Synthesis and characterization of the antibacterial potential of ZnO nanoparticles against extended-spectrum β-lactamases-producing <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> and <Emphasis Type="BoldItalic">Klebsiella pneumoniae</Emphasis> isolated from a tertiary care hospital of North India

The reemergence of infectious diseases and the continuous development of multidrug resistance among a variety of disease-causing bacteria in clinical setting pose a serious threat to public health worldwide. Extended-spectrum β-lactamases (ESBLs) that mediate resistance to third-generation cephalosporin are now observed all over the world in all species of Enterobacteriaceae, especially Escherichia coli and Klebsiella pneumoniae. In this work, ZnO nanoparticles (NPs) were synthesized by the sol–gel method and characterized by powder X-ray diffraction, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The image of synthesized ZnO NPs appeared spherical in SEM with a diameter of ≈19 nm and as hexagonal crystal in AFM. Clinical isolates were assessed for ESBL production and shown to be sensitive to ZnO NPs by different methods such as minimal inhibitory concentration (MIC) and minimal bactericidal concentration, time-dependent growth inhibition assay, well diffusion agar methods and estimation of colony forming units (CFU) of bacteria. The lowest MIC value for E. coli and K. pneumoniae was found to be 500 μg/ml. The results showed that ZnO NPs at 1,000 μg/ml completely inhibit the bacterial growth. The antibacterial effect of ZnO nanoparticles was gradual, but time- and concentration-dependent. The maximum inhibition zone at100 μg/ml for E. coli and K. pneumoniae was 22 and 20 mm, respectively. With the increasing ZnO NP loading, there is significant reduction in the numbers of CFU. At the concentration of 1,000 μg/ml, the decline in per cent survival of E. coli and K. pneumoniae was found to be 99.3% and 98.6%, respectively.     

Antioxidant and antimicrobial actions of the clubmoss <Emphasis Type="Italic">Lycopodium clavatum</Emphasis> L.

Purpose of the present study was to evaluate antioxidant, antibacterial, antifungal, and antiviral activities of the petroleum ether, chloroform, ethyl acetate and methanol extracts as well as the alkaloid fraction of Lycopodium clavatum L. (LC) from Lycopodiaceae growing in Turkey. Antioxidant activity of the LC extracts was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method at 0.2 mg/ml using microplate-reader assay. Antiviral assessment of LC extracts was evaluated towards the DNA virus Herpes simplex (HSV) and the RNA virus Parainfluenza (PI-3) using Madin-Darby Bovine Kidney (MDBK) and Vero cell lines. Antibacterial and antifungal activities of the extracts were tested against standard and isolated strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis as well as the fungi; Candida albicans and C. parapsilosis. All of the extracts possessed noteworthy activity against ATCC strain of S. aureus (4 μg/ml), while the LC extracts showed reasonable antifungal effect. On the other hand, we found that only the chloroform extract was active against HSV (16–8 μg/ml), while petroleum ether and alkaloid extracts inhibited potently PI-3 (16–4 μg/ml and 32–4 μg/ml, respectively). However, all of the extracts had insignificant antiradical effect on DPPH. In addition, we also analyzed the content of the alkaloid fraction of the plant by capillary gas chromatography-mass spectrometry (GC-MS) and identified lycopodine as the major alkaloid.     

Isolation and identification of antimicrobial agent-producing bacterium from <Emphasis Type="Italic">Taxus baccata</Emphasis> rhizosphere antagonistic against clinically significant microbes

A bacterium identified as Pseudomonas fluorescence was isolated from Taxus baccata rhizosphere. Ethyl acetate extract from its culture filtrate yielded an active antimicrobial compound that was purified by TLC. The active metabolites were resolved by column chromatography on silica gel (60–120 mesh). The compound was further characterized on the basis of spectral data (UV, IR and ¹HNMR), which indicated the presence of an aromatic ring and phenolic functionality. The compound showed significant antimicrobial activity against two-gram positive bacteria (B. subtilis and S. aureus), four-gram negative bacteria (E. coli, K. pneumoniae, S. flexneri and P. aeruginosa), and one pathogenic fungus (Candida albicans). The minimum inhibitory concentration (MIC) of the compound ranged between 75μg to 250 μg/ml.     

The acid tolerance response of Bacillus cereus ATCC14579 is dependent on culture pH, growth rate and intracellular pH

The food pathogen Bacillus cereus is likely to encounter acidic environments (i) in food when organic acids are added for preservation purposes, and (ii) during the stomachal transit of aliments. In order to characterise the acid stress response of B. cereus ATCC14579, cells were grown in chemostat at different pH values (pH_o from 9.0 to 5.5) and different growth rates (μ from 0.1 to 0.8 h⁻¹), and were submitted to acid shock at pH 4.0. Cells grown at low pH_o were adapted to acid media and induced a significant acid tolerance response (ATR). The ATR induced was modulated by both pH_o and μ, and the μ effect was more marked at pH_o 5.5. Intracellular pH (pH_i) was affected by both pH_o and μ. At a pH_o above 6, the pH_i decreased with the decrease of pH_o and the increase of μ. At pH_o 5.5, pH_i was higher compared to pH_o 6.0, suggesting that mechanisms of pH_i homeostasis were induced. The acid survival of B. cereus required protein neo-synthesis and the capacity of cells to maintain their pH_i and ΔpH (pH_i - pH_o). Haemolysin BL and non-haemolytic enterotoxin production were both influenced by pH_o and μ.     

Phosphate solubilization by <Emphasis Type="Italic">Rhizobium</Emphasis> strains

Forty-six Rhizobium isolates from legume root and stem nodules were examined for their phosphate-solubilizing ability on Pikovskaya’s agar medium. Rhizobium isolates from root nodules of Cassia absus, Vigna trilobata and three strains from Sesbania sesban showed zone of tricalcium phosphate (TCP) solubilization. The isolate from C. absus showed maximum solubilization (620 μg/ml) after 12 d of incubation, while the Rhizobium sp. strain 26 (from S. sesban) showed the least amount (150 μg/ml) of phosphate solubilization. Among the carbon sources tested for their ability to solubilize TCP, maximum solubilization (620 μg/ml) was observed in glucose by Rhizobium isolate from C. absus. Phosphate solubilization increased with increase in glucose concentration steeply up to 2% and slowly above this concentration in four isolates. Among the nitrogen sources tested, maximum solubilization (620 μg/ml) was observed in ammonium sulphate by Rhizobium isolate from C. absus.     

Amoxicillin and specific bacteriophage can be used together for eradication of biofilm of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> B5055

Despite the efficacy of antibiotics as well as bacteriophages in treatment of bacterial infections, their role in treatment of biofilm associated infections is still under consideration especially in case of older biofilms. Here, efficacy of bacteriophage alone or in combination with amoxicillin, for eradication of biofilm of Klebsiella pneumoniae B5055 has been assessed. Planktonic cells as well as biofilm of K. pneumoniae B5055 grown in 96-well microtiter plates were exposed to bacteriophage and amoxicillin at various Multiplicity of Infections (MoIs) as well as at three different antibiotic concentrations (512, 256 and 128 μg/ml), respectively. After exposure to 256 μg/ml (MIC) of amoxicillin, bacterial load of planktonic culture as well as 1-day-old biofilm was reduced by a log factor of 4.1 ± 0.31 (P = 0.008) and 1.24 ± 0.27 (P < 0.05), respectively but reduction in the bacterial load of mature biofilm (8-day-old) was insignificant (P = 0.23). When 8-day-old biofilm was exposed to higher antibiotic concentration (512 μg/ml) or phage alone (MoI = 0.01) a log reduction of 2.97 ± 0.11 (P = 0.182) and 3.51 ± 0.19 (P = 0.073), respectively was observed. While on exposing to a combination of both the amoxicillin and phage, a significant reduction (P < 0.01) in bacterial load of the biofilm was seen. Hence, when antibiotic was used in combination with specific bacteriophage a greater destruction of the biofilm structure suggested that the phages could be used successfully along with antibiotic therapy. An added advantage of the combination therapy would be its ability to check formation of resistant mutants that otherwise develop easily upon using phage or antibiotic alone.     

Morphometry and growth of three <Emphasis Type="Italic">Synechococcus</Emphasis>-like picoplanktic cyanobacteria at different culture conditions

Three phycocyanin-rich strains of Synechococcus-like picoplanktic cyanobacteria, isolated from the plankton of Czech oligotrophic to eutrophic freshwater reservoirs, were investigated in crossed gradients of light and temperature and in combination with two different culture media (BG-11 and WC). The strains exhibited similar growth and reproduction patterns and displayed overlapping ranges of cell size (1.5 × 0.8 μm) under standardized laboratory conditions (18 μmol m⁻² s⁻¹; 20°C). However, strains behavior differed in the crossed gradients. All strains preferred BG-11 medium, where also remarkable size changes could be observed. Length, width, cell abundance and growth rate of two strains were positively correlated with temperature and nutrients, whereas the impact of light intensity was insignificant. Maximum cell elongation (involution cells up to 19 μm) occurred in two strains only in BG-11 medium at highest temperature (28°C) and highest irradiance (53 μmol m⁻² s⁻¹). Cell dimensions in WC medium were constant under most conditions given. The third strain was influenced by all three factors, from which light and nutrients played pivotal role. The length of the lag-phase for all strains appeared to be temperature dependent (negative correlation). Despite the fact that the cell volume in all strains increased more than five times under the lowest light and low temperature (6 μmol m⁻² s⁻¹, <15°C) in both media, the length/width ratio remained unchanged. The strains differed in the degree of cell enlargement and cell division symmetry as well as in optimum temperature and light dependence. Based on this experimental work two strains could be identified as Synechococcus sp. and one as Cyanobium sp., which can be used as a support for the following genetical analyses.     

Activity of the Antimicrobial Peptide and Thanatin Analog S-thanatin on Clinical Isolates of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> Resistant to Conventional Antibiotics with Different Structures

The treatment of infections caused by bacteria resistant to the vast majority of antibiotics is a challenge worldwide. To evaluate the effect of S-thanatin (an analog of thanatin, a cationic antimicrobial peptide isolated from the hemipteran insect Podisus maculiventris) against microbial resistant to antibiotics, we studied its bactericidal kinetics, synergistic effect, resistance, and activity on clinical isolates of Klebsiella pneumoniae resistant to conventional antibiotics with different structures. The bactericidal rate of S-thanatin was more than 99% against K. pneumoniae ATCC 700603 when bacterial cultures were monitored for 60 min. The peptide was synergistic with β-lactam cefepime in most of the clinical MDR isolates tested (7/8). An average value of FIC was 0.3708. No synergy was found between the peptide and amoxicillin, gentamycin, tetracycline, or ciprofloxacin in all bacteria tested. A total of 48 isolates of K. pneumoniae with different resistance spectrum tested was susceptible to S-thanatin. The MICs were 6.25–25 μg/ml. No significant difference in the MICs of S-thanatin between the sensitive isolates and the resistant isolates to single antibiotic was observed (P > 0.05). The resistance of K. pneumoniae ATCC 700603 to S-thanatin was slightly higher, when cultured at sub-inhibitory concentration for 5 days. S-thanatin may be an attractive candidate for developing into an antimicrobial agent.     

Characterization and selection of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains for their effect on bile tolerance,taurocholate deconjugation and cholesterol removal

Fourteen Lactobacillus strains of six species were investigated with their characteristics of bile salt tolerance, deconjugation of sodium taurocholate and cholesterol removal in the spent broth. Meanwhile, a co-precipitation curve of cholesterol with cholic acid at concentrations ranged 0.0–6.0 μM/ml was involved in the evaluation of cholesterol removal. Results demonstrated that both co-precipitation and assimilation effects contributed to cholesterol removal during the incubation of these Lactobacillus strains. It was also indicated that the supplementation of bile salts influenced the cholesterol removal, not only as an essential factor related to co-precipitation but also a critical condition for cholesterol assimilation. Out of all strains tested, four L. plantarum strains LS12, LS31, Lp501 and Lp529 exhibited a high ability of cholesterol assimilation (maximum 20.76 μg/ml), deconjugation of sodium taurocholate (maximum 5.00 μM/ml) and bile tolerance. They could be further studied and used as potential probiotics strains to reduce serum cholesterol in humans     

Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli

The antibacterial activity and acting mechanism of silver nanoparticles (SNPs) on Escherichia coli ATCC 8739 were investigated in this study by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with SNPs. The experimental results indicated 10 μg/ml SNPs could completely inhibit the growth of 10⁷ cfu/ml E. coli cells in liquid Mueller–Hinton medium. Meanwhile, SNPs resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that SNPs were able to destroy the permeability of the bacterial membranes. When the cells of E. coli were exposed to 50 μg/ml SNPs, many pits and gaps were observed in bacterial cells by transmission electron microscopy and scanning electron microscopy, and the cell membrane was fragmentary, indicating the bacterial cells were damaged severely. After being exposed to 10 μg/ml SNPs, the membrane vesicles were dissolved and dispersed, and their membrane components became disorganized and scattered from their original ordered and close arrangement based on TEM observation. In conclusion, the combined results suggested that SNPs may damage the structure of bacterial cell membrane and depress the activity of some membranous enzymes, which cause E. coli bacteria to die eventually.     

Chymostatin can combine with pepstatin to eliminate extracellular protease activity in cultures of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL-3

Aspergillus strains are being considered as potential hosts for recombinant heterologous protein production because of their excellent extracellular enzyme production characteristics. However, Aspergillus proteases are problematic in that they modify and degrade the heterologous proteins in the extracellular medium. In previous studies we observed that media adjustments and maintenance of a filamentous morphology greatly reduced protease activity and that a low concentration of the aspartic protease inhibitor pepstatin inhibited the latter protease activity to the extent of approximately 90%. In this paper we report that when the serine protease inhibitor chymostatin is used in combination with pepstatin 99–100% of total protease activity in Aspergillus cultures is inhibited. In protease assays a concentration of 30 μM chymostatin combined with 0.075 μM pepstatin was required for maximum inhibition. Inhibitor concentrations of chymostatin and pepstatin of 120 and 0.3 μM, respectively, when added to Aspergillus cultures, has no significant effect on biomass production, glucose utilization or culture pH pattern. The potential of using these protease inhibitors in cultures of recombinant Aspergillus strains producing heterologous proteins will now be investigated to determine if the previously observed recombinant protein denaturing effects of Aspergillus proteases can be negated.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

A novel bacterial pathogen (Enterobacter sp.) isolated from the leaf roller, Caloptilia theivora of tea of Darjeeling foothills

Enterobacter sp. was isolated from the diseased and dead caterpillars of the tea leaf roller (Caloptilia theivora) from the Darjeeling foothill region. When the vegetative form of the bacterium was applied via food, mortality of C. theivora showed an LC₅₀ value at 363.1 μg/ml (bacterial wt./vol. of water) with fiducial limits 363.25 and 362.94 μg/ml respectively. The LT₅₀ values for C. theivora were 6 days for 100 μg/ml, 5.96 days for 300 μg/ml, 5.81 days for 500 μg/ml, 4.96 days for 750 μg/ml and 4.61 days for 1,000 μg/ml concentrations. The finding would enable one to contemplate development of a microbial pesticide using this novel Enterobacter sp. DD01 for control of the leaf rolling pest.     

Measurement of particle diameter of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> microcapsule by spray drying and analysis on its microstructure

Lactobacillus acidophilus, as a probiotic, is widely used in many functional food products. Microencapsulation not only increases the survival rate of L. acidophilus during storage and extends the shelf-life of its products, but also optimal size microcapsule makes L. acidophilus have an excellent dispersability in final products. In this paper, L. acidophilus was microencapsulated using spray drying (inlet air temperature of 170°C; outlet air temperature of 85–90°C). The wall materials used in this study were β-cyclodextrin and acacia gum in the proportion of 9:1 (w/w), and microcapsules were prepared at four levels of wall materials (15, 20, 25 and 30% [w/v]) with a core material concentration of 6% (v/v). The microcapsule diameters were measured by Malvern’s Mastersizer-2000 particle size analyzer. The results showed that the particle diameters of microcapsule were mostly within 6.607 μm and 60.256 μm and varied with 2.884–120.226 μm (the standard smaller microcapsule designated as <350 μm). Through comparison of microcapsule size and uniformity with different concentration of wall materials, we concluded that the optimal concentration of wall material was 20% (w/v), which gave microcapsule with a relatively uniform size (averaging 22.153 μm), and the number of surviving encapsulated L. acidophilus was 1.50 × 10⁹ c.f.u./ml. After 8 weeks storage at 4°C, the live bacterial number was above 10⁷ c.f.u./ml, compared with unencapsulated L. acidophilus, 10⁴–10⁵ c.f.u./ml. Through the observation of scanning electron microscopy, we found that the shapes of microcapsule were round and oval, and L. acidophilus cells located in the centre of microcapsule.     

Screening of diazotrophic bacteria Azopirillum spp. for nitrogen fixation and auxin production in multiple field sites in southern Brazil

Isolation of microorganisms, screening for desirable characters and selection of efficient strains are important steps to optimize high crop yields and improve the sustainability of the ecosystem. The objective of this study was isolate and identify Azopirillum spp. with enhanced potential to promote plant growth among the natural bacterial population associated with rhizosphere soil, roots and stem of maize collected from five maize-growing regions within the southern state of Rio Grande do Sul in Brazil. Diazotrophic microorganisms were isolated using semi-solid N-free and solid selective media NFb. In order to select the most efficient isolates as candidates for plant growth promotion, the purified bacterial strains were studied for cell morphology, and Gram staining, streptomycin resistance, as well as screened for their potential for nitrogen fixation and auxin production under sterile conditions. Among 224 isolates obtained 121 were able to fix nitrogen and produce auxin. The 30 most promising isolates produced indole-3-acetic acid (IAA) ranging in concentration from 3.51 μg to 246.69 μg IAA mg⁻¹. Nitrogen fixation ranged from 15.43 μg to 95.21 μg of N mg protein⁻¹ day⁻¹ From the 30 most productive isolates, chromosomal DNA was extracted and a portion of the nifH gene was amplified and sequenced. Twenty-nine isolates were found to be similar to the Azospirillum genus and one isolate was found to be similar to Herbaspirillum seropedicae. These bacterial isolates revealed potential to increase crop productivity, however field crop experiments in Rio Grande do Sul climatic conditions should be done in order to formulate recommendations for their use as inoculants.     

Decolorization of sulfonated azo dyes with two photosynthetic bacterial strains and a genetically engineered Escherichia coli strain

Sulfonated azo dyes were decolorized by two wild type photosynthetic bacterial (PSB) strains (Rhodobacter sphaeroides AS1.1737 and Rhodopseudomonas palustris AS1.2352) and a recombinant strain (Escherichia coli YB). The effects of environmental factors (dissolved oxygen, pH and temperature) on decolorization were investigated. All the strains could decolorize azo dye up to 900 mg l⁻¹, and the correlations between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Repeated batch operations were performed to study the persistence and stability of bacterial decolorization. Mixed azo dyes were also decolorized by the two PSB strains. Azoreductase was overexpressed in E. coli YB; however, the two PSB strains were better decolorizers for sulfonated azo dyes.     

Antibacterial activity of ZnO nanoparticles prepared via non-hydrolytic solution route

The antibacterial activity of ZnO nanoparticles has been investigated and presented in this paper. Nanoparticles were prepared via non-hydrolytic solution process using zinc acetate di-hydrate (Zn(CH₃COO)₂·2H₂O) and aniline (C₆H₅NH₂) in 6 h refluxing at ∼65 °C. In the presence of four pathogens such as Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae, the antibacterial study of zinc oxide nanoparticles were observed. The antibacterial activity of ZnO nanoparticles (ZnO-NPs) were studied by spectroscopic method taking different concentrations (5–45 μg/ml) of ZnO-NPs. Our investigation reveals that the lowest concentration of ZnO-NPs solution inhibiting the growth of microbial strain is found to be 5 μg/ml for K. pneumoniae, whereas for E. coli, S. aureus, and S. typhimurium, it was calculated to be 15 μg/ml. The diameter of each ZnO-NPs lies between “20 and 30 nm” as observed from FESEM and transmission electron microscopy images. The composition of synthesized material was analyzed by the Fourier transform infrared spectroscopy, and it shows the band of ZnO at 441 cm⁻¹. Additionally, on the basis of morphological and chemical observations, the chemical reaction mechanism of ZnO-NPs was also proposed.     

Antimicrobial susceptibility of <Emphasis Type="Italic">Lactobacillus</Emphasis> species isolated from commercial ethanol plants

Bacterial contamination of commercial fermentation cultures is a common and costly problem to the fuel ethanol industry. Antimicrobials such as virginiamycin (VIR) and penicillin (PEN) are frequently used to control contamination but there are little data available on the susceptibility of bacterial contaminants to these agents. A survey of bacterial contaminants from a wet-mill ethanol plant with no history of using antibiotics and a dry-grind facility that periodically doses with VIR found that the majority of contaminants were species of Lactobacillus. Thirty-seven isolates of Lactobacillus species from the wet-mill and 42 isolates from the dry-grind facility were tested for antimicrobial susceptibility using broth dilution and agar dilution methods. In general, the Lactobacillus isolates from the dry-grind plant had higher minimum inhibitory concentrations (MICs) for the tested agents than the isolates from the wet-mill facility. The MIC₉₀ for VIR was 4 μg/ml for the dry-grind isolates versus 0.25 μg/ml for the wet-mill isolates; and for PEN, the MIC₉₀’s were >8 and 2 μg/ml for the dry-grind and wet-mill isolates, respectively. Sixteen Lactobacillus isolates from the dry-grind plant but none from the wet-mill possessed vatE, a gene that encodes a streptogramin acetyltransferase associated with resistance to virginiamycin. Despite decreased susceptibility to virginiamycin, most dry-grind isolates had MICs lower than the maximal recommended application rate of 6 ppm. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Characterisation and bioactivity of protein-bound polysaccharides from submerged-culture fermentation of Coriolus versicolor Wr-74 and ATCC-20545 strains

The protein-bound polysaccharides of Coriolus versicolor (CPS) have been reported to stimulate overall immune functions against cancers and various infectious diseases by activating specific cell functions. A New Zealand isolate (Wr-74) and a patented strain (ATCC-20545) of C. versicolor were compared in this study. The fruit bodies of both strains were grown for visual verification. Both strains were grown in submerged-culture using an airlift fermentor with milk permeate as the base medium supplemented with glucose, yeast extract and salt. Metabolic profiles of both strains obtained over 7-day fermentation showed very similar trends in terms of biomass production (8.9–10.6 mg/ml), amounts of extracellular polysaccharide (EPS) from the culture medium (1150–1132 μg/ml), and intracellular polysaccharide (IPS) from the mycelium (80–100 μg/ml). Glucose was the dominant sugar in both EPS and IPS, and the polymers each consisted of three molecular weight fractions ranging from 2 × 10⁶ to 3 × 10³ Da. Both the EPS and IPS were able to significantly induce cytokine production (interleukin 12 and γ interferon) in murine splenocytes in vitro. Highest levels of interleukin 12 (291 pg/ml) and γ interferon (6,159 pg/ml) were obtained from samples containing Wr-74 IPS (0.06 μg/ml) and ATCC 20545 IPS (0.1 μg/ml), respectively. The results indicated that lower levels of EPS and IPS generally resulted in higher immune responses than did higher polymer concentrations.     

Bioactive metabolites from <Emphasis Type="Italic">Penicillium</Emphasis> sp., an endophytic fungus residing in <Emphasis Type="Italic">Hopea hainanensis</Emphasis>

The metabolites of endophytic fungus Penicillium sp. from the leaf of Hopea hainanensis were reported for the first time. By bioassay-guided fractionation, the EtOAc extract of a solid-matrix steady culture of this fungus afforded six compounds, which were identified through a combination of spectral and chemical methods (IR, MS, ¹H- and ¹³C-NMR) to be monomethylsulochrin (1), rhizoctonic acid (2), asperfumoid (3), physcion (4), 7,8-dimethyl-iso-alloxazine (5) and 3,5-dichloro-p-anisic acid (6). Compounds 2, 3 and 6 were obtained from Penicillium sp. for the first time. All of the six isolates were subjected to in vitro bioactive assays including antifungal action against three human pathogenic fungi Candida albicans, Trichophyton rubrum and Aspergillus niger and cytotoxic activity against the human nasopharyngeal epidermoid tumor KB cell line and human liver cancer HepG2 cell line. As a result, compounds 2–4 and 6 inhibited the growth of C. albicans with MICs of 40.0, 20.0, 50.0 and 15.0 μg/ml, respectively and the compound 6 showed growth inhibition against A. niger with MICs of 40.0 μg/ml. In addition, compounds 1–3 and 6 exhibited cytotoxic activity against KB cell line with IC₅₀ value of 30.0, 20.0, 20.0, 5.0 μg/ml, respectively and against HepG2 cell line with IC₅₀ value of 30.0, 25.0, 15.0, 10.0 μg/ml, respectively.