Development of Simplex-PCR assays for Accurate Identification of Nine Staphylococcal Species at Genus and Species Levels

Objective: Species identification of Staphylococcus is the prerequisite for precise assessment of microbial dynamics including their transmission and pathogenic significance in the dairy herd environment. The present study aimed to develop simplex PCR assays for rapid and specific identification of nine different Staphylococcus species. Methods: Specific primers targeting sodA gene for S. aureus, S. chromogenes, S. hominis, S. haemolyticus, S. hyicus; gap gene for S. sciuri, S. auricularis, S. simulans; and rdr gene for S. epidermidis were designed. The PCR assays were evaluated against 28 ATCC reference strains and 209 composite milk samples. Partial 16S rRNA sequencing was performed to reconfirm the results. Results: The PCR assays allowed species level identification of 348 staphylococcal field isolates recovered from 209 milk samples. The identification pattern was S. aureus (n=101), S. chromogenes (n=89), S. epidermidis (n=57), S. sciuri (n=43), S. haemolyticus (n=34), S. hyicus (n=13), S. hominis (n=5), S. auricularis (n=3) and S. simulans (n=3). The PCR based species identification was in 100% concordance with the partial 16S rRNA gene sequencing approach. Conclusion: The simplex PCR assays can be used as a precise tool for routine identification of Staphylococcus species from bovine milk as Staphylococcus species including coagulase-negative staphylococci is recognized a major cause of bovine mastitis in different parts of the world including India. J Microbiol Infect Dis 2018; 8(3):120-127