Search results for: Recombinant E.coli

Authors: Rosarin Rujananon, Poonsuk Prasertsan, Amornrat Phongdara, Tanate Panrat, Jibin Sun, Sugima Rappert, An-Ping Zeng

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In this study, a synthetic pathway was created by assembling genes from Clostridium butyricum and Escherichia coli in different combinations. Among the genes were dhaB1 and dhaB2 from C. butyricum VPI1718 coding for glycerol dehydratase (GDHt) and its activator (GDHtAc), respectively, involved in the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA). The yqhD gene from E.coli BL21 was also included which codes for an NADPHdependent 1,3-propanediol oxidoreductase isoenzyme (PDORI) reducing 3-HPA to 1,3-propanediol (1,3-PD). Molecular modeling analysis indicated that the conformation of fusion protein of YQHD and DHAB1 was favorable for direct molecular channeling of the intermediate 3-HPA. According to the simulation results, the yqhD and dhaB1 gene were assembled in the upstream of dhaB2 to express a fusion protein, yielding the recombinant strain E. coliBL21 (DE3)//pET22b+::yqhD-dhaB1_dhaB2 (strain BP41Y3). Strain BP41Y3 gave 10-fold higher 1,3-PD concentration than E. coliBL21 (DE3)//pET22b+::yqhD-dhaB1_dhaB2 (strain BP31Y2) expressing the recombinant enzymes simultaneously but in a non-fusion mode. This is the first report using a gene fusion approach to enhance the biological conversion of glycerol to the value added compound 1,3- PD.

Keywords: Recombinant E.coli, 1, 3-propanediol, glycerol, fusion protein.

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Authors: S. Banerjee, A. Apte Deshpande, N. Mandi, S. Padmanabhan

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We report a novel fusion tag for expressing recombinant proteins in E. coli. The fusion tag is the C-terminus part of the human GMCSF gene comprising 45 amino acids, which aid in over expression of otherwise non expressible genes. Expression of hIFN a2b with this fusion tag also escapes the requirement of rare codons for expression. This is also a first report of a small fusion tag of human origin having affinity to heparin sepharose column facilitating the purification of fusion protein.

Keywords: fusion tag, bacterial expression, rare codons, human GMCSF

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Authors: S. Liepina, A. Jemeljanovs

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Tasks of the work were study the possible E.coli contamination in red deer meat, identify pathogenic strains from isolated E.coli, determine their incidence in red deer meat and determine the presence of VT1, VT2 and eaeA genes for the pathogenic E.coli. 8 (10%) samples were randomly selected from 80 analysed isolates of E.coli and PCR reaction was performed on them. PCR was done both on initial materials – samples of red deer meat - and for already isolated liqueurs. Two of analysed venison samples contain verotoxin-producing strains of E. coli. It means that this meat is not safe to consumer. It was proven by the sequestration reaction of E. coli and by comparison of the obtained results with the database of microorganism genome available on the internet that the isolated culture corresponds to region 16S rDNS of E. coli thus presenting correctness of the microbiological methods.

Keywords: Deer meat, pathogenic Escherichia coli

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Authors: R. Majidzadeh Heravi, M. Sankian, H. Kermanshahi, M. R. Nassiri, A. Heravi Moussavi, S. A. Lari, A. R. Varasteh

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The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.

Keywords: Lactobacillus salivarus, Lactococcus lactis, recombinant, phytase, poultry.

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Authors: Marine A. Balayan, Susanna S. Mirzabekyan, Marine Isajanyan, Zaven S. Pepoyan, Аrmen H. Trchounian, Аstghik Z. Pepoyan, Helena Bujdakova

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It has been shown that pH 7,3 and 37 0C are the optimal condition for the growth of E. coli “ASAP". The cells grow well on Glucose, Lactose, D-Mannitol, D-Sorbitol, (+)-Xylose, L- (+)-Arabinose and Dulcitol. No growth has been observed on Sucrose, Inositol, Phenylalanine, and Tryptophan. The strain is sensitive to a range of antibiotics. The present study has demonstrated that E. coli “ASAP" inhibit the growth of S. enterica ATCC #700931 in vitro. The studies on conjugating activity has revealed no conjugant of E. coli “ASAP" with plasmid strains E. coli G35#59 and S. enterica ATCC #700931. On the other hand, the conjugants with low frequencies were obtained from E. coli “ASAP" with E. coli G35#61, and E. coli “ASAP" with randomly chosen isolate from healthy human gut microflora: E. coli E6. The results of present study have demonstrated improvements in gut microflora condition of patients with different diseases after the administration of “ASAP"

Keywords: E. coli, ASAP, Probiotic formula, gut microflora.

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Authors: Le Thuy Mai, Vu Nguyen Thanh

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β-Glucosidase is an important enzyme for production of ethanol from lignocellulose. With hydrolytic activity on cellooligosaccharides, especially cellobiose, β-glucosidase removes product inhibitory effect on cellulases and forms fermentable sugars. In this study, β-glucosidase encoding gene (BGL1) from traditional starter yeast Saccharomycosis fibuligera BMQ908 was cloned and expressed in Pichia pastoris. BGL1 of S. fibuligera BMQ 908 shared 98% nucleotide homology with the closest GenBank sequence (M22475) but identity in amino-acid sequences of catalytic domains. Recombinant plasmid pPICZαA/BGL1 containing the sequence encoding BGL1 mature protein and α-factor secretion signal was constructed and transformed into methylotrophic yeast P. pastoris by electroporation. The recombinant strain produced single extracellular protein with molecular weight of 120 kDa and cellobiase activity of 60 IU/ml. The optimum pH of the recombinant β-glucosidase was 5.0 and the optimum temperature was 50°C.

Keywords: β-Glucosidase, Pichia pastoris, Saccharomycopsisfibuligera, recombinant enzyme.

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Authors: Boci J., Çabeli P., Shtylla T., Kumbe I.

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The development of the poultry industry in Albania is mainly based on the existence of intensive modern farms with huge capacities, which often are mixed with other forms. Colibacillosis is commonly displayed regardless of the type of breeding, delivering high mortality in poultry industry. The mechanisms with which pathogen enterobacters are able to cause the infection in poultry are not yet clear. The routine diagnose in the field, followed by isolation of E. coli and species of Salmonella genres in reference laboratories cannot lead in classification or full recognition of circulative strains in a territory, if it is not performed a differentiation among the present microorganisms in intensive farms and those in rural areas. In this study were isolated 1.496 strains of E. coli and 378 Salmonella spp. This study, presents distribution of poultry pathogenosity of E.coli and Salmonella spp., based on the usage of innovative diagnostic methods.

Keywords: poultry, E.coli, Salmonella spp., Enterobacter

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Authors: Sang-Ho Baik

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D-erythro-cyclohexylserine (D chiral unnatural β-hydroxy amino acid expected for the synthesis of drug for AIDS treatment. To develop a continuous bioconversion system with whole cell biocatalyst of D-threonine aldolase (D genes for the D-erythro-CHS production, D-threonine aldolase gene was amplified from Ensifer arboris 100383 by direct PCR amplication using two degenerated oligonucleotide primers designed based on genomic sequence of Shinorhizobium meliloti Sequence analysis of the cloned DNA fragment revealed one open-reading frame of 1059 bp and 386 amino acids. This putative D-TA gene was cloned into NdeI and EcoRI (pEnsi His-tag sequence or BamHI (pEnsi-DTA[2]) sequence of the pET21(a) vector. The expression level of the cloned gene was extremely overexpressed by E. coli BL21(DE3) transformed with pEnsi-DTA[1] compared to E. coli BL21(DE3) transformed with pEnsi-DTA[2]. When the cells expressing the wild used for D-TA enzyme activity, 12 mM glycine was successfully detected in HPLC analysis. Moreover, the whole cells harbouring the recombinant D-TA was able to synthesize D-erythro of 0.6 mg/ml in a batch reaction.

Keywords: About four key words or phrases in alphabetical order, separated by commas.

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Authors: Tzyy Rong Jinn, Nguyen Tiep Khac, Tzong Yuan Wu

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Highly pathogenic avian influenza (HPAI) H5N1 viruses have created demand for a cost-effective vaccine to prevent a pandemic of the disease. Here, we report that Trichoplusia ni (T. ni) larvae can act as a cost-effective bioreactor to produce recombinant HA5 (rH5HA) proteins as an potential effective vaccine for chickens. To facilitate the recombinant virus identification, virus titer determination and access the infected larvae, we employed the internal ribosome entry site (IRES) derived from Perina nuda virus (PnV, belongs to insect picorna like Iflavirus genus) to construct a bi-cistronic baculovirus expression vector that can express the rH5HA protein and enhanced green fluorescent protein (EGFP) simultaneously. Western blot analysis revealed that the 70 kDa rH5HA protein and partially cleaved products (40 kDa H5HA1) were generated in T. ni larvae infected with recombinant baculovirus carrying the H5HA gene. These data suggest that the baculovirus-larvae recombinant protein expression system could be a cost-effective platform for H5N1 vaccine production.

Keywords: Avian Influenza, baculovirus, hemagglutinin, Trichoplusia ni larvae

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Authors: C.-C. Lin, S.-C. Kan, C.-W. Yeh, C.-I Chen, C.-J. Shieh, Y.-C. Liu

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In this study, lipid-deprived residuals of microalgae were hydrolyzed for the production of reducing sugars by using the recombinant Bacillus cellulosome, carrying eight genes from the Clostridium thermocellum ATCC27405. The obtained cellulosome was found to exist mostly in the broth supernatant with a cellulosome activity of 2.4 U/mL. Furthermore, the Michaelis-Menten constant (Km) and Vmax of cellulosome were found to be 14.832 g/L and 3.522 U/mL. The activation energy of the cellulosome to hydrolyze microalgae LDRs was calculated as 32.804 kJ/mol.

Keywords: Lipid-deprived residuals of microalgae, cellulosome, cellulose, reducing sugars, kinetics.

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Authors: Antonella Bandiera

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A new sythetic gene coding for a Human Elastin-Like Polypeptide was constructed and expressed. The recombinant product was tested as coating agent to realize a surface suitable for cell growth. Coatings showed peculiar features and different human cell lines were seeded and cultured. All cell lines tested showed to adhere and proliferate on this substrate that has been shown also to exert a specific effect on cells, depending on cell type.

Keywords: elastin, recombinant protein, coating, cell adhesion.

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Authors: Muhammad Ijaz Khan, Samina Jalali, Beenish Shahid, S. A. Shami, Muhammad Ikramullah

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In the present study, the response of Nili Ravi buffalo oocytes to recombinant human follicle stimulating hormone (rhFSH) (Organon) on meiotic maturation in vitro was examined. Oocytes were matured in vitro in medium containing either 0 or 0.05 IU/ ml rhFSH and the stage of nuclear maturation recorded after 24 hours. The percentage of oocytes in the control group undergoing germinal vesicle breakdown (GVBD) observed after 24 hours of culture was 29 % whereas as in rhFSH group the percentage was 10 % were at this stage (P< 0.001).Thus in the presence of rhFSH, a significantly greater number of oocytes had progressed to the more advanced stages of nuclear maturation. Indeed, the maturation of GV (Germinal Vesicle) stage oocytes to the metaphase II (M II) stage after 24 hours was significantly (P< 0.0001) increased by the addition of rhFSH (82 % VS 47 %). The percentage of degenerated oocytes after 24 hours of culture was 24 % in control group, whereas in rhFSH group the percentage was 8 % after 24 hours. Degeneration of the oocytes after 24 hours was not significantly (P = 0. 9361) decreased.

Keywords: Buffalo, in vitro, oocytes, recombinant FSH.

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Authors: Ibtissem Gammoudi, Ndeye Rokhaya Faye, Fabien Moroté, Daniel Moynet, Christine Grauby-Heywang, Touria Cohen-Bouhacina

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The objective of the present investigation was to evaluate the morphology of Escherchia coli bacteria in interaction with SiO2 nanoparticles. This study was made by atomic force microscopy and quartz crystal microbalance using SiO2 nanoparticles with 10nm, 50nm and 100nm diameter and bacteria immobilized on polyelectrolyte multilayer films obtained by spin coating or by “layer by layer” (LbL) method.

Keywords: Atomic Force Microscopy, Escherichia coli, Quartz Crystal Microbalance, polyelectrolyte, silica nanoparticle.

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Authors: Mohammad Reza Farzami Pour

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Determination of genetic variation is useful for plant breeding and hence production of more efficient plant species under different conditions, like drought stress. In this study a sample of 28 recombinant inbred lines (RILs) of wheat developed from the cross of Norstar and Zagross varieties, together with their parents, were evaluated for two years (2010-2012) under normal and water stress conditions using split plot design with three replications. Main plots included two irrigation treatments of 70 and 140 mm evaporation from Class A pan and sub-plots consisted of 30 genotypes. The effect of genotypes and interaction of genotypes with years and water regimes were significant for all characters. Significant genotypic effect implies the existence of genetic variation among the lines under study. Heritability estimates were high for 1000 grain weight (0.87). Biomass and grain yield showed the lowest heritability values (0.42 and 0.50, respectively). Highest genotypic and phenotypic coefficients of variation (GCV and PCV) belonged to harvest index. Moderate genetic advance for most of the traits suggested the feasibility of selection among the RILs under investigation. Some RILs were higher yielding than either parent at both environments.

Keywords: Wheat, genetic gain, heritability, recombinant inbred lines.

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Authors: M. H. Syakira, L. Brenda

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Antibacterial activity of Plumeria alba (Frangipani) petals methanolic extracts were evaluated against Escherichia coli, Proteus vulgaris,Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Enterococcus faecalis and Serratia marcescens by using disk diffusion method. Concentration extracts (80 %) showed the highest inhibition zone towards Escherichia coli (14.3 mm). Frangipani extract also showed high antibacterial activity against Staphylococcus saprophyticus, Proteus vulgaris and Serratia marcescens, but not more than the zones of the positive control used. Comparison between two broad specrum antibiotics to frangipani extracts showed that the 80 % concentration extracts produce the same zone of inhibition as Streptomycin. Frangipani extracts showed no bacterial activity towards Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterococcus faecalis. There are differences in the sensitivity of different bacteria to frangipani extracts, suggesting that frangipani-s potency varies between these bacteria. The present results indicate that frangipani showed significant antibacterial activity especially to Escherichia coli.

Keywords: Frangipani, Plumeria alba, anti microbial, Escherichia coli

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Authors: Samad Lotfollahzadeh

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Escherichia coli (E. coli) is the most isolated bacteria from blood circulation of septicemic calves. Given the prevalence of septicemia in animals and its economic importance in veterinary practice, better understanding of changes in clinical signs following disease, may contribute to early detection of disorder. The present study has been carried out to detect changes of clinical signs in induced sepsis in calves with E. coli. Colisepticemia has been induced in 10 twenty-day old healthy Holstein- Frisian calves with intravenous injection of 1.5 X 109 colony forming units (cfu) of O111:H8 strain of E. coli. Clinical signs including rectal temperature, heart rate, respiratory rate, shock, appetite, sucking reflex, feces consistency, general behavior, dehydration and standing ability were recorded in experimental calves during 24 hours after induction of colisepticemia. Blood culture was also carried out from calves four times during experiment. ANOVA with repeated measure is used to see changes of calves’ clinical signs to experimental colisepticemia, and values of P≤ 0.05 was considered statistically significant. Mean values of rectal temperature and heart rate as well as median values of respiratory rate, appetite, suckling reflex, standing ability and feces consistency of experimental calves increased significantly during study (P<0.05). In the present study median value of shock score was not significantly increased in experimental calves (P> 0.05). The results of present study showed that total score of clinical signs in calves with experimental colisepticemia increased significantly, although score of some clinical signs such as shock did not change significantly.

Keywords: Calves, Clinical signs scoring, E. coli O111:H8, Experimental colisepticemia,

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Authors: S. Techaoei, K. Jarmkom, P. Eakwaropas, W. Khobjai

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The objective of this research was focused on investigating in vitro antimicrobial activity of Phellinus linteus fruiting body extracts on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. Phellinus linteus fruiting body was extracted with ethanol and ethyl acetate and was vaporized. The disc diffusion assay was used to assess antimicrobial activity against tested bacterial strains. Primary screening of chemical profile of crude extract was determined by using thin layer chromatography. The positive control and the negative control were used as erythromycin and dimethyl sulfoxide, respectively. Initial screening of Phellinus linteus crude extract with the disc diffusion assay demonstrated that only ethanol had greater antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. The MIC assay showed that the lower MIC was observed with 0.5 mg/ml of Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus and 0.25 mg/ml. of Escherichia coli and Staphylococcus aureus, respectively. TLC chemical profile of extract was represented at R_f ≈ 0.71-0.76.

Keywords: Staphylococcus aureus, Phellinus linteus, methicillin-resistant Staphylococcus aureus, antimicrobial activity, Escherichia coli.

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Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

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Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA₄₄₀₄. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.

Keywords: Recombinant tissue plasminogen activator, plant cell comportment, leader signals, transgenic tobacco.

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Authors: Hazim Saadoon Aljewari, Mohammed Ibraheem Nader, Abdul Hussain M. Alfaisal, NatthidaWeerapreeyakul, Sahapat

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L-asparaginase was extracted from pathogenic Escherichia coli which was isolated from urinary tract infection patients. L-asparaginase was purified 96-fold by ultrafiltration, ion exchange and gel filtration giving 39.19% yield with final specific activity of 178.57 IU/mg. L-asparaginase showed 138,356±1,000 Dalton molecular weight with 31024±100 Dalton molecular mass. Kinetic properties of enzyme resulting 1.25×10-5 mM Km and 2.5×10-3 M/min Vmax. L-asparaginase showed a maximum activity at pH 7.5 when incubated at 37 ºC for 30 min and illustrated its full activity (100%) after 15 min incubation at 20-37 ºC, while 70% of its activity was lost when incubated at 60 ºC. L-asparaginase showed cytotoxicity to U937 cell line with IC50 0.5±0.19 IU/ml, and selectivity index (SI=7.6) about 8 time higher selectivity over the lymphocyte cells. Therefore, the local pathogenic E. coli strains may be used as a source of high yield of L-asparaginase to produce anti cancer agent with high selectivity.

Keywords: L-asparaginase, Purification, Cytotoxicity, selectivity index

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Authors: Prachi Singh

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This paper presents a low-cost, eco-friendly and reproducible microbe mediated biosynthesis of TiO₂ nanoparticles. TiO₂ nanoparticles synthesized using the bacterium, Bacillus subtilis, from titanium as a precursor, were confirmed by TEM analysis. The morphological characteristics state spherical shape, with the size of individual or aggregate nanoparticles, around 30-40 nm. Microbial resistance represents a challenge for the scientific community to develop new bioactive compounds. Here, the antibacterial effect of TiO₂ nanoparticles on Escherichia coli was investigated, which was confirmed by CFU (Colony-forming unit). Further, growth curve study of E. coli Hb101 in the presence and absence of TiO₂ nanoparticles was done. Optical density decrease was observed with the increase in the concentration of TiO₂. It could be attributed to the inactivation of cellular enzymes and DNA by binding to electron-donating groups such as carboxylates, amides, indoles, hydroxyls, thiols, etc. which cause little pores in bacterial cell walls, leading to increased permeability and cell death. This justifies that TiO₂ nanoparticles have efficient antibacterial effect and have potential to be used as an antibacterial agent for different purposes.

Keywords: Antibacterial effect, CFU, Escherichia coli Hb101, growth curve, TEM, TiO2 nanoparticle, toxicity, UV-Vis.

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Authors: Suleyman A. Muyibi, Munirat, A. Idris, Saedi Jami, Parveen Jamal, Mohd Ismail Abdul Karim

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In this study, statistical optimization design was used to study the optimum disinfection parameters using defatted crude Moringa oleifera seed extracts against Escherichia coli (E. coli) bacterial cells. The classical one-factor-at-a-time (OFAT) and response surface methodology (RSM) was used. The possible optimum range of dosage, contact time and mixing rate from the OFAT study were 25mg/l to 200mg/l, 30minutes to 240 minutes and 100rpm to 160rpm respectively. Analysis of variance (ANOVA) of the statistical optimization using faced centered central composite design showed that dosage, contact time and mixing rate were highly significant. The optimum disinfection range was 125mg/l, at contact time of 30 minutes with mixing rate of 120 rpm. 

Keywords: E.coli, disinfection, Moringa oleifera, response surface methodology.

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Authors: Igbakura I. Luga, Alex A. Adikwu

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A comparison of resistance to quinolones was carried out on isolates of Shiga toxin-producing Escherichia coliO157:H7 from cattle and mecA and nuc genes harbouring Staphylococcus aureus from pigs. The isolates were separately tested in the first and current decades of the 21^st century. The objective was to demonstrate the dissemination of resistance to this frontline class of antibiotic by bacteria from food animals and bring to the limelight the spread of antibiotic resistance in Nigeria. A total of 10 isolates of the E. coli O157:H7 and 9 of mecA and nuc genes harbouring S. aureus were obtained following isolation, biochemical testing, and serological identification using the Remel Wellcolex E. coli O157:H7 test. Shiga toxin-production screening in the E. coli O157:H7 using the verotoxin E. coli reverse passive latex agglutination (VTEC-RPLA) test; and molecular identification of the mecA and nuc genes in S. aureus. Detection of the mecA and nuc genes were carried out using the protocol by the Danish Technical University (DTU) using the following primers mecA-1:5'-GGGATCATAGCGTCATTATTC-3', mecA-2: 5'-AACGATTGTGACACGATAGCC-3', nuc-1: 5'-TCAGCAAATGCATCACAAACAG-3', nuc-2: 5'-CGTAAATGCACTTGCTTCAGG-3' for the mecA and nuc genes, respectively. The nuc genes confirm the S. aureus isolates and the mecA genes as being methicillin-resistant and so pathogenic to man. The fluoroquinolones used in the antibiotic resistance testing were norfloxacin (10 µg) and ciprofloxacin (5 µg) in the E. coli O157:H7 isolates and ciprofloxacin (5 µg) in the S. aureus isolates. Susceptibility was tested using the disk diffusion method on Muller-Hinton agar. Fluoroquinolone resistance was not detected from isolates of E. coli O157:H7 from cattle. However, 44% (4/9) of the S. aureus were resistant to ciprofloxacin. Resistance of up to 44% in isolates of mecA and nuc genes harbouring S. aureus is a compelling evidence for the rapid spread of antibiotic resistance from bacteria in food animals from Nigeria. Ciprofloxacin is the drug of choice for the treatment of Typhoid fever, therefore widespread resistance to it in pathogenic bacteria is of great public health significance. The study concludes that antibiotic resistance in bacteria from food animals is on the increase in Nigeria. The National Food and Drug Administration and Control (NAFDAC) agency in Nigeria should implement the World Health Organization (WHO) global action plan on antimicrobial resistance. A good starting point can be coordinating the WHO, Office of International Epizootics (OIE), Food and Agricultural Organization (FAO) tripartite draft antimicrobial resistance monitoring and evaluation (M&E) framework in Nigeria.

Keywords: Fluoroquinolone, Nigeria, resistance, Staphylococcus aureus.

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Authors: P. Pasomboon, P. Chumnanpuen, T. E-kobon

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Hyaluronic acid (HA) consists of linear heteropolysaccharides repeat of D-glucuronic acid and N-acetyl-D-glucosamine. HA has various useful properties to maintain skin elasticity and moisture, reduce inflammation, and lubricate the movement of various body parts without causing immunogenic allergy. HA can be found in several animal tissues as well as in the capsule component of some bacteria including Pasteurella multocida. This study aimed to modify a genome-scale metabolic model of Escherichia coli using computational simulation and flux analysis methods to predict HA productivity under different carbon sources and nitrogen supplement by the addition of two enzymes (GLMU2 and HYAD) from P. multocida to improve the HA production under the specified amount of carbon sources and nitrogen supplements. Result revealed that threonine and aspartate supplement raised the HA production by 12.186%. Our analyses proposed the genome-scale metabolic model is useful for improving the HA production and narrows the number of conditions to be tested further.

Keywords: Pasteurella multocida, Escherichia coli, hyaluronic acid, genome-scale metabolic model, bioinformatics.

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Authors: Kandpal M. , Gundampati R. K , Debnath M.

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Microbial contamination, most of which are fecal born in drinking water and food industry is a serious threat to humans. Escherichia coli is one of the most common and prevalent among them. We have developed a sensor for rapid and an early detection of contaminants, taking E.coli as a threat indicator organism. The sensor is based on co-polymerizations of aniline and formaldehyde in form of thin film over glass surface using the vacuum deposition technique. The particular doping combination of thin film with Fe-Al and Fe-Cu in different concentrations changes its non conducting properties to p- type semi conductor. This property is exploited to detect the different contaminants, believed to have the different surface charge. It was found through experiments that different microbes at same OD (0.600 at 600 nm) have different conductivity in solution. Also the doping concentration is found to be specific for attracting microbes on the basis of surface charge. This is a simple, cost effective and quick detection method which not only decreases the measurement time but also gives early warnings for highly contaminated samples.

Keywords: Sensor, Vacuum deposition technique, thin film, E.coli detection, doping concentration.

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Authors: M. Kaomek, J. R. Ketudat-Cairns

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The cDNA encoding the 326 amino acids of a Class I basic chitinase gene from Leucaena leucocephala de Wit (KB3, Genbank accession: AAM49597) was cloned under the control of CaMV35S promoter in pCAMBIA 1300 and transferred to Koshihikari. Calli of Koshihikari rice was transformed with agrobacterium with this construct expressing the chitinase and β- glucouronidase (GUS). The frequencies of calli 90 % has been obtained from rice seedlings cultured on NB medium. The high regeneration frequencies, 74% was obtained from calli cultured on regeneration medium containing 4 mg/l BAP, and 7 g/l phytagel at 25°C. Various factors were studied in order to establish a procedure for the transformation of Koshihikari Agrobacterium tumefaciens. Supplementation of 50 mM acetosyringone to the medium during coculivation was important to enhance the frequency to transient transformation. The 4 week-old scutellum-derived calli were excellent starting materials. Selection medium based on NB medium supplement with 40 mg/l hygromycin and 400 mg/l cefotaxime were an optimized medium for selection of transformed rice calli. The percentage of transformation 70 was obtained. Recombinant calli and regenerated rice plants were checked the expression of chitinase and gus by PCR, northern blot gel, southern blot gel, and gus assay. Chitinase and gus were expressed in all parts of recombinant rice. The rice line expressing the KB3 chiitnase was more resistant to the blast fungus Fusarium monoliforme than control line.

Keywords: chitinase, Leucaena leucocephala de Wit, Koshihikari, transgenic rice.

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Authors: H. Elsawy, A. Jeltsch

Abstract:

EcoDam is an adenine-N6 DNA methyltransferase that methylates the GATC sites in the Escherichia coli genome. DNA-adenine methylation is not present in higher eukaryotes including humans. These observations raise the possibility that dam inhibitors may be used as anti-microbial agents. Polyphosphate (Poly(P)) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. Here, by using gel retardation experiments to investigate the competition of DNA binding by EcoDam in the presence of polyphosphate, we found that Poly (P) strongly interferes with DNA binding by EcoDam, while same concentration of monophosphate does not. In addition, we demonstrated that Poly (P) binding inhibits the activity of EcoDam and our results suggest that Poly (P) led to strong inhibition of the EcoDam catalytic activity, while monophosphate had only moderate effect.

Keywords: Antibacterial drugs, EcoDam inhibitors, Polyphosphate.

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Authors: Jun Yop An, Kyoung Ryoung Park, Jung-Gyu Lee, Hyung-Seop Youn, Jung-Yeon Kang, Gil Bu Kang, Soo Hyun Eom

Abstract:

MinC plays an important role in bacterial cell division system by inhibiting FtsZ assembly. However, the molecular mechanism of the action is poorly understood. E. coli MinC Nterminus domain was purified and crystallized using 1.4 M sodium citrate pH 6.5 as a precipitant. X-ray diffraction data was collected and processed to 2.3 Å from a native crystal. The crystal belonged to space group P212121, with the unit cell parameters a = 52.7, b = 54.0, c = 64.7 Å. Assuming the presence of two molecules in the asymmetric unit, the Matthews coefficient value is 1.94 Å3 Da-1, which corresponds to a solvent content of 36.5%. The overall structure of MinCN is observed as a dimer form through anti-parallel ß-strand interaction.

Keywords: MinC, Cell division, Crystallization.

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Authors: Jackson H. O. Onyuka, Rose Kakai, David M. Onyango, Peter F. Arama, John Gichuki, Ayub V.O. Ofulla

Abstract:

A cross sectional study design and standard microbiological procedures were used to determine the prevalence and antimicrobial susceptibility patterns of Escherichia coli, Salmonella enterica serovar typhimurium and Vibrio cholerae O1 isolated from water and two fish species Rastrineobola argentea and Oreochromis niloticus collected from fish landing beaches and markets in the Lake Victoria Basin of western Kenya. Out of 162 samples analyzed, 133 (82.1%) were contaminated, with S. typhimurium as the most prevalent (49.6%), followed by E. coli (46.6%), and lastly V. cholerae (2.8%). All the bacteria isolates were sensitive to ciprofloxacin. E. coli isolates were resistant to ampicillin, tetracycline, cotrimoxazole, chloramphenical and gentamicin while S. typhimurium isolates exhibited resistance to ampicillin, tetracycline, and cotrimoxazole. The V. cholerae O1 isolates were resistant to tetracycline and ampicillin. The high prevalence of drug resistant enteric bacteria in water and fish from the study region needs public health intervention from the local government.

Keywords: Aquatic environments, Antimicrobial resistance, Enteric bacteria, Lake Victoria Basin

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Authors: G. Petruzzelli, F. Pedron, M. Grifoni, A. Pera, I. Rosellini, B. Pezzarossa

Abstract:

The addition of lime as Ca(OH)2 to sewage sludge to destroy pathogens (Escherichia coli), was evaluated also in relation to heavy metal bioavailability. The obtained results show that the use of calcium hydroxide at the dose of 3% effectively destroyed pathogens ensuring the stability at high pH values over long period and the duration of the sewage sludge stabilization. In general, lime addition decreased the total extractability of heavy metals indicating a reduced bioavailability of these elements. This is particularly important for a safe utilization in agricultural soils to reduce the possible transfer of heavy metals to the food chain.

Keywords: Biological sludge, Ca(OH)2, copper, pathogens, sanitation, zinc.

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Authors: Sabrin M. Al-Jafaeri, Nuri S. Madi, Mohamed H. Nahaisi

Abstract:

This study was conducted to investigate the incidence of pathogenic bacteria: Salmonella, Shigella, Escherichia coli O157 and Staphylococcus aureus in cakes and tarts collected from thirtyfive confectionery producing and selling premises located within Tripoli city, Libya. The results revealed an incidence of S. aureus with 94.4 and 48.0 %, E. coli O157 with 14.7 and 4.0 % and Salmonella sp. with 5.9 and 8.0 % in cakes and tarts samples respectively; while Shigella was not detected in all samples. In order to determine the source of these pathogenic bacteria, cotton swabs were taken from the hands of workers on the production line, the surfaces of preparation tables and cream whipping instruments. The results showed that the cotton swabs obtained from the hands of workers contained S. aureus and Salmonella sp. with an incidence of 42.9 and 2.9 %, the cotton swabs obtained from the surfaces of preparation tables 22.9 and 2.9 % and the cotton swabs obtained from the cream whipping instruments 14.3 and 0.0 % respectively; while E. coli O157 and Shigella sp. were not detected in all swabs. Additionally, other bacteria were isolated from the hands of workers and the Surfaces of producing equipments included: Aeromonas sp., Pseudomonas sp., E. coli, Klebsiella sp., Enterobacter sp., Citrobacter sp., Proteus sp., Serratia sp. and Acinetobacter sp. These results indicate that some of the cakes and tarts might pose threat to consumer's health. Meanwhile, occurrences of pathogenic bacteria on the hands of those who are working in production line and the surfaces of equipments reflect poor hygienic practices at most confectionery premises examined in this study. Thus, firm and continuous surveillance of these premises is needed to insure the consumer's health and safety.

Keywords: Pathogenic bacteria, Cakes, Tarts, Confectionery producing and selling premises.

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