 mg/ml) were lower than the corresponding disc-diffusion MIC determined values (6.25 mg/ml-25.00 mg/ml). Bacteriocidal and bacteriostatic effect varied with, solvent type of extract, concentration of the plant extract and the method of the test adopted. The active components of the plant have no antifungal effect on the tested yeast (Candida albicans). These findings are discussed in relation to plant chemicals as a means of disease control and also to the problem of microbial resistance to synthetic drugs.     

Resveratrol lacks antifungal activity against Candida albicans

The putative candicidal activity of resveratrol is currently a matter of controversy. Here, the antifungal activity as well as the antioxidant response of resveratrol against Candida albicans, have been tested in a set of strains with a well-established genetic background At the doses usually employed in antifungal tests (10-40 μg/ml), resveratrol has no effect on the exponential growth of the C. albicans CAI.4 strain, a tenfold increase (400 μg/ml) was required in order to record a certain degree of cell killing, which was negligible in comparison with the strong antifungal effect caused by the addition of amphotericin B (5 μg/ml). An identical pattern was recorded in the prototrophic strains of C. albicans SC5314 and RM-100, whereas the oxidative sensitive trehalose-deficient mutant (tps1/tps1 strain) was totally refractory to the presence of resveratrol. In turn, the serum-induced yeast-to-hypha transition remained unaffected upon addition of different concentrations of resveratrol. Determination of endogenous trehalose and catalase activity, two antioxidant markers in C. albicans; revealed no significant changes in their basal contents induced by resveratrol. Collectively, our results seem to dismiss a main antifungal role as well as the therapeutic application of resveratrol against the infections caused by C. albicans.     

Evaluation of Systemic Antifungal Agents in X-Irradiated Mice

The effect of X irradiation on the survival time of animals experimentally infected with pathogenic fungi was studied, and the activity of antifungal agents in pre-irradiated hosts was evaluated. A 24-hr preinfection dose of X irradiation decreased the survival time of mice infected with Cryptococcus neoformans and Histoplasma capsulatum to a greater extent than Candida albicans or Blastomyces dermatitidis infections. Exposure to 400 r caused a significant reduction in the variation (S(2)) survival time of C. albicans or H. capsulatum mouse infections. A single 100-mg/kg dose of 5-fluorocytosine or amphotericin B administered within 24 hr postinfection significantly extended the survival time of mice infected with C. albicans. Delayed treatment with amphotericin B was effective against C. neoformans infections. Four 50-mg/kg doses of 5-fluorocytosine were more effective than a single 200-mg/kg dose against C. neoformans infections. A single dose of amphotericin B provided significant protection when administered 48 hr postinfection against B. dermatitidis in preirradiated mice. A single dose of saramycetin 48 hr postinfection was highly effective against H. capsulatum mouse infections. A 100-mg/kg dose of amphotericin B was only effective against this fungal pathogen when administered within 8 hr postinfection. In vivo activity of the antifungal agents studied was detected within 8 to 14 days. The relative in vivo activity of several antifungal agents indicated the importance of considering their individual pharmacological properties for optimum effectiveness. The experimental model used in this study should be useful for the detection and for the preclinical evaluation of new antifungal agents.     

Antifungal and fungicidal activities of tea extract and catechin against Trichophyton

We examined tea extract, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antifungal and fungicidal activities against Trichophyton mentagrophytes, T. rubrum, Candida albicans and Cryptococcus neoformans. Tea extract (2.5%) inhibited completely the growth of both T. mentagrophytes and T. rubrum. EGCg at 2.5 mg/ml failed to inhibit their growth, whereas TF3 at 0.5 mg/ml inhibited the growth. EGCg (1mg/ml) showed no fungicidal activity against Trichophyton. TF3 (1mg/ml) killed Trichophyton by a long time contact (72-96 hrs). Tea extract showed a fungicidal activity against Trichophyton in a dose- and contact time-dependent manner. It did not inhibit the growth of C. albicans, but at a high concentration, inhibited slightly the growth of C. neoformans. It had no fungicidal activity against C. albicans or C. neoformans.     

Oral Candida flora from Brazilian human immunodeficiency virus-infected patients in the highly active antiretroviral therapy era

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90% of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, S?o Paulo. Of the isolates, 57.8% were identified as Candida albicans and 42.1% as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5%), C. lusitaniae (5.2%), C. tropicalis (4.6%), C. parapsilosis (4.6%), C. glabrata (2.8%), C. kefyr (1.7%), C. guilliermondii (1.7%), C. intermedia (1.1%), C. norvegensis (0.5%), and Rhodotorula rubra (1.7%). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (> 64 micro g/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.     

Antifungal and new metabolites of Myrothecium sp. Z16, a fungus associated with white croaker Argyrosomus argentatus

AIMS: Fungal infection is still a life-threatening risk for the immunocompromised population such as AIDS patients and those who receive treatments with immunosuppressors and/or frequent administrations of wide-spectrum antibiotics, which inevitably lead to the drug resistance and unbalanced microflora populations. The present work was accordingly performed to characterize more potent antifungal metabolites from various cultures of marine fungi residing in white croaker Argyrosomus argentatus. METHODS AND RESULTS: The three most common opportunistic human pathogens Candida albicans (CCCCM ID 00148), Aspergillus niger (CCCCM ACCC 30005) and Trichophyton rubrum (CCCCM ID 00001) were selected as test fungi. A total of 16 cultivable fungal strains were isolated from the variant tissues of Ar. argentatus collected from the Yellow Sea, followed by preliminary antifungal screenings of the EtOAc extracts of the corresponding cultures. As a result, the inhibition of the three target fungi, plus being allergic to isolators' skin, were discerned with the EtOAc extract of the fungus under the isolation number Z16 that was identified subsequently as Myrothecium sp. by a combination of morphological and 18S rDNA finger-typing characteristics. A follow-up bioassay fractionation of the EtOAc extract, in conjunction with spectral analyses [MS, (1)H-NMR, (13)C-NMR, distortionless enhancement by polarization transfer (DEPT), heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond resonance (HMBC)] wherever required, afforded eventually the characterization of a new acid (compound 1: 4,5-ditridecyl-octanedioic acid), three macrocyclic trichothecenes including roridin A (compound 2), verrucarin A (compound 3) and 8beta-acetoxy-roridin H (compound 4), (22E,24R)-cerevisterol (compound 5) and N-phenyl-beta-amino-naphthalene (compound 6). In vitro antifungal tests showed that the three trichothecenes were active against A. niger, T. rubrum and C. albicans with MICs of 31.25, 62.5 and 125 microg ml(-1) for compound 2, 250, 125 and 31.25 microg ml(-1) for compound 3 as well as 125, 62.5 and 125 microg ml(-1) for compound 4 respectively. The MICs of ketoconazole (co-assayed herewith as a positive reference) against A. niger, T. rubrum and C. albicans were 31.25, 250, 31.25 microg ml(-1) respectively. A preliminary structure-activity relationship of the antifungal trichothecenes was highlighted in brief. CONCLUSIONS: The present investigation demonstrated that marine fungus Myrothecium sp. Z16 associated with white croaker (Ar. argentatus), was an efficient producer of a new acid and antifungal trichothecenes, the latter presumably being also the allergic substances in the culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The title marine fungus was investigated to be a resource of new aliphatic acid, and trichothecenes with potent antifungal and dermal toxic actions.     

The genetic basis of fluconazole resistance development in Candida albicans

Infections by the opportunistic fungal pathogen Candida albicans are widely treated with the antifungal agent fluconazole that inhibits the biosynthesis of ergosterol, the major sterol in the fungal plasma membrane. The emergence of fluconazole-resistant C. albicans strains is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis (OPC) in acquired immunodeficiency syndrome (AIDS) patients. Resistance can be caused by alterations in sterol biosynthesis, by mutations in the drug target enzyme, sterol 14alpha-demethylase (14DM), which lower its affinity for fluconazole, by increased expression of the ERG11 gene encoding 14DM, or by overexpression of genes coding for membrane transport proteins of the ABC transporter (CDR1/CDR2) or the major facilitator (MDR1) superfamilies. Different mechanisms are frequently combined to result in a stepwise development of fluconazole resistance over time. The MDR1 gene is not or barely transcribed during growth in vitro in fluconazole-susceptible C. albicans strains, but overexpressed in many fluconazole-resistant clinical isolates, resulting in reduced intracellular fluconazole accumulation. The activation of the gene in resistant isolates is caused by mutations in as yet unknown trans-regulatory factors, and the resulting constitutive high level of MDR1 expression causes resistance to other toxic compounds in addition to fluconazole. Disruption of both alleles of the MDR1 gene in resistant C. albicans isolates abolishes their resistance to these drugs, providing genetic evidence that MDR1 mediates multidrug resistance in C. albicans.     

26种滇产植物提取物的体外抗耐药菌作用研究

研究26种云南滇东南产植物80%乙醇提取物的体外抗耐药菌作用。制备植物的醇提物,用琼脂打孔法对其进行抗菌活性的筛选。通过微量稀释法测定提取物的最低抑菌浓度[minimum inhibitory concentration(MIC)]、最低杀细菌浓度[minimum bactericidal concentration(MBC)]及最低杀真菌浓度[minimum fungicidal concentration(MFC)]。结果表明:26种提取物中有9种对金黄色葡萄球菌(简称金葡菌)、大肠埃希菌、铜绿假单胞菌、白色念珠菌有不同程度的抑制活性;其中以红花荷、马蹄参、百花贝母兰、二色大包兰和光序肉实树提取物对耐甲氧西林金葡菌的抑菌作用最强,其MIC/MBC(mg/L)范围分别为512/2048～>2048,512/2048～>2048,256-512/2048～>2048,512～1024/1024～>2048和512～1024/>2048。另外,肋果茶提取物对铜绿假单胞菌及白色念珠菌有较好的抑制作用,其对铜绿假单胞菌及其耐药菌株的MIC/MBC为512～2048/>2048mg/L;对白色念珠菌及其耐药菌株的MIC/MFC均为2048/>2048mg/L。26种植物提取物对大肠埃希菌的抑制作用均较弱。     

Antifungal Activities of Different Extracts of Marine Macroalgae Against Dermatophytes and Candida Species

Algae are bioactive natural resources, and due to the medical importance of superficial mycoses, we focused the action of macroalgae extracts against dermatophytes and Candida species. Seaweed obtained from the Riacho Doce beach, Alagoas (Brazil), were screened for the antifungal activity, through crude extracts using dichloromethane, chloroform, methanol, ethanol, water and chloroform and hexane fractions of green, brown and red algae in assays with standard strains of the dermatophytes Trichophyton rubrum, T. tonsurans, T. mentagrophytes, Microsporum canis, M. gypseum and yeasts Candida albicans, C. krusei, C. guilliermondi and C. parapsilosis. The M44-A and M27-A2/M38A manuals by CLSI were followed, and the minimum inhibitory concentration (MIC) ranged from 0.03 to 16.00 μg ml(-1), and an inhibition halo of 10.00-25.00 mm was observed for dermatophytes, while for yeast, it was from 8.00 to 16.00 μg ml(-1) and 10.00-15.00 mm. M. canis showed MIC of 0.03 μg ml(-1) and the largest inhibition halo in T. rubrum (25.00 mm) through the use of the methanol extract. For C. albicans, dichloromethane, methanol and ethanol extracts formed the largest inhibition halo. The ethanol extract was shown to be the best inhibiting fungi growth, and chloroform and hexane fractions of H. musciformis inhibited the growth of all dermatophytes and C. albicans, yielding the conclusion that apolar extracts obtained from algae presented the best activity against important pathogenic fungi.     

Antifungal effects of hydrolysable tannins and related compounds on dermatophytes, mould fungi and yeasts

A series of hydrolysable tannins and related compounds was evaluated for antifungal activities against filamentous fungi (Epidermophyton floccosum; Microsporum canis; Microsporum gypseum; Trichophyton mentagrophytes; Trichophyton rubrum; Trichophyton tonsurans; Trichophyton terrestre; Penicillium italicum; Aspergillus fumigatus; Mucor racemosus; Rhizopus nigricans) and opportunistic yeasts (Candida albicans; Candida glabrata; Candidata krusei; Cryptococcus neoformans), using the agar dilution method. While all samples had no activity against the filamentous fungi in concentrations of 1.1-5.9 microM (1000 microg/ml), the phenolic compounds displayed significant potencies against all the opportunistic yeasts tested but C. albicans, with minimum inhibitory concentrations ranging from 0.02 to 0.1 microM (16-125 microg/ml). Although the presence of galloyl groups in flavonoids did not necessarily produce activity, this structural element, an HHDP moiety or its oxidatively modified entity proved to be an important structural feature of hydrolysable tannins. Comparison of dilution methods provided strong evidence of dependence of MIC values on the test method. Employing the microdilution broth method, the ellagitannin corilagin (MIC 0.8 nM) was found to be similarly potentially active as amphotericin B (MIC 0.5 nM) and sertaconazole (MIC 0.9 nM) against Candida glabrata strains. The order of effectiveness observed being 64- and 4-8-fold increased for corilagin and the reference compounds respectively, when compared with that of the agar dilution test.     

Evaluation of several tree species for activity against the animal fungal pathogen Aspergillus fumigatus

Aspergillus fumigatus causes severe problems in poultry production systems. Seven South African tree species were selected from the database of the Phytomedicine Programme based on its antifungal activity against the fungus Cryptococcus neoformans. The acetone leaf extracts of the selected species had minimum inhibitory concentrations (MICs) of 0.16 mg/ml and lower in the preliminary screening. The antibacterial and antifungal activities of hexane, dichloromethane, acetone and methanol extracts of the leaves were determined using a two-fold serial microdilution method against a range of commonly encountered animal pathogenic fungi (A. fumigatus, Candida albicans, C. neoformans, Microsporum canis and Sporothrix schenckii) and four nosocomial bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa). The plant species investigated were Combretum vendae (A.E. van Wyk) (Combretaceae), Commiphora harveyi (Engl.) Engl. (Burseraceae), Khaya anthotheca (Welm.) C.DC (Meliaceae), Kirkia wilmsii Engl. (Kirkiaceae), Loxostylis alata A. Spreng. ex Rchb. (Anacardiaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae) and Protorhus longifolia (Bernh.) Engl. (Anacardiaceae). All the extracts had activity against at least one of the test organisms over an incubation period of 24 or 48 h. The MIC values of the non-polar and intermediate polarity extracts of O. natalitia, K. anthotheca, C. vendae, C. harveyi, and P. longifolia had MICs as low as 0.08 mg/ml against at least one of the tested bacteria. Furthermore, the acetone extracts of L. alata, K. wilmsii, O. natalitia and C. vendae had antifungal activities with MIC values ranging from 0.04 to 0.08 mg/ml against at least one of the tested fungi. The average MIC values of the plant extracts against the different bacteria ranged from 0.17 to 2.11 mg/ml, while the range was 0.23–1.98 mg/ml for fungi. The Gram-positive bacteria (S. aureus and E. faecalis) were more susceptible to the plant extracts than the Gram-negative bacteria (E. coli and P. aeruginosa). E. faecalis was the most susceptible microbe and C. vendae extracts were the most active against nearly all the bacteria tested. The acetone extract of L. alata was the most active against fungal pathogens, with activity against at least 3 fungal organisms. L. alata was selected for further work to isolate compounds active against A. fumigatus and other fungal pathogens.     

Antimicrobial activities of Conyzolide and Conyzoflavone from Conyza canadensis

Antibacterial and antifungal activities of the two isolated compounds from Conyza canadensis have been reported in the current study. The two isolated compounds i.e. Conyzolide (1) and Conyzoflavone (2) were tested against six bacterial and five fungal strains, employing hole diffusion and macrodilution methods. Both the compounds showed significant activities against the tested pathogens with special reference to E. coli, P. aeruginosa, S. aureus, Trichophytom longifusus, C. albicans, and C. glaberata. Conyzolide revealed comparatively better antibacterial activity against E. coli (minimum inhibitory concentration (MIC): 25 μg/mL) in comparison to Conyzoflavone. However, in case of antifungal activities, Conyzoflavone exhibited superior antifungal activity against C. albicans (MIC: 10 μg/mL) as compared to Conyzolide.     

Evaluation of phytochemical and antimicrobial properties of leaf extract of Tapinanthus sessilifolius (P. Beauv) van Tiegh

Leaf extracts of T. sessilifolius growing on five different host plants (Psidium guajava, Citrus lemon, Vernonia amygdalina, Persea americana and Jatropa curcas) were evaluated for antimicrobial activity of the plant. Powdered leaves of T. sessilifolius collected from each host plant was divided into two portions. One portion was used for aqueous infusion and the other portion was successively extracted with hexane, ethylacetate and methanol. Infusion of aqueous extract of powdered leaves did not show antimicrobial effect even at the concentration of 1000 and 2000 microg/ml on test microorganisms (Staph. aureus, E. coli, Bacillus subtilis, Pseudomonas aeruginosa and Candida albicans). However in broth culture, methanolic and hexane extract had MIC range of 62.5-500 microg/ml and ethylacetate extract had 250-500 microg/ml. Phytochemical screening of leaf samples of T. sessilifolius collected from different host plants showed positive test for hydrolysable tannins, saponins, flavonoids, terpenes, cardiac glycoside, reducing sugars and proteins. LD50 concentration was found to be > 1.500 mg/kg for samples from P. guajava; 489.89 mg/kg for J. curcas and C. lemon; and 692 mg/kg for V. amydalina in mice.     

Evaluation of Amphotericin B and Chloramphenicol as Alternative Drugs for Treatment of Chytridiomycosis and Their Impacts on Innate Skin Defenses

Chytridiomycosis, an amphibian skin disease caused by the emerging fungal pathogen Batrachochytrium dendrobatidis, has been implicated in catastrophic global amphibian declines. The result is an alarming decrease in amphibian diversity that is a great concern for the scientific community. Clinical trials testing potential antifungal drugs are needed to identify alternative treatments for amphibians infected with this pathogen. In this study, we quantified the MICs of chloramphenicol (800 μg/ml), amphotericin B (0.8 to 1.6 μg/ml), and itraconazole (Sporanox) (20 ng/ml) against B. dendrobatidis. Both chloramphenicol and amphotericin B significantly reduced B. dendrobatidis infection in naturally infected southern leopard frogs (Rana [Lithobates] sphenocephala), although neither drug was capable of complete fungal clearance. Long-term exposure of R. sphenocephala to these drugs did not inhibit antimicrobial peptide (AMP) synthesis, indicating that neither drug is detrimental to this important innate skin defense. However, we observed that chloramphenicol, but not amphotericin B or itraconazole, inhibited the growth of multiple R. sphenocephala skin bacterial isolates in vitro at concentrations below the MIC against B. dendrobatidis. These results indicate that treatment with chloramphenicol might dramatically alter the protective natural skin microbiome when used as an antifungal agent. This study represents the first examination of the effects of alternative antifungal drug treatments on amphibian innate skin defenses, a crucial step to validating these treatments for practical applications.