الفهرس 
List of Abbreviation
Section IOnly 14 pages are availabe for public view
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Abstract
In this study two pathogenic bacterial isolates (Gram-positive and Gram- negative) were obtained from clinical sources. The two isolates were identified by using Microscan Walk A way system and the results proved that two isolates were Staphylococcus sciuri and Klebsiella pneumoniae. The two bacterial isolates were subjected to detection for β-lactamase production by some phenotypic methods and molecular detection by PCR. The PCR assays of S. sciuri indicated that this strain harbored the blaZ gene, whereas of K. pneumoniae harbored two MBLs genes (blaIMP and blaVIM). Beta-lactamase production by Staphylococcus sciuri and Klebsiella pneumoniae were investigated. The two media nutrient broth and brain heart infusion broth were used. The second medium proved to be optimal medium for enzyme production, it was better than the first since the activity of the enzyme was higher. The β-lactamase production increased with increasing the inoculum size in a concentration-dependent manner. The optimal temperature was 35 ºC, 40 ºC for β-lactamase production from S. sciuri and K. pneumoniae, respectively. The optimal pH for β-lactamase production was 7, 6 from S. sciuri and K. pneumoniae, respectively. The best incubation time of enzyme production from S. sciuri and K. pneumoniae was 4 h and 6h, respectively. The optimal concentration of the substrate for enzyme production was 400μg ml -1 from both bacteria. Beta-lactamase was isolated and purified from S. sciuri and K.pneumoniae by several steps included precipitation with ammonium sulphate at 80% saturation, DEAE-Cellulose and gel filtration on Sephadex G-200 column. The characterization of the purified β-lactamase showed that the molecular weight was about 30 KDa for purified S. sciuri β-lactamase, and about 28 KDa for purified K. pneumonae β-lactamase as estimated by sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme from S. sciuri and K. pneumonae.