Synthesis of secondary metabolites by Cladosporium resinae (NRL-6437) under different growth media and chemical inducers and their pharmaceutical activity.

The role of different growth media and chemical enhancer on synthesis of secondary metabolites Cladosporium resinae (NRL-6437) was investigated for their in vitro biological activities. Cladosporium resinae (NRL-6437) were grown in various nutrient media (Czapeak-dox Broth (CB), Czapeak Yeast-extract Broth (CYB), Yeast Extract Sucrose (YES), Potato Dextrose Broth (PDB) and Czapeak-dox (supplemented with glucose and starch) Broth (CGSB) for the production of metabolites. Two chemical epigenetic modifiers (suberoyl-anilide hydroxamic acid (SAHA) and 5-azacytidine (5-AZA) were also used for the expression of silent genes for secondary metabolite production. Our results indicated that among different media, Czapeak yeast extract broth produced more secondary metabolites. Application of 15mM of both modifiers was effective for the expressions of silent genes resulting in an increased metabolites production. Secondary metabolites extracted in ethyl acetate and fractionized in n-Hexane were also tested for their biological activity. The secondary metabolites revealed varying degrees of growth inhibitions of the tested organisms. Similarly, these metabolites were also active against brine shrimps and Lemna.

Characterization of pathogens involved in ventilator associated pneumonia in surgical and medical intensive care units - A single center experience.

In the present study 60 samples were collected from lower respiratory tract of patients suffering from Ventilator Associated Pneumonia (VAP) admitted in surgical and medical Intensive Care Units (ICUs) of Khyber Teaching Hospital, Peshawar, Pakistan. Recovered pathogens were characterized and their susceptibility pattern against commonly used antibacterial agents investigated. Most frequent bacterial pathogen found was methicillin- resistant Staphylococcus aureus (MRSA) (40%) followed by members of Enterobacteriaceae (22%; of which Escherichia coli (50%), Klebsiella pneumonia (30%), Enterobacter cloacae (10%) and Citrobacter freundii (10%), Pseudomonas aeruginosa 20% and Acinetobacter baumannii 18%. Majority of the specimens yielded polymicrobial growth (85.75% polymicrobial growth compared to 14.25% specimens yielding monomicrobial growth). The susceptibility pattern showed that A. baumannii was the most resistant bacterial pathogen. Based on the results of susceptibility pattern obtained in the present study, combination of linezolid with meropenem and colistin has been found to be the best combination option for empirical therapy for VAP pathogens in this region.

In vitro activity of antimicrobial agents against streptococcus pyogenes isolated from different regions of Khyber Pakhtun Khwa Pakistan.

The present study investigates the antibiotic resistance of S. pyogenes of 600 isolates collected from different body parts including throat and sputum were analyzed for their antimicrobial susceptibility to 5 antibiotics using the Kirby Bauer disc diffusion method. Based on different identification tests including, gram staining, beta hemolysis, catalase test and bacitracin sensitivity test, a total of 138 isolates were confirmed as S. pyogenes. The prevalence of S. pyogenes was 80% in sore throat and 29% in sputum samples. These isolates were further tested for antibiotics resistance using disk diffusion method. Out of 138 isolates, 49.27% isolates showed resistance towards cefixime, 28.98% towards cefotaxime and 17.39% towards ciprofloxacin, 17.39% towards ampicillin, 17.39% towards erythromycin, 15.94% towards streptomycin, 0.724% isolates towards chloromphenicol and 0% towards penicillin. Among the resistant isolates of S. pyogenes, 60.71% showed resistance towards cefixime, 57.14% towards ciprofloxacin, 57.14% towards streptomycin, 50% towards erythromycin and 25% towards cefotaxime.

Fungi as chemical industries and genetic engineering for the production of biologically active secondary metabolites

Fungi is somewhere in between the micro and macro organisms which is a good source of producing biologically active secondary metabolites. Fungi have been used as tool for producing different types of secondary metabolites by providing different nutrients at different laboratory conditions. The fungi have been engineered for the desired secondary metabolites by using different laboratory techniques, for example, homologous and heterologous expressions. This review reported how the fungi are used as chemical industry for the production of secondary metabolites and how they are engineered in laboratory for the production of desirable metabolites;also the biosynthetic pathways of the bio-organic-molecules were reported.

Cloning and characterization of novel methylsalicylic acid synthase gene involved in the biosynthesis of isoasperlactone and asperlactone in Aspergillus westerdijkiae.

Aspergillus westerdijkiae is the main producer of several biologically active polyketide metabolites including isoasperlactone and asperlactone. A 5298bp polyketide synthase gene "aomsas" has been cloned in Aspergillus westerdijkiae by using gene walking approach and RACE-PCR. The predicted amino acid sequence of aomsas shows an identity of 40-56% with different methylsalicylic acid synthase genes found in Byssochlamys nivea, P. patulum, A. terreus and Streptomyces viridochromogenes. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aomsas expression was found to be associated with the biosynthesis of isoasperlactone and asperlactone. Moreover an aomsas knockout mutant "aoDeltamsas" of A. westerdijkiae, not only lost the capacity to produce isoasperlactone and asperlactone, but also 6-methylsalicylic acid. The genetically complemented mutant ao+msas restored the biosynthesis of all the missing metabolites. Chemical complementation through the addition of 6-methylsalicylic acid, aspyrone and diepoxide to growing culture of aoDeltamsas mutant revealed that these compounds play intermediate roles in the biosynthesis of asperlactone and isoasperlactone.

Aspergillus westerdijkiae polyketide synthase gene "aoks1" is involved in the biosynthesis of ochratoxin A.

Ochratoxin A (OTA) is a potential nephrotoxic, teratogenic, immunogenic, hepatotoxic and carcinogenic mycotoxin, produced by Aspergillus westerdijkiae NRRL 3174. Herein we describe the characterization of a putative OTA-polyketide synthase gene "aoks1", cloned by using gene walking approach. The predicted amino acid sequence of the 2kb clone display 34-60% similarities to different polyketide synthase genes including lovastatine biosynthesis gene "lovb" in A. terreus, compactin biosynthesis gene "mlcA" in Penicillium citrinum and OTA biosynthesis gene "otapksPN" in P. nordicum. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aoks1 expression was found to be associated with OTA biosynthesis. Further a mutant, in which the aoks1 gene was inactivated by Escherichia coli hygromycin B phosphotransferase gene, lost the capacity to produce OTA, but still producing mellein. To our knowledge this report describes for the first time characterization of a gene involved in OTA biosynthesis, with the information about mellein which was proposed in the literature to be an intermediate OTA. This study also suggests that aoks1 may be the second polyketide synthase gene required for OTA biosynthesis in A. westerdijkiae NRRL 3174.

Regulation and role of a STE12-like transcription factor from the plant pathogen Colletotrichum lindemuthianum.

In phytopathogenic fungi, STE12-like genes encode transcription factors essential for appressorium-mediated host penetration. However, their regulation and downstream targets are still unknown. In the present study, a STE12-like gene (CLSTE12) from Colletotrichum lindemuthianum was isolated. We identified a spliced variant whose expression was negatively regulated during early stages of pathogenesis, whereas the correctly spliced mRNA remained expressed up to the penetration step, suggesting distinct roles for these two transcripts. Indeed, the full-length sequence was able to complement a yeast STE12 mutant, whereas overexpression of the transcript variant had a dominant-negative effect on yeast invasive growth and C. lindemuthianum pathogenicity. To further investigate the downstream genes that could be regulated by CLSTE12, disruption mutants were generated. Phenotypic analyses of the mutants revealed reduced pectinase activity and conidial adhesion to polystyrene. Analysis of cell surface proteins allowed the identification of a major protein, Clsp1p, which was absent from the mutants. Clsp1p belongs to a new family of wall-associated proteins only found in euascomycetous fungi. Overall, these results suggest that the activity of CLSTE12 can be modulated by a regulated alternative splicing mechanism and that this factor is involved in the production of cell surface proteins and host cell wall degrading enzymes.

Soil solarization: a safe, affective and practicable technique for the control of soil born fungi and nematodes.

A technique i.e., Soil Solarization and Amendments (neem, chicken farmyard manure, farmyard manure and biokhad viz synthetic bio fertilizer), towards the natural cropping system has been evaluated for its effectiveness and practicability at the National Agricultural Research Center Islamabad Pakistan. Soil solarization and amendments were analyzed as a control measure against soil born fungi and nematodes. Eight weeks of solarization resulted in about 11 degrees C increase in the soil temperature. This increase in soil temperature caused a reduction of about 70 to 80% in the fungal population and about 99% in nematode population at various depths. Neem and Biokhad amendments were proved synergistic for solarization and also improved the properties of soil in the benefit of crop plants. Fusarium sp., Macrophomina phyaseolina and Verticillium sp. of fungi and Tylenchus sp., Haplolaimus sp., Xiphenema sp. and almost all of the parasitic nematodes were significantly (p<0.01) controlled. It was found that even after 40 days the solarized plots contain significantly less number of fungi and nematodes as compared to the nonsolarized plots, which confirmed the durability of this process.