Quantitative RT-PCR Analysis of spa and hla Virulence Gene Expression in Nasal Isolates of Staphylococcus aureus: Association Between Virulence Transcription and Clinical Symptoms

Background: Nasal colonization by Staphylococcus aureus is common in humans and represents a major reservoir for subsequent infection. However, not all colonizing strains cause disease, suggesting that differences in virulence gene expression may contribute to the transition from asymptomatic carriage to symptomatic infection. Aim: The present study investigated the transcriptional profile of two key virulence genes, spa (protein A) and hla (alpha-hemolysin), in nasal swab isolates from healthy carriers and symptomatic patients using quantitative reverse transcription PCR (RT-qPCR). Patients and Methods: A total of 100 nasal swab samples were collected from two groups: 50 healthy carriers and 50 symptomatic individuals presenting with nasal or respiratory infection. Bacterial RNA was extracted from confirmed S. aureus isolates and converted to cDNA. Quantitative RT-PCR was performed to measure transcription levels of spa and hla, with gyrB serving as the internal reference gene. Gene expression levels were calculated using the ΔΔCt method. Statistical comparisons between groups were performed using independent two-tailed Student’s t-tests. Effect sizes and statistical power were also evaluated. Results: Both virulence genes demonstrated significantly lower Ct values in symptomatic patients. The mean Ct value of spa was 28.5 ± 1.4 in healthy carriers compared with 25.9 ± 1.2 in symptomatic patients (P < 0.0001). Similarly, hla showed Ct values of 30.1 ± 1.7 in healthy carriers and 26.8 ± 1.3 in symptomatic individuals (P < 0.0001). Normalized expression analysis revealed substantial differences between groups. The mean ΔCt value for spa was 7.4 ± 1.1 in healthy carriers and 4.6 ± 1.0 in symptomatic patients, producing a ΔΔCt value of −2.8 and corresponding to a 6.96-fold increase in expression. For the hla gene, the mean ΔCt values were 9.0 ± 1.3 in healthy carriers and 5.5 ± 1.2 in symptomatic patients, generating a ΔΔCt of −3.5 and an 11.31-fold increase in expression among symptomatic individuals. Statistical analysis confirmed highly significant differences between groups, with t-values of 13.2 for spa and 14.8 for hla (both P < 0.0001). Effect size analysis indicated very large differences in expression, with Cohen’s d values of 2.6 for spa and 2.9 for hla. Conclusions: The findings demonstrate that nasal isolates from symptomatic individuals exhibit markedly increased transcription of key virulence determinants compared with isolates from healthy carriers. The observed 6.96-fold increase in spa expression and 11.31-fold increase in hla expression suggest that activation of virulence gene transcription plays an important role in the transition from asymptomatic colonization to clinical infection. Quantitative profiling of virulence gene expression using RT-qPCR may therefore represent a valuable molecular approach for identifying potentially pathogenic nasal strains and improving early detection of infection-associated bacterial phenotypes.