Screening of terrestrial and freshwater halotolerant cyanobacteria for antifungal activities

Cyanobacterial cultures tolerating 200 mmol l⁻¹ sodium chloride isolated from terrestrial and freshwater habitats of North Maharashtra region of India were evaluated for antifungal activity. Aqueous, methanol, n-propanol, and petroleum ether extracts of 40 cyanobacterial isolates belonging to nine genera were examined for inhibitory activity against five fungal pathogens. Eighteen isolates belonging to genus Oscillatoria dominated the population of halotolerant cyanobacterial cultures. Four antifungal bioassays viz. double layer agar method, disc diffusion assay, silica gel method, and minimum inhibitory concentration (MIC) were used to screen the cultures for antifungal activity. Among the solvents used, methanol extracts showed 34.9% inhibition followed by n-propanol, petroleum ether, and water exhibiting 30.2%, 18.6% and 16.2% inhibition, respectively. The double agar layer method was found to be a suitable method in preliminary screening for handling large number of cultures without extraction of compounds. However, in later screening experiments, silica gel method was seen to be advantageous over MIC and agar disc diffusion methods.     

The ability of white-rot fungi to degrade the endocrine-disrupting compound nonylphenol

Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Bjerkandera sp. BOL13 were tested for their ability to degrade the endocrine-disrupting compound nonylphenol at an initial concentration of 100 mg l^–1. The highest removals were achieved with T. versicolor and Bjerkandera sp. BOL13, which were able to degrade 97 mg l^–1 and 99 mg l^–1 of nonylphenol in 25 days of incubation, respectively. Nonylphenol removal was associated with the production of laccase by T. versicolor, but the levels of laccase, manganese peroxidase and lignin peroxidase produced by Bjerkandera sp. BOL13 were very low. At 14°C, T. versicolor and Bjerkandera sp. BOL13 sustained the removal of 88 mg l^–1 and 79 mg l^–1 of nonylphenol, respectively. No pollutant removal was recorded at 4°C, although both fungi could grow at this temperature in the absence of nonylphenol. A microtoxicity assay showed that the fungi produced compounds that were toxic to Vibrio fischerii; and thus a reduction in toxicity could not be correlated with nonylphenol metabolism. T. versicolor and Bjerkandera sp. BOL13 were capable of colonizing soil artificially contaminated with 430 mg kg^–1 of nonylphenol. Only 1.3±0.1% of nonylphenol remained in the soil after 5 weeks of incubation.     

Acute toxicity tests on three species of the genus Lecane (Rotifera: Monogononta)

Three rotifer species, Lecane hamata L. luna, and L. quadridentata, were submitted to acute toxicity tests to compare their susceptibility to 11 toxicants. In acute tests with 48-h exposure of neonates of less than 24 h old, copper was most toxic with LC₅₀ values in the range of 0.06–0.33 mg l^–1, while acetone was the least toxic with LC₅₀ values in the range of 5000–7000 mg l^–1. Differences in LC₅₀ value of up to 22-fold were found in the susceptibility to lead between the three species. These data indicate large differences in toxicity among members of the same genus, and point out that it is necessary to submit several species to toxicity tests in order to assess the potential effects of toxicants to rotifers. The commonly used Brachionus calyciflorus cannot be considered representative of all freshwater rotifers in this respect.     

Transgenic cucumber fruits that produce elevated level of an anti-aging superoxide dismutase

Superoxide dismutase (SOD) plays an important role in cellular defense against oxidative stress in aerobic organisms. To generate cucumber (Cucumis sativus L.) fruits producing high yields of SOD for an anti-aging cosmetic material as a plant bioreactor, the CuZnSOD cDNA (mSOD1) from cassava was introduced into cucumber fruits by Agrobacterium-mediated transformation using the ascorbate oxidase promoter with high expression in fruits. The bialaphos-resistant shoots were selected on medium containing MS basal salts, 2 mg l^–1 BA, 0.1 mg l^–1 IAA, 300 mg l^–1 claforan, and 2 mg l^–1 bialaphos. After 6 weeks of culture on the selection medium, the shoots were transferred to MS medium containing 1 mg l^–1 IAA, 300 mg l^–1 claforan, 2 mg l^–1 bialaphos to induce roots. Southern blot analysis confirmed that the mSOD1 gene was properly integrated into the nuclear genomes of three cucumber plants tested. The mSOD1 gene was highly expressed in the transgenic cucumber fruits, whereas it was expressed at a low level in the transgenic leaves. The SOD specific activity (units/mg protein) in transgenic fruits was approximately 3 times higher than in those of non-transgenic plants.     

In vitro susceptibility to 9 antifungal agents of 14 strains of Zygomycetes isolated from clinical specimens

Fourteen clinical isolates of Zygomycetes were tested for their in vitro susceptibility to nine antifungal agents. Susceptibility assessment was performed using a microtiter broth dilution method. Synthetic broth with YNB and glucose was used for 5-fluorocytosine and BHI broth for all the other antimycotics. Amphotericin B exhibited the strongest activity against all isolates tested. MIC values of other two polyenes — nystatin and pimaricin — ranged within the susceptibility limits, with a little pronounced higher activity of pimaricin. The isolates of the genusAbsidia andSyncephalastrum were well sensitive to all antimycotics with the exception of 5-fluorocytosine and naftifine. A very weak or zero growth inhibitory effect against all members of the generaMucor andRhizopus was found in azoles, 5-fluorocytosine and naftifine.     

Performance of different methods for testing polymyxin B: comparison of broth microdilution,agar dilution and MIC test strip in mcr-1 positive and negative Escherichia coli

Antimicrobial susceptibility testing with the last-resort antibiotics polymyxins (polymyxin B and colistin) is associated with several methodological issues. Currently, broth microdilution (BMD) is recommended for colistin and polymyxin B. BMD is laborious and the utility of alternative methods needs to be evaluated for polymyxin B susceptibility testing. In this study, using BMD as a reference method, the performance of agar dilution (AD) and MIC test strips (MTS) were evaluated in polymyxin B susceptibility testing. BMD, AD and MTS were used to determine MICs of 193 clinical isolates of Escherichia coli. Seventy-nine were positive for the polymyxin resistance gene mcr-1. Method performances were evaluated based on pair-wise agreements with the reference method (BMD) and statistical testing. AD and MTS showed an unacceptable number of very major errors (VMEs) compared with BMD, 9·3 and 10·7%, respectively. The essential agreement (EA) was low for AD (49·7%), but high for MTS (97·8%). However, statistical testing showed that MTS tended to yield a one-step lower MIC (P < 0·01) compared with BMD. The discordances observed with MTS and AD in comparison with BMD for polymyxin B susceptibility testing for E. coli suggest their inapplicability in routine testing. A large number of isolates clustered around the susceptibility breakpoint (2–4 mg l⁻¹) and several mcr-1 positive isolates (17%) were determined as susceptible with BMD. A screening breakpoint for mcr-1 of 2 mg l⁻¹ should also be considered.     

Can soil Chytridiomycota survive and grow in different osmotic potentials?

Twenty isolates from soil in the orders Spizellomycetales, Blastocladiales and Chytridiales (Chytridiomycota) grew on complex solid media supplemented with 10 g l⁻¹ sodium chloride. In a synthetic liquid medium, 4.4 g l⁻¹ sodium chloride strongly inhibited growth in three of the five isolates, possibly because of the effect of the ions or osmolarity of the solution. The maximum concentration for growth in synthetic liquid medium with different osmotic potentials using polyethylene glycol (PEG) varied considerably amongst the isolates. Three patterns of growth with increasing concentrations of PEG were evident among isolates within the genus Rhizophydium. Up to the concentration where growth ceased, the dry weight of each isolate either decreased, remained constant, or in one case, increased. Most of the fungi survived when incubated at room temperature for 7 d in complex liquid media supplemented with 35 g l⁻¹ sodium chloride or 300 g l⁻¹ PEG. These data indicate that soil Chytridiomycota can survive various osmotic potentials that may occur during the wetting and drying phases in soils.     

Standardization of hyphal growth inhibition rate as a means of evaluating Microsporum spp. in vitro susceptibility to terbinafine, griseofulvin, and ciclopiroxolamine

Reference methods for antifungal susceptibility tests recommend the use of conidia as inoculum. However, some isolates produce few conidia, while the invasive form of filamentous fungi in general is hyphae making susceptibility tests infeaseble. These facts suggest that other than conidia broth dilution method is required for susceptibility tests. The aim of this study was to clarify if the hyphal growth inhibition rate could be used as a method of determining the antifungal susceptibility of genus Microsporum. For this reason, a method which traces hyphal tips automatically and measures their growth rate was standardized for Microsporum spp. Control growth curves and test growth curves obtained by real-time observation of the hyphae groups responses to different concentrations of terbinafine, griseofulvin, and ciclopiroxolamine were used to compare with minimum inhibitory concentrations (MICs) obtained by conidia broth microdilution method. A visible reduction in the growth inhibition rate was observed when hyphal activity was evaluated using the third or fourth serial two-fold dilution below the MIC determined by broth microdilution for terbinafine and ciclopiroxolamine. For griseofulvin, this reduction occurred after the fifth dilution below the MIC. This study highlights the importance of the inoculum type used to determine the in vitro susceptibility of Microsporum strains. We conclude that measurement of hyphal growth inhibition, despite being time consuming, could be a suitable method for evaluating antifungal susceptibility, particularly for fungi as Microsporum spp. that produce a small (or not at all) number of conidia.     

In vitro activities of voriconazole, fluconazole, itraconazole and amphotericin B against non Candida albicans yeast isolates

Antifungal susceptibility testing was performed on 197 yeast isolates from the BCCM/IHEM biomedical fungi and yeasts collection (Belgian Co-ordinated Collections of Micro-organisms / IPH-Mycology) to study the in vitro activity of voriconazole against fluconazole, itraconazole and amphotericin B. MICs of the four antifungal agents were determined by an adapted NCCLS M27-A microdilution reference method. MIC readings were visually and spectrophotometrically determined. Optical density data were used for calculation of the MIC endpoints. For amphotericin B, the MIC endpoint was defined as the minimal antifungal concentration that exerts 90% inhibition, compared to the control growth. The azoles endpoints were determined at 50% inhibition of growth. The MIC distribution of voriconazole susceptibilities showed that 193 isolates had a MIC < or = 2 microg/ml and 185 a MIC < or = 1 microg/ml. Cross-tabulation of voriconazole, fluconazole, and itraconazole MICs indicated that voriconazole MICs raised with fluconazole and itraconazole MICs. The in vitro data obtained in this study suggest that voriconazole may also be effective treating yeast infection in patients infected with fluconazole or itraconazole resistant isolates.     

Inhibitory effect of garlic on bacterial pathogens from spices

An unconventional technique for primary screening of bacterial susceptibility to garlic (Allium sativum Linn.), using a slice from its clove, was described. Aqueous extracts of garlic were found to possess a potent bacteriostatic principle against Gram-positive as well as Gram-negative foodborne bacterial pathogens. In agar medium, the minimum inhibitory concentrations (MICs) of garlic were 6–10 mg ml^–1 for Bacillus cereus, 30–40 mg ml^–1 for Staphylococcus aureus (excepting the isolate from garlic, where the MIC was 100 mg ml^–1), 20–30 mg ml^–1 for Clostridium perfringens, 10 mg ml^–1 for Escherichia coli (30 mg ml^–1 for the garlic isolate), 40–100 mg ml^–1 for Salmonella, and 10–40 mg ml^–1 for Shigella. It inhibited the growth of all these strains, which were resistant to some commonly used antibiotics. Most of the tested strains were resistant to penicillins, although sensitive to garlic. While the growth of B. cereus and Cl. perfringens was completely inhibited at 10 and 70 mg garlic, respectively, ml^–1 test broth, their respective enterotoxin production ceased at 10 and 50 mg garlic ml^–1.     

Bioconversion of compactin into pravastatin by Streptomyces sp.

Streptomyces sp. Y-110, isolated from soil, modified compactin to pravastatin, a therapeutic agent for hypercholesterolemia. In a batch culture, the highest production of pravastatin was 340 mg l^–1 from 750 mg compactin l^–1 in 24 h. By intermittent feeding of compactin into the culture medium, both the compactin concentration and its conversion increased to 2000 mg l^–1 and 1000 mg pravastatin l^–1, respectively, with the conversion rate of 10 mg l^–1 h^–1. Continuous feeding of compactin increased production of pravastatin to 15 mg l^–1 h^–1.     

Production potential of eicosapentaenoic acid by the diatom Nitzschia laevis

To investigate the production potential of eicosapentaenoic acid (EPA) by the diatom Nitzschia laevis, the growth characteristics and fatty acid composition of the cells were studied under photoautotrophic, mixotrophic and heterotrophic conditions of growth. The specific growth rate and maximum biomass concentration were respectively 0.466 d^–1 and 2.27 g l^–1 for mixotrophic culture, 0.344 d^–1 and 2.04 g l^–1 for heterotrophic culture, and 0.167 d^–1 and 0.5 g l^–1 for photoautotrophic culture, respectively. As for EPA production, the yield and productivity were respectively 52.32 mg l^–1 and 10.46 mg l^–1 d for mixotrophic culture, 35.08 mg l^–1 and 6.37 mg l^–1 d for heterotrophic culture, and 6.78 mg l^–1 and 3.39 mg l^–1 d for photoautotrophic culture, respectively. Results suggest that mixotrophic culture is the most suitable growth mode for the production of EPA by the diatom Nitzschia laevis. The results are useful for the development of a cost-effective fermentation process for EPA production by Nitzschia laevis.     

Enhancement of paclitaxel production by abscisic acid in cell suspension cultures of Taxus chinensis

Addition of 5 mg abscisic acid l^–1 after 12 days' growth of Taxus chinensis suspension culture gave the greatest paclitaxel accumulation at 11 mg l^–1, which was almost 5 times that of the control culture. The highest paclitaxel production, 18 mg l^–1, was obtained using 5 mg abscisic acid l^–1 and 20 mg methyl jasmonate l^–1.     

Antibiotic susceptibility testing ofCandida albicans by flow cytometry

A flow cytofluorometric susceptibility test (FCST) for in vitro antifungal drug testing ofCandida albicans was developed. Membrane damage was indicated by increased cellular fluorescence owing to propidium iodide or rose bengal uptake. Ketoconazole caused an exponential dose-response effect, best defined by E_max, at therapeutically achievable concentrations (0.02–0.2 µg/ml). This effect was not comparable to the conventional minimum inhibitory concentration (MIC) effect elicited by higher antibiotic concentrations. Amphotericin B, on the other hand, did not elicit E_max, but caused a >1 log dose-response effect which did correspond to the MIC. 5-Fluorocytosine susceptibility was also measurable. Other cytometric data indicating abnormal growth and growth inhibition, as well as conventional growth inhibition testing, confirmed that the 10-h FCST measured useful parameters of in vitro susceptibility.     

In vitro activity of seven azole compounds against some clinical isolates of non-dermatophytic filamentous fungi and some dermatophytes

The in vitro activity of seven azole compounds viz clotrimozole, isoconazole, bifanazole, fluconazole oxyconazole, Bay n 7133 and Bay L 9139 was investigated against 47 clinical isolates of pathogenic non-dermatophytic filamentous fungi and dermatophytic fungi. The isolates included Hendersonula toruloidea-26, Scytalidium hyalinium-5, Scytalidium japonicum-1, Trichophyton rubrum-5, Trichophyton tonsurans-3, Trichophyton mentagrophytes var. mentagrophytes-4, Epidemophyton floccosum-2, Microporum gypseum-2 isolates. The drugs were significantly more active against the dermatophytes (MIC range 0.025–1.56 g/ml) than non-dermatophytes (MIC range 0.39–6.25 g/ml). Isoconazole showed more activity than the rest of the azole compound tested. Clotrimazole, fluconazole, oxyconazole, bifonazole were comparable in their inhibitory activity against both dermatophytes and non-dermatophytes. The azole derivatives, Bay n 7133 and Bay L 9139 showed higher MIC range i.e. gave a range of 0.39–1.56 g/ml for dermatophytes and 1.56–6.25 g/ml for non-dermatophytic filamentous fungi. The minimal fungicidal concentration (MFC) of all the drugs tested were mostly within 2–8 times their MIC values.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Efficient plant regeneration in vitro in buckwheat

An in vitro highly efficient plant regeneration system was established from hypocotyl segments in buckwheat (Fagopyrum esculentum Moench.). Calli were induced on Murashige–Skoog (MS) medium containing 1.0 mg l^–1 to 2.0 mg l^–1 2,4-dichlorophenoxyacetic acid and 1.5 mg l^–1 6-benzylaminopurine. Shoot buds were formed on subcultured pieces of callus. A high frequency (over 80%) of shoot differentiation was obtained on MS medium supplemented with 2.0 mg l^–1 6-benzylaminopurine and 1.0 mg l^–1 6-furfurylaminopurine. The regenerated shoots rooted readily on MS medium plus 0.2 mg l^–1naphthaleneacetic acid and 0.2 mg l^–1 indole butyric acid. The regenerated plantlets were acclimatized and successfully transferred to pots. Chromosome examination showed that the regenerated plants had normal chromosome number (2n=16).     

Improved alkaloid production in Catharanthus roseus suspension cell cultures by various chemicals

Fourteen chemicals were used to treat Catharanthus roseussuspension cell cultures to improve ajmalicine, catharanthine or serpentine biosynthesis. Ajmalicine production was increased by betaine (to 55 mg l^–1), n-propyl gallate (to 27 mg l^–1), succinic acid (to 31 mg l^–1), malic acid (to 60 mg l^–1) and tetramethyl ammonium bromide (to 64 mg l^–1). Ajmalicine and catharanthine yields were about 5–6 fold higher than the control. A large portion (up to 50–85%) of total indole alkaloids was released into the medium. For maximal catharanthine production, the optimal doses of malic acid and tetramethyl ammonium bromide were 50 mg l^–1and 120 mg l^–1, respectively. The mechanisms which may be responsible for these treatment effects are discussed.     

Degradation of sodium anthraquinone sulphonate by free and immobilized bacterial cultures

Aerobic biodegradation of a xenobiotic recalcitrant compound sodium anthraquinone-2-sulphonate (SAS), was investigated using as an inoculum a mixed microbial culture, which was activated sludge from industrial and domestic waste-water treatment plants. The difference in SAS degradation was examined using two main systems: (1) suspended cells and (2) immobilized cells, both in batch and in continuous culture. In the suspended cell system, under continuous culture conditions using SAS as a unique source of carbon and energy, it was possible to degrade about 95% of this substrate after 6 days. Maximal SAS removal rates in the suspended-cell system were 593 mg SAS l^–1 h^–1 and 88.7 mg SAS l^–1 h^–1 for dilution rates (D) of 0.05 h^–1 and 0.075 h^–1, respectively. In the immobilized-cell system, almost all SAS was degraded in 6 days and the maximal removal rate reached 88.7 mg SAS l^–1 h^–1 at D=0.05 h^–1. Application of a continuous-flow enrichment procedure resulted in selection of several kinds of micro-organisms and led to a progressive elimination of some species of Aeromonas. A stable microbial community of 11 strains has been established and characterized at D=0.075 h^–1. Most of them were Gram-negative and belonged to the genus Pseudomonas.     

Effect of brassinolide on callus growth and regeneration in Spartina patens (Poaceae)

The effect of brassinolide (BL) on cultured calluses of Spartina patens (Ait.) Muhl. (Poaceae), a halophyte monocot was studied. BL at 0.03–0.04 mg l^–1 at fixed concentrations of IAA (0.2 mg l^–1) and BA (3.0 mg l^–1) added in MS medium increased the ratio for fresh weight (CIRFW) to dry weight (CIRDW) by 96–111% and 235–326%. Similarly, in callus regeneration capacity, BL at 0.03 mg l^–1 was most effective, increasing the shoot regeneration ratio (SRR) by 425%. BL at 0.04 mg l^–1 had not such an increasing effect as BL at 0.03 mg l^–1, which increased SRR by 79%. However, BL at 0.005 mg l^–1 promoted regenerated shoot growth most significantly, increasing the shoot height increasing ratio (SHIR) by 395% after a 40-day culture. BL at 0.05 mg l^–1 was least effective in the callus regeneration and regenerated shoot growth, decreasing SRR by 27% and SHIR by 52%. Present results suggest that BL at 0.03 mg l^–1 is suitable for the callus growth and shoot regeneration, while BL at 0.005 mg l^–1 effectively enhanced the regenerated shoot growth.