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Updated: May 4, 2026
Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR
Published on: November 4, 2016
Molecular Characterization and Toxicity Confirmation of LukM/F'-PV Producing Staphylococcus aureus Isolated from
1Department of Food Microbiology, CSIR-Central Food Technological Research Institute, Mysore, 570020 Karnataka India.
Abstract:
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.
Insights
Subclinical bovine mastitis is linked to Staphylococcus aureus producing leukotoxin. This study identified and characterized these toxin-producing bacteria, confirming their pathogenic traits and leukotoxicity for improved diagnostics.
Area of Science:
- Veterinary Microbiology
- Bovine Pathology
- Bacterial Toxinology
Background:
- Subclinical bovine mastitis is a significant economic issue in dairy farming.
- Staphylococcus aureus strains producing leukotoxin M/F'-PV are frequently implicated in mastitis.
- Accurate identification of pathogenic S. aureus is crucial for effective mastitis control.
Purpose of the Study:
- To profile leukotoxin-producing Staphylococcus aureus in mastitis-infected cattle.
- To correlate bacterial identification with their leukotoxicity.
- To develop improved diagnostic methods for subclinical bovine mastitis.
Main Methods:
- Isolation and identification of staphylococcal isolates from mastitis-infected Holstein-Friesian cattle.
- Phenotypic characterization: vancomycin resistance, double zone hemolysis, coagulase test.
- Molecular confirmation: RAPD-PCR, 16S rDNA-RFLP, and leukotoxin (lukM) gene specific PCR.
- Quantification of leukotoxin activity using MTT assay and determination of LD50 on bovine neutrophils.
Main Results:
- Eight staphylococcal isolates identified as S. aureus in 20% of samples.
- Isolates exhibited intermediate vancomycin resistance and characteristic pathogenic attributes.
- Molecular methods confirmed genotypic relatedness and presence of the lukM gene.
- Secreted leukotoxin showed significant cytotoxic activity (223 toxic units, LD50) on bovine neutrophils.
Conclusions:
- The study successfully profiled leukotoxin-producing Staphylococcus aureus in subclinical bovine mastitis.
- Integrated conventional and molecular methods provide accurate identification and characterization.
- The findings support the development of enhanced diagnostic strategies for early detection of bovine mastitis.
