In-vitro characterization of thrombolytic activity of staphylokinase produced by Staphylococcus aureus S1

Abstract

Cardiovascular diseases (CVDs) are one of the leading causes of death worldwide, and thrombosis is the most common of the three major cardiovascular diseases: ischemic heart disease (acute coronary syndrome), stroke, and venous thromboembolism (VTE). In this study, an attempt to investigate a new potential pathogenic bacterium from clinical source (throat swab) that produced fibrinolytic enzymes and the activity of the enzymes on blood clot formation was made. For the bacterial screening, two different media were used, which are blood agar and skimmed milk agar. Next, for the primary identification of bacterial genus, gram staining, and catalase test was done, followed by 16S rRNA to identify the isolated bacteria rapidly and accurately, which include genomic DNA (gDNA) isolation, PCR amplification, nucleotide sequencing, gel electrophoresis, nucleotide sequence analysis, and phylogenetic tree construction. Apart from that, protein assay was done by Lowry's method, and the determination of the proteolytic activity was done by the caseinolytic analysis, followed by the in vitro clot lysis activity of the enzymes. Initially, three main colonies were found, which was differed in the pigmentation of the colony. However, only YS1 strain showed a positive ß-hemolytic pattern on blood agar and clear hydrolysis zones on skimming milk agar. Therefore, YS1 strain was chosen for further fibrinolytic analysis. Moreover, bacterial sequencing confirmed the YS1 strain gene sequence was found 100 % similarity with the members of the highly associated genera Staphylococcus aureus and was designated as Staphylococcus aureus S1. The total amount of protein in S. aureus S1 was 0.57 (mg/ml), while the caseinolytic activity and the specific activity were 1.43 (U/ml). The highest 46% of clot lysis by staphylokinase after 4 hrs. incubation was observed.