32 isolates of S. aureus (S1-S32) were collected from different sources (wounds, burns, and throat) obtained from Mansoura university hospitals, Egypt. This work was done after approval of the administrative authorities (Research Ethics Committee) in the Faculty of Pharmacy, Mansoura University, Egypt. The isolates were purified and confirmed as S. aureus by Gram staining and biochemical reactions (Mannitol salt agar, catalase, and coagulase) and using S. aureus Newman as a reference strain [20] that was kindly provided by Dr. Mohammed Youssef Ali (Associate professor of microbiology and immunology, faculty of pharmacy, Mansoura university). All bacterial strains were stored in brain heart infusion (BHI) broth medium containing 20% glycerol at −80˚C, until further analyses were performed.

Overnight cultures of different isolates in BHI broth were incubated at 37˚C with shaking at 200 rpm. Later, subculturing into fresh BHI broth medium (starting OD_{630 nm} equivalent to 1.7 × 10⁷ CFU/ml) was performed with shaking at 200 rpm, and 37˚C. Samples (1 ml) were taken at the required time point, and centrifuged at 3200 x g for 5 min. Spent media (supernatant containing the produced HysA enzyme) were filter sterilized and stored at −80˚C till further analyses.

The procedure of [8] was followed with minor modifications. Sterile glass plates (12 cm diameter) containing 1% agarose, 1% bovine serum albumin (BSA), and 0.4 mg/ml of HA (Sigma, H-1504) in 0.3 M sodium phosphate buffer (pH 5.3) were prepared. After solidification of the agarose medium, wells (7 mm diameter) were made aseptically, and 100 μl of spent media were added into each well.

Plates were incubated for 24 h at 37˚C before flooding with 2 M acetic acid. Clear zones were observed against a background of opaque precipitated BSA conjugated to the undigested HA and their diameters were measured in millimeters. Hyaluronidase (Sanofi, 1500 IU) was used as a standard positive control and for construction of standard curve after dissolving in 500 µl BHI broth to obtain a final concentration of 3000 IU/ml, where two fold serial dilutions from the concentration 3000 IU/ml till 5.8 IU/ml were prepared in BHI broth. Later, 100 µl from each dilution was examined for its hyaluronidase as indicated above. The standard curve was constructed from plotting of the logarithmic concentration of standard hyaluronidase (3000 - 5.8 IU/ml) and the clear zones of HA hydrolysis subtracted from the cup diameter (Figure 1). The hyaluronidase activity present in the spent medium of each isolate was expressed as IU/ml according to the standard hyaluronidase (Figure 1).

Hyaluronidase DNA sequences of 41 S. aureus strains were obtained from the GenBank database and aligned by using the multisequence alignment tool (Clustal omega) that is provided by EMBL-EBI website (Figure S1). For qPCR analysis for hyaluronidase expression, forward (Hyalq-F) and reverse (Hyalq-R) primers (Table 1) were precisely designed from the conserved regions of the aligned sequences and with the consideration of the optimum GC% (40% - 60%).

For qPCR analysis of hysA expression, the optimum time for RNA extraction was investigated by measurement of HysA activity in the spent media and the OD630nm at different time points (3, 5, 9, 24 h). For such optimization, two

F: Foward primer, R: Reverse primer.

strains were selected as representative samples; Newman, and S10.

Cell pellets were harvested from aliquots of 1.5 ml at each specified time for RNA isolation and centrifuged at 3200 x g and 4˚C for 5 min. Glass beads (150 mg, 0.25 - 0.5 mm diameter) [Roth, Carlsruhe, Germany] and 300 µl of triazole reagent (Zymo research) were added to the cell pellet. The mixture was subjected to alternating intervals of vortexing (30 sec) and immersion in ice (30 sec) for total of 10 min. Later, freezing at −80˚C (3 min) and thawing at 37˚C (3 min) was performed 3 times. The disrupted cells were centrifuged at 3200 x g and 4˚C for 5 min. RNA was precipitated according to the protocol of the triazole reagent company for isolation of RNA by traizole method. The precipitated RNA was dried, dissolved in 20 µl RNAase free water, and stored at −80˚C till further analysis. The quality of RNA was checked by agarose gel electrophoresis and RNA concentration was determined using nanodrop.

The synthesis of cDNA from the purified RNA of different isolates was performed using the Quantitect Reverse Transcription kit (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. Real-time PCR was performed in 20 μl reaction volumes using the Rotor-Gene Q (Qiagen) and 5x HOT FIREPol EvaGreen HRM Mix according to the manufacturers’ instructions. Each reaction volume contained 4 μl 5x HOT FIREPol EvaGreen HRM, 0.5 μl forward (250 nM final), 0.5 μl reverse primer (250 nM final), 13 μl sterile water (PCR grade), and 2 μl of template cDNA. The gene encoding GyrB was used as a reference gene and amplified using the GyrB-F and GyrB-R primers (Table 1). Negative control reactions without cDNA template were included in each PCR run. Additionally, strain S32 was included in each run as a control for the strains that were negative for HysA production by phenotypic assay. The PCR was performed using the following conditions: initial activation at 95˚C for 12 min, followed by 40 cycles of denaturation (95˚C for 12 sec), annealing (59˚C for 20 sec), and extension (72˚C for 20 sec).

Melting curves were analyzed to check PCR specificity, and the absence of primer dimers. The PCR efficiency (E) of each primer pair was determined by making dilution series (undiluted, 1/5, 1/10, 1/50, and 1/100) using cDNA of S. aureus strain Newman as a template. The cDNA sample of S. aureus strain Newman was used as a calibrator sample in all qPCR runs to minimize run-to-run variations. The threshold cycle (CT) values were calculated by the Rotor-Gene Q software, and exported to Excel for relative quantification analysis. To ensure the accuracy of amplification, all generated amplicons were separated by agarose gel electrophoresis in a 2% agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator.

Results were statistically analyzed using Excel program. The values for either concentration/OD630nm (Phenotypic assay) or the fold of change in RNA expression (Genotypic assay) were calculated as the average mean ± SD of 3 independent experiments. The correlation between the phenotypic production of HysA, as indicated by plate assay, and the qPCR analysis of hysA expression, was performed using the Pearson correlation analysis test.

Twenty four strains (S1-S24) out of 32 strains were able to produce HysA as indicated by the clear zones obtained from the plate assay. The concentration of hyaluronidase in the spent media was obtained from the standard curve of hyaluronidase enzyme (Figure 1), which was then normalized to the OD_{630 nm} of each individual culture (Concentration/OD_{630 nm}). The highest HysA producing strain was S10, followed by Newman as indicated in Table 2.

As indicated in Figure 2, the highest production of HysA was detected after 3 h cultivation of subcultures of the strains; Newman and S10. Therefore, for qPCR analysis of hysA mRNA expression, the RNA was isolated from the subcultures of the HysA producing isolates of S. aureus after 3 h cultivation.

The efficiency of amplification of hysA and gyrB genes were 1.7 and 1.87 respectively, as determined by the software of Rotor gene Q. To correct for the difference between amplification efficiencies of both genes, the Paffl method [22] was used in this study to determine the relative expression of hysA gene in different isolates using Newman as a calibrator strain. The fold of change (FC) in expression of hysA in different clinical isolates relative to hysA expression by Newman is shown as mean ± SD of three independent experiments (Table 2).