DETERMINATION GENETIC FACTORS CONTROLLING Β-LACTEMASE ENZYME RELATED TO BLA TEM AND BLA SHV FAMILIES USING THE POLYMERASE CHAIN REACTION

Clinical isolates (75) of Gram- negative bacteria were isolated from patients with different infections at Al-Kadhumia Educational hospital in Baghdad. These isolates were diagnosed using different morphological and biochemical test followed by the complementary api 20E and api 20NE test. They were distributed as Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa (25 isolates for each). β-lactemase production was investigated using the rapid iodometric method and the results revealed that 48 isolates out of 75(64%) gave a positive result .These isolates were, 21 from E.coli (84%),17 isolates for P. mirabilis (68%) and 10 of Ps. aeruginosa(40%).The double disc approximation test was used to detect Extended spectrum β-lactemases (ESBLs)and the result showed that 28 isolates out of 48(58.33%)gave positive result as 19,3,6 isolates for E. coli, P. mirabilis and Ps. aeruginosa respectively. Polymerase chain reaction(PCR) technique was performed to detect the genetic factors controlling β-lactemases related to bla TEM and bla SHV families and template DNA was prepared either by the boiling method in which the total genomic DNA will serve as a template or by using the plasmid DNA as a template and the later was prepared by the alkaline lysis method. Results of using total genomic DNA showed that 27 isolates out of 28(96.42)were harboring bla TEM gene based on the presence of 950 bp bands in 1% agarose gel .By using the plasmid DNA as a template ,only 10 isolates (35.7%) gave clear band which may indicate that β-lactemases of these isolates may be are plasmid mediated .For bla SHV gene , the results of using the first template showed that only 2 E. coli isolates out of 28(4.14%)gave a positive result represented by presence of 800 bp band in the aga rose gel while no band was observed when the plasmid template was used which may indicate that the enzymes here under chromosomal control.