Purification and characterization of the cold-active killer toxin from the psychrotolerant yeast Mrakia frigida isolated from sea sediments in Antarctica

The psychrotolerant yeast Mrakia frigida 2E00797 isolated from sea sediments in Antarctica was found to be able to produce killer toxin against Metschnikowia bicuspidata, Candida tropicalis and Candida albicans. In the present study, the killer toxin was purified and characterized. The molecular weight of the purified killer toxin was estimated to be 55.6 kDa and the purified killer toxin shared 35.1% sequence homology with a protein kinase. The purified killer toxin's optimal temperature and pH for killing activity were 16 °C and 4.5, respectively, and it was stable in the temperature range from 10 to 25 °C at pH 4.5. The toxin's highest killing activity was observed in the presence of 3.0 g/100 ml NaCl. The purified killer toxin was able to actively kill whole cells of M. bicuspidata but could not kill the protoplast of the sensitive yeast. Of the eight yeast species tested in this study, the killer toxin was able to kill C. tropicalis and C. albicans in addition to M. bicuspidata.     

A novel killer toxin produced by the marine-derived yeast Wickerhamomyces anomalus YF07b

In our previous study, it was found that the killer toxin produced by the marine-derived yeast Wickerhamomyces anomalus YF07b has both killing activity and β-1,3-glucanase activity and the molecular mass of it is 47.0 kDa. In this study, the same yeast strain was found to produce another killer toxin which only had killing activity against some yeast strains, but had no β-1,3-glucanase activity and the molecular mass of the purified killer toxin was 67.0 kDa. The optimal pH, temperature and NaCl concentration for action of the purified killer toxin were 3.5, 16 °C and 4.0 % (w/v), respectively. The purified killer toxin could be bound by the whole sensitive yeast cells, but was not bound by manann, chitin and β-1,3-glucan. The purified killer toxin had killing activity against Yarrowia lipolytica, Saccharomyces cerevisiae, Metschnikowia bicuspidata WCY, Candida tropicalis, Candida albicans and Kluyveromyces aestuartii. Lethality of the sensitive cells treated by the newly purified killer toxin from W. anomalus YF07b involved disruption of cellular integrity by permeabilizing cytoplasmic membrane function.     

Antifungal Activity of Chitosan-Coated Poly(lactic-co-glycolic) Acid Nanoparticles Containing Amphotericin B

Amphotericin B (AmB) is one of the most used drugs for the treatment of systemic fungal infections; however, the treatment causes several toxic manifestations, including nephrotoxicity and hemolytic anemia. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. In this work, the antifungal efficacy of chitosan-coated PLGA nanoparticles containing AmB was evaluated in 20 strains of fungus isolates from patients with vulvovaginal candidiasis (01 Candida glabrata and 03 Candida albicans), bloodstream infections (04 C. albicans and 01 C. tropicalis) and patients with urinary tract infection (04 Candida albicans, 02 Trichosporon asahii, 01 C. guilhermondii, 03 C. glabrata) and 01 Candida albicans ATCC 90028. Moreover, the cytotoxicity over erythrocytes was evaluated. The single-emulsion solvent evaporation method was suitable for obtaining chitosan-coated PGLA nanoparticles containing AmB. Nanoparticles were spherical in shape, presented mean particle size about 460 nm, positive zeta potential and encapsulation efficiency of 42%. Moreover, nanoparticles prolonged the AmB release. All the strains were susceptible to plain AmB and nanostructured AmB, according to EUCAST breakpoint version 8.1 (resistant > 1 μg/mL), using broth microdilution method. In C. albicans (urine, blood, and vulvovaginal secretion isolates, and 1 ATCC), the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.5 μg/mL. In urine and vulvovaginal secretion isolates of C. glabrata, the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.015 μg/mL. In urine isolates of C. guilhermondii, the MIC value of AmB-loaded nanoparticles was 0.12 μg/mL and EUCAST was 0.06 μg/mL. In blood isolates of C. tropicalis, the MIC value of AmB-loaded nanoparticles was 0.5 μg/mL and EUCAST was 0.25 μg/mL. Finally, in urine isolates of T asahii, the MIC value of AmB-loaded nanoparticles was 1 μg/mL and EUCAST varied from 0.5 to 1 μg/mL. In the cytotoxicity assay, plain AmB was highly hemolytic (100% in 24 h) while AmB-loaded chitosan/PLGA nanoparticles presented negligible hemolysis.     

Species-specific activation of Cu/Zn SOD by its CCS copper chaperone in the pathogenic yeast Candida albicans

Candida albicans is a pathogenic yeast of important public health relevance. Virulence of C. albicans requires a copper and zinc containing superoxide dismutase (SOD1), but the biology of C. albicans SOD1 is poorly understood. To this end, C. albicans SOD1 activation was examined in baker’s yeast (Saccharomyces cerevisiae), a eukaryotic expression system that has proven fruitful for the study of SOD1 enzymes from invertebrates, plants, and mammals. In spite of the 80 % similarity between S. cerevisiae and C. albicans SOD1 molecules, C. albicans SOD1 is not active in S. cerevisiae. The SOD1 appears incapable of productive interactions with the copper chaperone for SOD1 (CCS1) of S. cerevisiae. C. albicans SOD1 contains a proline at position 144 predicted to dictate dependence on CCS1. By mutation of this proline, C. albicans SOD1 gained activity in S. cerevisiae, and this activity was independent of CCS1. We identified a putative CCS1 gene in C. albicans and created heterozygous and homozygous gene deletions at this locus. Loss of CCS1 resulted in loss of SOD1 activity, consistent with its role as a copper chaperone. C. albicans CCS1 also restored activity to C. albicans SOD1 expressed in S. cerevisiae. C. albicans CCS1 is well adapted for activating its partner SOD1 from C. albicans, but not SOD1 from S. cerevisiae. In spite of the high degree of homology between the SOD1 and CCS1 molecules in these two fungal species, there exists a species-specific barrier in CCS–SOD interactions which may reflect the vastly different lifestyles of the pathogenic versus the noninfectious yeast.     

Cellulose Acetate Containing Toluidine Blue and Rose Bengal Is an Effective Antimicrobial Coating when Exposed to White Light

Simple methods of reducing the microbial load on surfaces in hospitals are needed to reduce the risk of hospital-associated infections. Here we report on the ability of a cellulose acetate coating containing the photosensitizers toluidine blue and rose bengal to kill microbes (Staphylococcus aureus, Escherichia coli, Clostridium difficile, a bacteriophage, and Candida albicans) on its surface when illuminated with white light.     

Antifungal activity of magnoflorine against <Emphasis Type="Italic">Candida</Emphasis> strains

Candida albicans is a major invasive pathogen, and the development of strains resistant to conventional antifungal agents has been reported in recent years. We evaluated the antifungal activity of 44 compounds against Candida strains. Magnoflorine showed the highest growth inhibitory activity of the tested Candida strains, with a minimum inhibitory concentration (MIC) of 50 μg/mL based on microdilution antifungal susceptibility testing. Disk diffusion assay confirmed the antifungal activity of magnoflorine and revealed that this activity was stable over 3 days compared to those of berberine and cinnamaldehyde. Cytotoxicity testing showed that magnoflorine could potentially be used in a clinical setting because it didn’t have any toxicity to HaCaT cells even in 200 μg/mL of treatment. Magnoflorine at 50 μg/mL inhibited 55.91?±?7.17% of alpha-glucosidase activity which is required for normal cell wall composition and virulence of Candida albicans. Magnoflorine also reduced the formation of C. albicans’ biofilm. Combined treatment with magnoflorine and miconazole decreased the amount of miconazole required to kill various Candida albicans. Therefore, magnoflorine is a good candidate lead compound for novel antifungal agents.     

Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3

Marine microbes are a rich source of bioactive compounds, such as drugs, enzymes, and biosurfactants. To explore the bioactive compounds from our marine natural product library, an oil emulsification assay was applied to discover biosurfactants and bioemulsifiers. A spore-forming bacterial strain from sea mud was found to produce bioemulsifiers with good biosurfactant activity and a broad spectrum of antimicrobial properties. It was identified as Bacillus velezensis H3 using genomic and phenotypic data analysis. This strain was able to produce biosurfactants with an optimum emulsification activity at pH 6.0 and 2% NaCl by using starch as the carbon source and ammonium sulfate as the nitrogen source. The emulsification-guided isolation and purification procedure led to the discovery of the biosurfactant components, which were mainly composed of nC₁₄-surfactin and anteisoC₁₅-surfactin as determined by NMR and MS spectra. These compounds can reduce the surface tension of phosphate-buffered saline (PBS) from 71.8 to 24.8 mN/m. The critical micelle concentrations (CMCs) of C₁₄-surfactin and C₁₅-surfactin in 0.1 M PBS (pH 8.0) were determined to be 3.06?×?10^-5 and 2.03?×?10^-5?mol/L, respectively. The surface tension values at CMCs for C₁₄-surfactin and C₁₅-surfactin were 25.7 and 27.0 mM/m, respectively. In addition, the H3 biosurfactant exhibited antimicrobial activities against Staphyloccocus aureus, Mycobacterium, Klebsiella peneumoniae, Pseudomonas aeruginosa, and Candida albicans. Thus B. velezensis H3 is an alternative surfactin producer with potential application as an industrial strain for the lipopeptide production.     

Differentially-expressed genes in Candida albicans exposed to ε-poly-l-lysine

Candida albicans is an opportunistic human pathogen whose disinfection is a challenge. ε-Poly-l-lysine (ε-PL), an antagonistic agent, can disrupt cell membranes and inhibit the growth of C. albicans. Genes that were differentially-expressed in response to ε-PL were isolated from C. albicans and identified by suppression subtractive hybridization. Ten subtracted clones, that share >98 % homology with known genes of C. albicans, were isolated. Among these, four genes encoded cell wall-associated proteins. Real-time quantitative PCR and northern blot hybridization suggest that these genes are involved in the response to ε-PL. These findings will help to determine the mechanism of the antimicrobial activity of ε-PL against C. albicans.     

Epidemiology and Antifungal Susceptibilities of Yeasts Causing Vulvovaginitis in a Teaching Hospital

Vulvovaginal candidiasis is one of the most common mycosis. However, the information about antifungal susceptibilities of the yeasts causing this infection is scant. We studied 121 yeasts isolated from 118 patients with vulvovaginal candidiasis. The isolates were identified by phenotypic and molecular methods, including four phenotypic methods described to differentiate Candida albicans from C. dubliniensis. Antifungal susceptibility testing was performed according to CLSI documents M27A3 and M27S4 using the drugs available as treatment option in the hospital. Diabetes, any antibacterial and amoxicillin treatment were statistically linked with vulvovaginal candidiasis, while oral contraceptives were not considered a risk factor. Previous azole-based over-the-counter antifungal treatment was statistically associated with non-C.albicans yeasts infections. The most common isolated yeast species was C. albicans (85.2 %) followed by C. glabrata (5 %), Saccharomyces cerevisiae (3.3 %), and C. dubliniensis (2.5 %). Fluconazole- and itraconazole-reduced susceptibility was observed in ten and in only one C. albicans strains, respectively. All the C. glabrata isolates showed low fluconazole MICs. Clotrimazole showed excellent potency against all but seven isolates (three C. glabrata, two S. cerevisiae, one C. albicans and one Picchia anomala). Any of the strains showed nystatin reduced susceptibility. On the other hand, terbinafine was the less potent drug. Antifungal resistance is still a rare phenomenon supporting the use of azole antifungals as empirical treatment of vulvovaginal candidiasis.     

Isolation and characterization of an antimicrobial peptide from bovine hemoglobin ��-subunit

In this study, a novel 18-residue linear antimicrobial peptide derived from the central part of the bovine hemoglobin ??-subunit was identified. The peptide was purified by a combination of cationic exchange and reversed-phase high-performance liquid chromatography. The sequence was determined to be VNFKLLSHSLLVTLASHL. The theoretical molecular weight of this peptide was calculated to be 1992.38 Da, which is the same as that determined (1992.401 Da) by matrix-assisted laser desorption ionization mass spectrometry. Sequence analysis showed that there is a high degree of homology in this peptide among hemoglobin ??-subunits of bovine, sheep, deer, porcine, and human. In a radial-diffusion plate assay, this purified peptide exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans.     

Efficient biotransformation for preparation of pharmaceutically active ginsenoside Compound K by Penicillium oxalicum sp. 68

Pathogenic fungus Penicillium oxalicum sp. 68 was screened from soil and identified by ITS sequencing. The strain was found to be able to transform protopanaxadiol-type ginsenosides to produce a series of bioactive metabolites. Glycosidase from the culture of P. oxalicum sp. 68 was partially purified with a simple two-step procedure consisting of DEAE-cellulose chromatography and ammonium sulfate precipitation. Bioactive ginsenoside Compound K was prepared selectively and efficiently by biotransformation of ginsenosides Rb1, Rb2, Rc and Rd using the partially purified glycosidase. The optimal conditions for transforming Rb1 into Compound K were pH 4.0, 55 °C and 0.5 mg mL^?1 Rb1. The sole product is Compound K and the maximum yield reached 87.7 % (molar ratio). The transformation pathways of Rb1, Rb2, Rc and Rd are Rb1→Rd→F2→Compound K, Rb2→CO→CY→Compound K, Rc→Mb→Mc→Compound K and Rd→F2→Compound K, respectively. This biotransformation method showed great potential for preparing minor bioactive ginsenosides, especially Compound K, in the pharmaceutical industry because of its high specificity and favorable environmental compatibility.     

Pseudomembranous Candidiasis in HIV/AIDS Patients in Cali, Colombia

Candida albicans is the most frequently isolated yeast from the oral cavity of HIV/AIDS individuals. The use of fluconazole has increased the number of resistant or less-sensitive Candida species different than C. albicans. The purpose of this study was to identify the Candida species producing pseudomembranous candidiasis in patients suffering from AIDS, their relationship with CD4⁺ counts and their sensitivity to fluconazole and itraconazole. We studied 71 patients at a hospital in the city of Cali. Samples of white plaque were seeded on CHROMagar Candida, yeast identification was done with API 20C Aux, and susceptibility testing was determined by E test. Ninety-three yeast isolates were obtained, 52 single and 41 mixed. C. albicans was the most isolated, followed by C. glabrata. An increased frequency of isolates and variety of Candida species occurred in patients with a CD4⁺ cell count ≤100 cells/mm³ without significant differences (p = 0.29). The susceptibility study showed that 8 (8.6 %) isolates were resistant to fluconazole and 11 (11.8 %) to itraconazole, while 6 (8.8 %) C. albicans were simultaneously resistant. No association was found between the isolates of C. albicans or Candida species different than C. albicans and the use of fluconazole (p = 0.21). The results of this study indicate that in the tested population, fluconazole continues to be the best treatment option for oropharyngeal candidiasis in patients suffering from AIDS (HIV/AIDS); however, susceptibility tests are necessary in patients who present therapeutic failure.     

Characterization of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) lectin for biological activity

Lectins are proteins that are subject of intense investigations. Information on lectin from chickpea (Cicer arietinum L.) with respect to its biological activities are very limited. In this study, we purified lectin from the seeds of chickpea employing DEAE-cellulose and SP-Sephadex ion exchange chromatography and identified its molecular subunit mass as 35 kDa. The free radical scavenging activity of lectin measured by the DPPH assay has IC₅₀ of 0.88 µg/mL. Lectin exerted antifungal activity against Candida krusei, Fusarium oxysporium oxysporium, Saccharomyces cerevisiae and Candida albicans, while antibacterial activity against E. coli, B. subtilis, S. marcescens and P. aeruginosa. The minimum inhibitory concentrations were 200, 240, 160 and 140 µg for C. krusei, F. oxysporium, S. cerevisiae and C. albicans respectively. Lectin was further examined for its antiproliferative potential against cancerous cell line. The cell viability assay indicated a high inhibition activity on Ishikawa, HepG2, MCF-7 and MDA-MB-231 with IC₅₀ value of 46.67, 44.20, 53.58 and 37.46?µg/mL respectively. These results can provide a background for future research into the benefits of chickpea lectin to pharmacological perspective.     

Isolation and identification of antimicrobial secondary metabolites from Bacillus cereus associated with a rhabditid entomopathogenic nematode

The cell-free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain six bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (LCMS, FABMS, ¹H NMR, ¹³C NMR, ¹H ?¹H COSY, ¹H ?¹³C HMBC) and Marfey’s method. The compounds were identified as cyclo(D-Pro-D-Leu), cyclo(L-Pro-D-Met), cyclo (L-Pro-D-Phe), cyclo (L-Pro-L-Val), 3,5-dihydroxy-4-ethyl-trans-stilbene, and 3,5-dihydroxy-4-isopropylstilbene, respectively. Compounds recorded antibacterial activity against all four tested bacteria strains of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. 3,5-dihydroxy-4-isopropylstilbene recorded activity only against Gram-positive bacteria while cyclo(L-Pro-L-Val) recorded no antibacterial activity. Best antibacterial activity was recorded by 3,5-dihydroxy-4-ethyl-trans-stilbene (4 μg/ml) against Escherichia coli. The six compounds recorded significant antifungal activities against five fungal strains tested (Aspergillus flavus, Candida albicans, Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum) and they were more effective than bavistin, the standard fungicide. The activity of cyclo(D-Pro-D-Leu), cyclo(L-Pro-D-Met), 3,5-dihydroxy-4-ethyl-trans-stilbene, and 3,5-dihydroxy-4-isopropylstilbene against Candida albicans was better than amphotericin B. To the best of our knowledge, this is the first report of antifungal activity of the bioactive compounds against the plant pathogenic fungi Fusarium oxysporum, Rhizoctonia solani, and Penicillium expansum. We conclude that the Bacillus cereus strain associated with entomopathogenic nematode is a promising source of natural bioactive secondary metabolites which may receive great benefit as potential sources of new drugs in the agricultural and pharmacological industry.     

Candida species in tchapalo and bangui, two traditional alcoholic beverages from Côte d'Ivoire

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while β-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.     

Anti‐Adhesion Activity of Tannins Isolated from the Mangrove Laguncularia racemosa

In the search of new compounds with biofilm‐inhibiting properties, mangroves with their richness of secondary metabolites can be a valuable resource. Crude methanolic leaf extracts from the mangrove Laguncularia racemosa enriched in phenolic substances cause a reduction in initial cell adhesion of Candida glabrata and Candida albicans, but not on Escherichia coli. LC/MS‐guided fractionation of the phenolic compounds resulted in 19 fractions, of which ten were analyzed for their bioactivity against cell adhesion. Effects on cell adhesion and planktonic growth of Escherichia coli, Candida glabrata and Candida albicans were measured in 96‐well microtiter plates in the presence of 0.2 mg ml^?1 of the isolated fractions. Two fractions caused a reduction of cell adhesion of Candida albicans. These fractions containing bioactive compounds were analyzed by LC/MS and NMR spectroscopy. Casuarinin and digalloyl‐hexahydroxydiphenoyl‐glucose were identified in the active fractions, in addition to three signals of ellagitannins. These results indicate a specific mode of action of hydrolysable tannins against cell adhesion of Candida albicans, which needs to be further analyzed.     

Species Distribution and Virulence Factors of Candida spp. Isolated from the Oral Cavity of Kidney Transplant Recipients in Brazil

Although yeasts belonging to the genus Candida are frequently seen as commensals in the oral cavity, they possess virulence attributes that contribute for pathogenicity. The aims of the present study were to study the prevalence of Candida spp. isolated from the oral cavity of renal transplant recipients and to analyze strains virulence factors. We isolated a total of 70 Candida strains from 111 transplant recipients, and Candida albicans was the most prevalent species (82.86 %). Oral candidiasis was diagnosed in 14.4 % kidney transplant patients, while 11 isolates (15.7 %) corresponded to non-Candida albicans Candida (NCAC) species. C. albicans adhered to a higher extension than NCAC strains. Some isolates of Candida tropicalis were markedly adherent to human buccal epithelial cells and highly biofilm-forming strains. Regarding proteinase activity, Candida orthopsilosis was more proteolytic than Candida metapsilosis. Candida glabrata and Candida dubliniensis showed very low ability to form biofilm on polystyrene microtiter plates. We have demonstrated here diverse peculiarities of different Candida species regarding the ability to express virulence factors. This study will contribute for the understanding of the natural history and pathogenesis of yeasts belonging to the genus Candida in the oral cavity of patients who were submitted to kidney transplant and are under immunosuppressive therapies.     

Lipopeptides from Bacillus strain AR2 inhibits biofilm formation by Candida albicans

The ability of the human fungal pathogen Candida albicans to reversibly switch between different morphological forms and establish biofilms is crucial for establishing infection. Targeting phenotypic plasticity and biofilm formation in C. albicans represents a new concept for antifungal drug discovery. The present study evaluated the influence of cyclic lipopeptide biosurfactant produced by Bacillus amyloliquefaciens strain AR2 on C. albicans biofilms. The biosurfactant was characterized as a mixture of iturin and fengycin by MALDI-TOF and amino acid analysis. The biosurfactant exhibited concentration dependent growth inhibition and fungicidal activity. The biosurfactant at sub-minimum growth inhibition concentration decreased cell surface hydrophobicity, hindered germ tube formation and reduced the mRNA expression of hyphae-specific gene HWP1 and ALS3 without exhibiting significant growth inhibition. The biosurfactants inhibited biofilm formation in the range of 46–100 % depending upon the concentration and Candida strains. The biosurfactant treatment dislodged 25–100 % of preformed biofilm from polystyrene plates. The biosurfactant retained its antifungal and antibiofilm activity even after exposure to extreme temperature. By virtue of the ability to inhibit germ tube and biofilm formation, two important traits of C. albicans involved in establishing infection, lipopeptides from strain AR2 may represent a potential candidate for developing heat stable anti-Candida drugs.     

The Diversity and Antimicrobial Activity of Preussia sp. Endophytes Isolated from Australian Dry Rainforests

Limited knowledge currently exists regarding species diversity and antimicrobial activity of endophytic isolates of Preussia within Australia. This report describes endophytic Preussia species that were identified through molecular analysis of the internal transcribed spacer region. Screening for antimicrobial secondary metabolites was determined by testing crude ethyl acetate (EtOAc) extracts derived from fungal mycelia against a panel of ATCC type strains which included Bacillus cereus, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Serratia marcescens, methicillin-resistant Staphylococcus aureus (MRSA) and the opportunist yeast pathogen Candida albicans. Subsequently, high-performance liquid chromatography generated fractions of bioactive EtOAc extracts which were subject to confirmatory testing using the Clinical and Laboratory Standards Institute reference microdilution antimicrobial activity assay. A total of 18 Preussia were isolated from nine host plants with 6/18 having a <97 % sequence similarity to other known species in Genbank, suggesting that they are new species. In preliminary screening, 13/18 Preussia isolates revealed antimicrobial activity against at least one of the microbes tested, whilst 6/18 isolates, including 4/6 putative new species showed specific antimicrobial activity against MRSA and C. albicans. These results highlight the antimicrobial potential of Australian Preussia spp. and also the importance of Australian dry rainforests as an untapped repository of potentially significant bioactive compounds.     

Killing Rates for Caspofungin Against Candida albicans After Brief and Continuous Caspofungin Exposure in the Presence and Absence of Serum

It was previously demonstrated that brief (≤1 h) exposures to echinocandins are as effective to kill Candida albicans cells as continuous 24-h exposure. However, killing rates after continuous and short (1 h) echinocandin exposures to C. albicans have not yet been evaluated in RPMI-1640 with and without 50 % serum. We evaluated four echinocandin susceptible C. albicans bloodstream isolates, ATCC 10231 type strain and an echinocandin-resistant isolate (DPL20, FKS F645P). Caspofungin MICs, time-kill and postantifungal effect (PAFE) tests were performed in RPMI-1640 with and without 50 % serum. Killing rates (k values) in time-kill and PAFE experiments were determined for each strain and concentration. In time-kill experiments, colony count decreases were isolate- and concentration-dependent at 0.25, 1, 4, 8, 16 and 32 mg/L in RPMI-1640, but concentration-independent at 1, 4, 8, 16 and 32 mg/L in 50 % serum. One-hour caspofungin exposure at 4, 16 and 32 mg/L resulted in CFU decreases comparable with the results obtained in time-kill experiments in RPMI-1640, but 50 % serum at 4, 16 and 32 mg/L allowed growth of all isolates (k values were negative) (P < 0.05–0.001). PAFE in 50 % serum decreased markedly at 4, 16 and 32 mg/L. Killing rates remained high and concentration-independent in 50 % serum in case of continuous but not in case of brief caspofungin exposure. As only a short growth inhibition without killing was observed in 50 % serum, clinical relevance of caspofungin PAFE in vivo is questionable.