Transformation of Lactobacillus acidophilus with Hygromycin Gene Originated from Streptomyces hygroscopicus

           To Investigate the ability of Streptomyces isolate to produce hygromycin B antibiotic as antithelmantic agent in poultry feed and selecting a suitable expression vector encoded hygromycin B phosphotransferase resistance gene and transforming Lactobacillus isolateby electroporation , from a total of 53 Streptomyces isolates, DNA was extracted and screened for the presence of hygomycin B gene using specific designed primer sets in Polymerase Chain Reaction (PCR) technique.  Only three of the isolates were positive for the existence of such a gene which is responsible for the production of this antibiotic.To select the most efficient one, the three isolates were subjected to the antimicrobial activity evaluation. Then, the selected isolate was identified and characterized morphologically and biochemically. The gene of hygromycin B phosphotransferase (hph) was also detected in the Streptomyces isolate using specific designed primers. When the hygromycin B was extracted by ethyl acetate, which separates organic phase from aqueous phase in the broth culture filtrate, only the aqueous phase showed a significant antimicrobial activity by using agar well diffusion technique. At a concentration of 25mg/ml (as crude extract), this phase excreted its activity against the test microorganisms which include; one G(+) bacteria (Staphylococcus aureus), five G(–) bacteria (Pseudomonas aeruginosa , Proteus mirabilis, Escherichia coli , Klebsiella pneumoniae, Salmonella typhi) and one yeast (Saccharomyces  cerevisiae). After detecting the aminoglycoside hygromycin B by the Thin Layer Chromatography (TLC) method to ensure presence of the antibiotic, the same flow rate (R ) value (0.357), as that of the standard hygromycin B ,was obtained. Results of the optimization conditions showed that the highest  antimicrobial activity of hygromycin B was obtained at a medium pH of 8 and incubation temperature of 35°C for 10 days. When the toxicity of hygromycin B crude extract under such conditions was examined on mice liver, mild effects appeared. Lactobacillus acidophilus isolated from a yoghurt sample was identified conventionally, by API 50 kit, and also by using 16S ribosomal gene technique. Cloning vector containing the (hph) gene was extracted from E.coli bacteria and used to transform cells of Lactobacillus acidophilus by using electroporation technique,that give transformation efficiency of  1.75x10 8 cfu/100ng of plasmid DNA then the transformed cells were tested by specific hph–designed primer and give positive result in gel electrophoresis technique.



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