Biofilm-Associated Genes Detection

Genomic DNA isolation from the S. haemolyticus strain used in our experiment as well as 19 other clinical S. haemolyticus strains were carried out as previously described (Frebourg et al., 2000; Zhou et al., 2019). Standard strains of S. haemolyticus (ATCC29970) and S. epidermidis (ATCC12228) were used as controls. The presence of icaA and icaD genes (main component of PIA biofilm) along with a variety of significant biofilm-associated genes [encoding cell surface attachment proteins (ebpS, cbp, fbp, and srtA), quorum sensing (agrA, agrB, agrD, and luxS), and eDNA release (cidA, cidB, lrgA, lrgB, and atlE)] were detected in all the S. haemolyticus strains by polymerase chain reaction (PCR). Amplification conditions (20 μl volume) were as follows: denaturation for 3 min at 94°C, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with final extension at 72°C for 5 min. The primer sequences of PCR used in this study are shown in Supplementary Table S2 (Panda and Singh, 2018). The amplified PCR products were analyzed on 1.5% agarose gel and were visualized using a UV trans-illuminator² (FR980A, Furi Technology, China). In addition, the PCR products were sequenced to confirm the product identity.