Pseudomonas aeruginosa, a prevalent opportunistic bacterium, leads to several diseases., like wounds, sputum, throat, urine, and blood infections. This research aimed to assess the presence of the pilS2 gene and its correlation with the P. aeruginosa plasmid pKLC102 and the PAPI-1 pathogenicity island in P. aeruginosa isolates. Patient samples were obtained from individuals with various infections, including wounds, sputum, throat, urine, and blood cultures. DNA extraction from these samples utilized a DNA extraction kit for subsequent PCR analysis. PCR analysis involved the use of two primer pairs, SojR/4541F and intF/sojR, to detect the presence of PAPI-1. Additionally, the detection of pKLC102 was accomplished using the primer pair cp44F/cp44R. Forty-three isolates of P. aeruginosa were collected from urine (16), wounds (12), sputum (7), throats (2), and blood (6). According to the higher resistance of samples to the different antibiotic discs, we selected 15 samples: wound (4), urine (6), sputum (4), and blood (1). PCR results from 15 samples revealed that the pilS2 gene was present in 7 strains, accounting for 46% of the samples. Among the isolates, 8 showed integration of PAPI-1-attB, constituting 53.3%, while the circular form of PAPI-1 was identified in 6 isolates, representing 40%. Furthermore, pKLC102 tested positive in 5 strains, totaling 33%. The qRT-PCR analysis showed significant decreases in gene expression for three genes in the disease groups. Pils2 gene expression decreased by 0.045-fold (P-value=0.037), Cp44 gene expression decreased by 0.032-fold (P-value=0.013), and Soj/4541 gene expression decreased by 0.059-fold (P-value=0.041) compared to controls. We found the majority of the virulence may be related to the presence of pils2 genes, plasmid PKLC102, and Soj/4541 genes of PAPI-1, which showed a significant down-regulation of gene expression in the disease group of tested Pseudomonas aeruginosa isolates that showed higher antibiotic resistance levels relative to the control group using qRT-PCR analysis.