Sequences and expression of the active lysostaphin gene from staphylococcus simulans isolated of bovine mastitis and its bactericidal effect on staphylococcus aureus

Abstract EN

Sixty milk samples were recovered from cows suffering from clinical mastitis in Basra.

Among these 55 (91.66%) gave good growth in blood agar.

After microscopical and biochemical tests, 49 isolates have been diagnosed as G+ bacteria staphylococci.

Thirty (50%) isolates of S.

aureus are coagulase positive staphylococci (CoPS) and 19 (31.66%) isolates were coagulase negative staphylococci (CoNS).

Among the recovered isolates, two isolates are S.

simulans and only one gave good antimicrobial activity against S.

aureus on the basis of inhibition zone.

The active lysostaphin gene with determined size and sequences are 738 bp was isolated and then cloned to E.

coli.

A lysostaphin gene was prepared with terminal Histidine group in order to isolate and purify lysostaphin protein by used special primers.

His-tag column was chosen for isolation and purification of protein with in short time.

A high fidelity in running, one band of protein in polyacrylamide gel was seen in comparison with standard protein from Sigma which gave more than two bands, after running in polyacrylamide gel, the molecular weight was about 27 KDa.

The antibacterial effect of lysostaphin against S.

aureus was studied in Vitro which gave good inhibition zone on sold media.

The LD50 of lysostaphin was determined and there were no effects of lysostaphin on mice with all concentrations used.

The effect of lysostaphin against S.

aureus in Vivo was studied by inducible infection in mammary glands (mastitis).

The antibacterial activity of lysostaphin showed significant effect.

Finally the histological studies of mammary glands showed the significant activities of lysostaphin to inhibit the growth of S.

aureus.

To eliminate the antibiotics resistant building this study is conducted.

The aims of the study: Isolation and identification of Staphylococcus simulans as a source of lysostaphin producer from mastitis cows.

Cloning the lysostaphin gene, using expression of this gene as bactericidal effect on S.

aureus in plate.

Screening the effect of lysostaphin on induced infected mice (locally) with S.

aureus.