Lysis of mastitis pathogens isolated from dairy cow milk samples by purified recombinant lysostaphin

Abstract

To avoid the use of broad spectrum antibiotics which can induce antibiotic resistance, we developed a recombinant lysostaphin with high purity against mastitis pathogens isolated from dairy cow milk. The gene encoding for mature lysostaphin peptide was cloned into an Escherichia coli expression systems, pET28a, to produce recombinant lysostaphin. This recombinant lysostaphin was purified by a His60 Ni Gravity Column to obtain milligram amounts of recombinant lysostaphin protein with high purity. Staphylococcal mastitis pathogens, which were isolated from 30 milk samples from cows diagnosed with mastitis, were identified on the basis of colony characteristics, Gram staining, pigment production, hemolysis on 5% bovine blood agar and tube coagulase test. The antibiotic resistance of the isolates was determined by disc diffusion assay. Most of the tested isolates were resistant to some antibiotics. All the tested strains were lysed by the purified recombinant lysostaphin in turbidity reduction assay. The recombinant lysostaphin was found capable of killing Staphylococci pathogens in vitro within an hour (minimum inhibitory concentration (MIC) 2.5 to 16 µg/ml) and was active in a milk-like environment. Thus, the recombinant lysostaphin from the pET system is effective and is expected to complement clinical treatment of cow mastitis.

Key words: Lysostaphin, Staphylococcus aureus, bovine mastitis, expression, purification.