Phenotypic and Molecular Characterization of Heme Release in Selected Clinical Isolates of Staphylococcus aureus: Expression Analysis of PSMA and HLD Genes by RT-PCR and Quantification by HPLC

This study evaluated whether the expression of psmα and hld is associated with HPLC-detected heme release from human erythrocytes after exposure to clinical S. aureus isolates. A total of 90 human clinical samples were examined, including wound swabs, pus aspirates, blood-culture isolates and catheter-tip isolates. S. aureus was identified using culture, Gram staining, catalase, coagulase, mannitol fermentation and automated biochemical confirmation. Thirty confirmed S. aureus isolates were selected for the erythrocyte-exposure experiment. Bacterial isolates were incubated with washed human erythrocytes for 2 h at 37°C. Total RNA was extracted from bacterial pellets, converted into cDNA and analyzed by RT-qPCR for psmα and hld, using gyrB as the housekeeping gene. Free heme released into the supernatant was quantified by reverse-phase HPLC using a hemin chloride calibration curve. S. aureus was recovered from 42/90 samples, giving an isolation rate of 46.7%. Among the 30 selected isolates, 15 were classified as high heme-release isolates and 15 as low heme-release isolates. High heme-release isolates showed significantly higher psmα expression than low heme-release isolates, with mean fold expression of 3.41±1.02 versus 1.48±0.51, respectively. The hld gene was also significantly upregulated in high heme-release isolates, with mean fold expression of 2.87±0.94 versus 1.26±0.42. HPLC analysis showed significantly higher free heme concentration in the high heme-release group than in the low heme-release group, 38.6±9.4 µM versus 12.4±5.1 µM. psmα expression correlated positively with HPLC-detected heme concentration, while hld expression also showed a significant positive correlation. Clinical S. aureus isolates with increased psmα and hld expression showed greater HPLC-detected erythrocyte heme release.