Evaluation of carcinogenic activities and sperm abnormalities of Gram-negative bacterial metabolites isolated from cancer patients after subcutaneous injection in albino rats.

Microbial pathogens drive tumorigenesis in 20% of cancer cases, so the present study is aimed to evaluate the carcinogenic activities, sperm abnormalities and other dangerous effects of the subcutaneous injection of extracts obtained from various clinical Gram-negative bacteria derived from cancer patients using albino rats. We isolated, identified and extracted of their secondary metabolites of carbapenem resistant Gram-negative bacteria derived from cancer patients. Various methods have been used to determine hepatotoxicity, nephrotoxicity, tumorigenesis, inflammatory and sperm abnormalities in the albino rats injected with extracts. In comparison with the normal animals group, all extracts induced hepatotoxicity which was evidenced by the significant elevation in the activity of the serum alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase and alkaline phosphatase; also, nephrotoxicity that was indicated through the marked increase in the serum urea and creatinine levels; tumorigenesis was achieved from the sharp elevation in serum levels of alpha fetoprotein, carcinoembryonic antigen and lactate dehydrogenase values as tumor markers; as well as severe inflammatory characteristics were monitored from the marked raise of tumor necrosis factor alpha and interleukin-1beta. Furthermore, the proportion of micronuclei in polychromatic erythrocytes and sperm abnormalities were statistically significant in all groups compared to control group. Various kinds of head abnormalities and coiled tail were noted. Histopathological examination of hepatic tissue came in line with the biochemical and cytological findings. It could conclude that the extracts of Serratia sp. Esraa 1, Stenotrophomonas sp. Esraa 2, Acinetobacter sp. Esraa 3, Escherichia sp. Esraa 4 and Pseudomonas sp. Esraa 5 were able to initiate cytotoxicity and tumorigenesis in rats.

Carcinogenic Activities and Sperm Abnormalities of Methicillin Resistance Staphylococcus aureus and Inhibition of Their Virulence Potentials by Ayamycin.

This investigation aimed to study the in vivo harmful effects of the subcutaneous injection of different methicillin resistance Staphylococcus aureus extracts (MRSA2, MRSA4, MRSA10, MRSA69, MRSA70, MRSA76, and MRSA78). Such strains represented the highest minimum inhibition concentration toward methicillin with various multidrug-resistant patterns. The obtained results revealed that rats injected with the MRSA4 extract died immediately after the last dose indicating the high cytotoxicity of MRSA4 strain (100% mortality). While the mortalities in other groups injected by the other MRSA extracts ranged from 50 to 75%. In comparison with the normal animal group, all MRSA extracts induced a hepatotoxic effect which was indicated from the significant (p < 0.01) increases in the activities of the serum alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) enzymes. Moreover, alkaline phosphatase (ALP) combined with a partial nephrotoxicity that was monitored from the significant elevation of serum urea concentration. While serum creatinine levels did not affect. Similarly, a significant elevation was recorded in serum levels of tumor biomarkers (alpha fetoprotein; AFP, carcinoembryonic antigen; CEA, and lactate dehydrogenase; LDH) reflecting their carcinogenic potential. On the other hand, the percentage of micronuclei (MN) in polychromatic erythrocytes from bone marrow cells was statistically significant in all groups as compared to the control group. The percentage of sperm abnormalities was statistically significant compared to the control. Different types of head abnormalities and coiled tail were recorded. Consequently, the current study focused on fighting MRSA virulence factors by the new compound ayamycin, which proved to be potent anti-virulence factor against all MRSA strains under study by significant decreasing of their streptokinase activities, hemolysin synthesis, biofilm formation, and their cell surface hydrophobicity.

Production and evaluation of antimycotic and antihepatitis C virus potential of fusant MERV6270 derived from mangrove endophytic fungi using novel substrates of agroindustrial wastes.

Among forty endophytic fungal isolates derived from the mangrove plant Avicennia marina, thirty-seven isolates (92.5 %) shown vary antimycotic activity against clinical Trichophyton, Microsporum, and Epidermophyton isolates. The hyperactive wild antagonistic strains Acremonium sp. MERV1 and Chaetomium sp. MERV7 were subjected to intergeneric protoplast fusion technique, and out of 20 fusants obtained, the fusant MERV6270 showed the highest antimycotic activity with the broadest spectrum against all dermatophytes under study. Solid-state fermentation (SSF) showed its superiority for antimycotic/antiviral metabolite production using cost-effective agroindustrial residues. Low-cost novel fermentation medium containing inexpensive substrate mixture of molokhia stalk, lemon peel, pomegranate peel, peanut peel (2:1:1:1) moistened with potato, and meat processing wastewaters (2:1, at moisture content of 60 %) provided a high antimycotic metabolite yield, 33.25 mg/gds, by the fusant MERV6270. The optimal parameters for antimycotic productivity under SSF were incubation period (4 days), incubation temperature (27.5-30 °C), initial pH (6), initial moisture level (60 %), substrate particle size (1.0 mm), and inoculum size (2 × 10(6) spores/gds), which elucidated antimycotic activity to 44.19 mg/gds. Interestingly, wild mangrove Acremonium sp. MERV1 and Chaetomium sp. MERV7 strains and their fusant MERV6270 showed significant inhibition of hepatitis C virus with viral knockdown percent of -82.48, -82.44, and -97.37 %, respectively, compared to the control (100 %), which open a new era in combat epidemic viral diseases.

Overproduction and biological activity of prodigiosin-like pigments from recombinant fusant of endophytic marine Streptomyces species.

Thirty-four endophytic marine Actinomycetes isolates were recovered from the Egyptian marine sponge Latrunculia corticata, out of them 5 isolates (14.7 %) showed red single colonies on yeast-CzAPEK plates. Isolates under the isolation code NRC50 and NRC51 were observed with the strongest red biomass. After application of protoplast fusion between NRC50 and NRC51 isolates, 26 fusants were selected and produced widely different amounts of prodigiosin-like pigments (PLPs) on different fermentation media. Among them fusant NRCF69 produced 79 and 160.4 % PLPs more than parental strains NRC50 and NRC51, respectively. According to the analysis of 16S rDNA sequence (amplified, sequenced, and submitted to GenBank under Accession no. JN232405 and JN232406, respectively), together with their morphological and biochemical characteristics, parental strains NRC50 (P1) and NRC51 (P2) were identified as Streptomyces sp. and designated as Streptomyces sp. NRC50 and Streptomyces sp. NRC51. This study describes a low cost, effective production media by using peanut seed broth, sunflower oil broth or dairy processing wastewater broth alone, or supplemented with 0.5 % mannitol that supports the production of PLPs by the Streptomyces fusant NRCF69 under study (42.03, 40.11, 36.7 and 47 g L(-1), respectively). PLPs compounds exhibited significant cytotoxic activities against three human cancer cell lines: colon cancer cell line (HCT-116), liver cancer cell line (HEPG-2) and breast cancer cell line (MCF-7) and antimycotic activity against clinical dermatophyte isolates of Trichophyton, Microsporum and Epidermophyton.

Molecular identification using ITS sequences and genome shuffling to improve 2-deoxyglucose tolerance and xylanase activity of marine-derived fungus, Aspergillus sp. NRCF5.

During the screening of xylanolytic enzyme from marine-derived fungi isolated from the inner tissue of Egyptian soft coral Rhytisma sp., one strain, NRCF5, exhibited high enzyme activity with 0.1 % (w/v) antimetabolite 2-deoxyglucose (2DG) tolerance. This fungal strain was identified as Aspergillus sp. NRCF5 based on its morphological characteristics and internal transcribed spacer (ITS) sequences. The ITS region of hyperactive xylanolytic strain (NRCF5) was amplified, sequenced, and submitted to GenBank (accession no. JQ277356). To apply the fundamental principles of genome shuffling in breeding of xylanase-producing fungi, marine-derived fungus Aspergillus sp. NRCF5 was used as starting strain in this work and applied for induction of genetic variability using different combinations and doses of mutagens. Five mutants with high xylanase activity and 0.25 % (w/v) antimetabolite 2DG tolerance were obtained from the populations generated by the mutation of combination between ultraviolet irradiation (UV, 5 min) and N-methyl-N-nitro-N-nitrosoguanidine (NTG, 100 µg/ml) for 30 (UNA) and 60 (UNB)min as well as NTG (100 µg/ml) and ethidium bromide (250 µg/ml) for 30 (NEA) and 60 (NEB)min. Then, they were subjected for recursive protoplast fusion. Seven hereditarily stable recombinants with high xylanase activity and 1.0 % (w/v) 2DG tolerance were obtained by four rounds of genome shuffling. Among them, a high xylanase-producing recombinant, R4/31, was obtained, which produced 427.5 U/ml xylanase. This value is 6.13-fold higher than that of the starting strain NRCF5 and 2.48-fold higher than that of the parent strain (mutant NEA51). The subculture experiments indicated that the high producer of marine Aspergillus sp. R4/31 fusant was stable.

Cellulase production from agricultural residues by recombinant fusant strain of a fungal endophyte of the marine sponge Latrunculia corticata for production of ethanol.

Several fungal endophytes of the Egyptian marine sponge Latrunculia corticata were isolated, including strains Trichoderma sp. Merv6, Penicillium sp. Merv2 and Aspergillus sp. Merv70. These fungi exhibited high cellulase activity using different lignocellulosic substrates in solid state fermentations (SSF). By applying mutagenesis and intergeneric protoplast fusion, we have obtained a recombinant strain (Tahrir-25) that overproduced cellulases (exo-ß-1,4-glucanase, endo-ß-1,4-glucanase and ß-1,4-glucosidase) that facilitated complete cellulolysis of agricultural residues. The process parameters for cellulase production by strain Tahrir-25 were optimized in SSF. The highest cellulase recovery from fermentation slurries was achieved with 0.2% Tween 80 as leaching agent. Enzyme production was optimized under the following conditions: initial moisture content of 60% (v/w), inoculum size of 10(6) spores ml(-1), average substrate particle size of 1.0 mm, mixture of sugarcane bagasse and corncob (2:1) as the carbon source supplemented with carboxymethyl cellulose (CMC) and corn steep solids, fermentation time of 7 days, medium pH of 5.5 at 30°C. These optimized conditions yielded 450, 191, and 225 units/gram dry substrate (U gds(-1)) of carboxylmethyl cellulase, filter-paperase (FPase), and ß-glucosidase, respectively. Subsequent fermentation by the yeast, Saccharomyces cerevisiae NRC2, using lignocellulose hydrolysates obtained from the optimized cellulase process produced the highest amount of ethanol (58 g l(-1)). This study has revealed the potential of exploiting marine fungi for cost-effective production of cellulases for second generation bioethanol processes.

Genome shuffling of marine derived bacterium Nocardia sp. ALAA 2000 for improved ayamycin production.

Genome shuffling is a recent development in microbiology. The advantage of this technique is that genetic changes can be made in a microorganism without knowing its genetic background. Genome shuffling was applied to the marine derived bacterium Nocardia sp. ALAA 2000 to achieve rapid improvement of ayamycin production. The initial mutant population was generated by treatment with ethyl methane sulfonate (EMS) combined with UV irradiation of the spores, resulting in an improved population (AL/11, AL/136, AL/213 and AL/277) producing tenfold (150 µg/ml) more ayamycin than the original strain. These mutants were used as the starting strains for three rounds of genome shuffling and after each round improved strains were screened and selected based on their ayamycin productivity. The population after three rounds of genome shuffling exhibited an improved ayamycin yield. Strain F3/22 yielded 285 µg/ml of ayamycin, which was 19-fold higher than that of the initial strain and 1.9-fold higher than the mutants used as the starting point for genome shuffling. We evaluated the genetic effect of UV + EMS-mutagenesis and three rounds of genome shuffling on the nucleotide sequence by random amplified polymorphic DNA (RAPD) analysis. Many differences were noticed in mutant and recombinant strains compared to the wild type strain. These differences in RAPD profiles confirmed the presence of genetic variations in the Nocardia genome after mutagenesis and genome shuffling.

Keratinolytic activity from new recombinant fusant AYA2000, derived from endophytic Micromonospora strains.

Two different endophytic strains, ESRAA1997 and ALAA2000, were isolated from the Egyptian herbal plant Anastatica hierochuntica. The 2 strains produced alkaline serine protease and were identified based on their phenotypic and chemotypic characteristics as different strains of Micromonospora spp. Both strains grew and produced keratinase, using different keratinous waste substances as the sole source of carbon and nitrogen. In our study, the activity and properties of keratinase enzymes of the wild strains ESRAA1997 and ALAA2000 were altered by genetic recombination through protoplast fusion between them, leading to a potent keratinolytic fusant Micromonospora strain AYA2000 with improved properties (activity, stability, specificity, and tolerance to inhibitors). Using a mixture of yeast extract, peptone, and malt extract as a supplement to the bovine hair medium increased keratinase production by 48%, and addition of 1% glucose suppressed enzyme production by Micromonospora strain AYA2000. The enzyme was purified by ammonium sulphate precipitation and DEAE-cellulose chromatography followed by gel filtration. The molecular weight, estimated using SDS-PAGE, was 39 kDa. The enzyme exhibited remarkable activity towards all keratinous wastes used and could also adapt to a broad range of pH and temperatures, with optima at pH 11 and 60 °C. The enzyme was not influenced by chelating reagents, metal ions, or alcohols. These properties make AYA2000 keratinase an ideal candidate for biotechnological application.

Production and genetic improvement of a novel antimycotic agent, saadamycin, against dermatophytes and other clinical fungi from endophytic Streptomyces sp. Hedaya48.

As a part of our ongoing efforts towards finding novel antimycotic agents from marine microflora of the Red Sea, vanillin, 5,7-dimethoxy-4-p-methoxylphenylcoumarin and the new antimycotic compound saadamycin were isolated from endophytic Streptomyces sp. Hedaya48. The producing strain was isolated from the Egyptian sponge Aplysina fistularis and subjected to different UV irradiation doses. A mutant strain Ah22 with 10.5-fold (420 mg/l as compared to 40 mg/l produced by the parental strain) improved saadamycin production was isolated. Production of saadamycin from mutant Ah22 was enhanced to 2.26-fold (950 mg/l) and 2.38-fold (1000 mg/l) under optimized culture conditions in batch culture and bioreactors, respectively. Both saadamycin and 5,7-dimethoxy-4-p-methoxylphenylcoumarin exhibited significant antimycotic activity against dermatophytes and other clinical fungi.

Essramycin: a first triazolopyrimidine antibiotic isolated from nature.

In the course of our screening program for new bio-active compounds, a novel triazolopyrimidine antibiotic, essramycin (1), was obtained from the culture broth of the marine Streptomyces sp., isolate Merv8102. Structure 1 was established by intensive NMR studies and by mass spectra. The compound is antibacterially active with MIC of 2 to 8 microg/ml against Gram-positive and Gram-negative bacteria, while it showed no antifungal activity. The fermentation and isolation, as well as the structure elucidation and biological activity of 1 are described.

Gene transfer between different Trichoderma species and Aspergillus niger through intergeneric protoplast fusion to convert ground rice straw to citric acid and cellulases.

Single-stage direct bioconversion of cellulosic materials to citric acid using intergeneric hybrids obtained from three different Trichoderma species and Aspergillus niger was carried out. The recent results were obtained on the basis of either resistance or sensitivity to one or more of five metal ions, two catabolite repressors, and five antifungal agents, which were used in this study at different concentrations. Sixty-six fusants were isolated after using the three intergeneric protoplast fusion experiments, belonging to two types of intergeneric fusants. Fusants of the first type are heterokaryons (35 fusants). On the other hand, those of the second type are haploids (31 fusants), i.e., they were stable. The present study can be successfully applied in the construction of 14 new genetic fusants, which produced at least 100% more citric acid than the citric acid producer strain A. niger. Out of the fusants, three (1/18, 2/13 and 2/15) showed about a threefold increase of citric acid production in comparison with the parent A. niger strain. Furthermore, studies on DNA content showed that this finding may be submitted on the evidence that citric acid and cellulases production was not correlated with DNA content; however, the productivity depends on specific DNA content.