High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections

The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of approximate to 20,000 microbial extracts, 12 hits were identified with broadspectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.

Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 +/- 0.07 mol H-2/mol glucose (mean +/- S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 +/- 0.03 mol H-2/mol glucose, 0.17 +/- 0.01 mol H-2/mol glucose, 0.11 +/- 0.01 mol H-2/mol glucose and 0.20 +/- 0.04 mol H-2/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp., However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

Comparative growth and viability of hybrids between two populations of Chinese shrimp (Fennropenaeus chinensis)

Fenneropenaeus chinensis is confined to the Yellow Sea and Bohai Sea in China and the West Coast of the Korean Peninsula. Intra- and intercross populations were produced between Rushany (YP) and Korean (KN) populations. Seven traits were recorded. The heterosis of hybrids was computed and comparison between treatments was performed by ANOVA. At the fourth month after post-larvae, six indexes of growth trait and viability showed a range of heterosis, ranging from 0.514% to 14.950%. At the fifth month after post-larvae, six indexes of growth trait and viability ranged from -9.000% to 19.090%. The negative heterosis was observed in CL, HST and viability. The heterosis of KN female xYP male tended generally to increase as the age of the Chinese shrimp increased while the heterosis of YP female xKN male tended to decrease. The results indicated that the viability of reciprocal hybrids were not significantly different (P > 0.05) from their parents during the experiment. The result of ANOVA indicated that the F1 hybrids were significantly different (P < 0.05) in WST and TW at the fourth month. The multiple comparisons of LSD test indicated that the hybrids of YP female xKN male were significantly different (P < 0.05) from their parents in TW. The hybrids of YP female xKN male were significantly different (P < 0.05) from the other three combinations in WST. At the fifth months, the F1 hybrids had significant difference (P < 0.05) in body weight while other traits showed no significant differences (P > 0.05) from their parents. The multiple comparisons of LSD test indicated that the hybrids of KN female xYP male were significantly different (P < 0.05) from the KN parents in TW. The results indicate that in experimental conditions, the F-1 hybrids created from two populations of Chinese shrimp showed a certain level of heterosis for growth performance and viability. The crossing scheme may improve growth performance and viability in Chinese shrimp, but the improvement may be limited because effective crossbreeding requires the maintenance of pure, preferably inbred, lines and possibly involves specialized sire and dam lines. Therefore, the exploitation of heterosis through single crossbreeding in Chinese shrimp is of limited utility in practical commercial shrimp aquaculture in spite of the potential of significant heterosis. The crossbreeding of different populations can be applied in the establishment of base populations.

Microbial modulation in the biomass and toxin production of a red-tide causing alga

The effect of S-10, a strain of marine bacteria isolated from sediment in the Western Xiamen Sea, on the growth and paralytic shellfish poison (PSP) production in the alga Alexandrium tamarense (A. tamarense) was studied under controlled experimental conditions. The results of these experiments have shown that the growth of A. tamarense is obviously inhibited by S-10 at high concentrations, however no evident effect on its growth was observed at low concentrations. Its PSP production was also inhibited by S 10 at different concentrations, especially at low concentrations. The toxicity of this strain of A. tamarense is about (0.9512.14) x 10(-6) MU/cell, a peak toxicity value of 12.14 x 10(-6) MU/cell appeared on the 14th day, after which levels decreased gradually. The alga grew well in conditions of pH 6-8 and salinities of 20-34 parts per thousand. The toxicity of the alga varied markedly at different pH and salinity levels. Toxicity decreased as pH increased, while it increased with salinity and reached a peak value at a salinity of 30 parts per thousand, after which it declined gradually. S-10 at a concentration of 1.02 x 10(9) cells/ml inhibited growth and the PSP production of A. tamarense at different pH and salinity levels. S-10 had the strongest inhibitory function on the growth of A. tamarense under conditions of pH 7 and a salinity of 34 parts per thousand. The best inhibitory effect on PSP production by A. tamarense was at pH 7, this inhibitory effect on PSP production did not relate to salinity. Interactions between marine bacteria and A. tamarense were also investigated using the flow cytometer technique (FCM) as well as direct microscope counting. S-10 was identitied as being a member of the genus Bacillus, the difference in 16S rDNA between S-10 and Bacillus halmapalus was only 2%. The mechanism involved in the inhibition of growth and PSP production of A. tamarense by this strain of marine bacteria, and the prospect of using it and other marine bacteria in the biocontrol of red-tides was discussed. (c) 2005 Elsevier Ltd. All rights reserved.

Intra-habitat heterogeneity of microbial food web structure under the regime of eutrophication and sediment resuspension in the large subtropical shallow Lake Taihu, China

Planktonic microbial community structure and classical food web were investigated in the large shallow eutrophic Lake Taihu (2338 km(2), mean depth 1.9 m) located in subtropical Southeast China. The water column of the lake was sampled biweekly at two sites located 22 km apart over a period of twelve month. Site 1 is under the regime of heavy eutrophication while Site 2 is governed by wind-driven sediment resuspension. Within-lake comparison indicates that phosphorus enrichment resulted in increased abundance of microbial components. However, the coupling between total phosphorus and abundance of microbial components was different between the two sites. Much stronger coupling was observed at Site 1 than at Site 2. The weak coupling at Site 2 was mainly caused by strong sediment resuspension, which limited growth of phytoplankton and, consequently, growth of bacterioplankton and other microbial components. High percentages of attached bacteria, which were strongly correlated with the biomass of phytoplankton, especially Microcystis spp., were found at Site 1 during summer and early autumn, but no such correlation was observed at Site 2. This potentially leads to differences in carbon flow through microbial food web at different locations. Overall, significant heterogeneity of microbial food web structure between the two sites was observed. Site-specific differences in nutrient enrichment (i.e. nitrogen and phosphorus) and sediment resuspension were identified as driving forces of the observed intra-habitat differences in food web structure.

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus)

In this study, the intestinal microbiota of kuruma shrimp (Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.

Genetic improvement on Chinese shrimp (Fenneropenaeus chinensis): growth and viability performance in F1 hybrids of different populations

Fenneropenaeus chinensis distributed in the Yellow Sea and Bohai Sea of China and the west coast of the Korean Peninsula. Different geographical populations represent potentially different genetic resources. To learn further the characteristics of different geographical population, crosses among two wild and three farmed populations were produced. The two wild populations were from the Yellow Sea and Bohai Sea (WYP), and the west coast of the Korean Peninsula and coast (WKN). The three farmed populations included the offspring of first generation of wild shrimp from coast in Korea (FKN), the Huang Hai (the Yellow Sea in Chinese) No.1 (HH1), and JK98. The phenotypes growth and survival rates of these populations were compared to confirm the feasibility for crossbreeding. The body length (BL), carapace length (CL), carapace width (CW), height of the second and third abdominal segment (HST), width of the second and third abdominal segment (WST), length of the first abdominal segment (LF), length of the last abdominal segment (LL), live body weight (BW), and survival rate were measured. Different combinations were statistically performed with ANOVA and Duncan's Multiple Range Test. The results show that the survival rate of JK98(a (TM) Euro)xWKN(a (TM),) was the highest, followed by WYP(a (TM) Euro)xWKN(a (TM),), FKN(a (TM) Euro)xWYP(a (TM),), FKN(a (TM) Euro)xHH1(a (TM),) and WYP(a (TM) Euro)xFKN(a (TM),); the body weight of FKN(a (TM) Euro)sxHH1(a (TM),) was the highest, followed by FKN(a (TM) Euro)xWYP(a (TM),), WYP(a (TM) Euro)xWKN(a (TM),), WYP(a (TM) Euro)xFKN(a (TM),) and JK98(a (TM) Euro)xWKN(a (TM),); the total length had the same ranking as the body weight. All growth traits in hybrids JK98(a (TM) Euro)xWKN(a (TM),) were the lowest among all combinations. F1 hybrids had significant difference (P < 0.05) in BL, CL, HST, LL, and BW; and insignificant difference (P > 0.05) in other growth traits and survival rate. The results of Duncan's Multiple Range Test are that BL and CL of JK98(a (TM) Euro)xWKN(a (TM),) were significantly different from the other combinations; HST different from the combination of FKN(a (TM) Euro)xWYP(a (TM),), FKN(a (TM) Euro)xHH1(a (TM),) and WYP(a (TM) Euro)xWKN(a (TM),); and BW different from FKN(a (TM) Euro)xWYP(a (TM),) and FKN(a (TM) Euro)xHH1(a (TM),). As a whole, the results indicate that the FKN(a (TM) Euro)xHH1(a (TM),) was the best combination in all growth traits. Therefore, hybridization can introduce the variation to base populations. The systematic selection program based on additive genetic performance may be more effective than crossbreeding.

Monitoring microbial populations of sulfate-reducing bacteria using an impedimetric immunosensor based on agglutination assay

An impedimetric immunosensor was fabricated for rapid and non-labeled detection of sulfate-reducing bacteria, Desulforibrio caledoiensis (SRB) by immobilizing lectin-Concanavalin A using an agglutination assay. The immobilization of lectin was conducted using amine coupling on the surface of a gold (Au) electrode assembled with 11-Mercaptounclecanoic acid. Electrochemical impedance spectroscopy (EIS) was used to verify the stepwise assembly of the sensor system. The work conditions of the impedimetric immunosensor, such as pH of the buffer solutions and the incubation time of lectin, were optimized. Faradic impedance spectra for charge transfer for the redox probe Fe(CN)(6)(3-/4-) were measured to determine SRB concentrations. The diameter of the Nyquist diagram that is equal to the charge-transfer resistance (RI) increased with increasing SRB concentration. A linear relationship between R-ct and SRB concentration was obtained in SRB concentration range of 1.8 to 1.8 x 10(7) cfu/ml. The variation of the SRB population during the growth process was also monitored using the impedimetric immunosensor. This approach has great potential for simple, low-cost. and time-saving monitoring of microbial populations. (C) 2009 Elsevier B.V. All rights reserved.