Molecular Characterization and Toxicity Confirmation of LukM/F′-PV Producing Staphylococcus aureus Isolated from Bovine Mastitis Samples in Mysore India

摘要

The widespread status of subclinical condition of bovine mastitis is often associated with the production of leukotoxin M/F′-PV producing Staphylococcus aureus. The present study aims for the profiling of such leukotoxin producers through conventional and molecular methods in parallel to their leukotoxicity. The incidence of this particular pathogen was assessed in mastitis infected Holstein–Friesian cattle, where eight isolates of staphylococci were found to be present in 20 % of collected samples. Being intermediately resistant to vancomycin, they showed characteristic double zone hemolysis on 7 % sheep blood agar and typical type II reaction for coagulase test indicating the pathogenic attributes. Further with RAPD-PCR and 16S rDNA-RFLP, epidemiological specificity and genotypic relatedness of isolates to S. aureus was confirmed. Subsequently, the presence of leukotoxin (lukM) gene in native isolates was detected by leukotoxin gene specific PCR. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay evaluated for secreted leukotoxin in cell free supernatant was estimated to be 223 toxic units which had an LD50 cytotoxic activity on bovine neutrophil. Thus, the data acquired during study can be of prime diagnostic method for timely and accurate analysis of subclinical mastitis samples which goes undetected at consumer level.