Molecular Characterization of rhlR and fliC Genes in Biofilm-Forming, Multidrug-Resistant Pseudomonas Aeruginosa Isolated from Pediatric Clinical Specimens

Background:  Quorum sensing (QS) and motility are vital for biofilm development and pathogenicity of Pseudomonas aeruginosa. The rhlR gene regulates the synthesis of rhamnolipids and biofilm maturity, whereas the fliC gene codes for flagellin, which is indispensable for cell adhesion to surfaces. In this project, we will investigate (a) the frequency of rhlR and fliC genes in pediatric isolates using PCR; (b) genetic diversity by DNA sequencing; (c) susceptibility to antimicrobials; and (d) correlations between gene load, biofilm development, and antibiotic resistance. Methodology: This study analyzed 100 isolates of P. aeruginosa from pediatric patients (6 months to 15 years old) with different infections in three Iraqi cities. The TCP test was used to quantify biofilm formation. rhlR and fliC were detected in 30 selected strains using PCR and subsequently sequenced (three amplicons for each gene). The antibiotic susceptibility test was performed against nine antipseudomonal antibiotics. Results: In total, 70% of strains were found to produce moderate-to-strong biofilm. rhlR was identified in 43.3% and fliC in 33.3% of strains. Sequence analysis showed that rhlR had an identity value of 96.3-97.7%, with 18 reliable SNPs and a stop-gained mutation in codon 49. In contrast, there was a high variability in the fliC gene, showing an identity percentage of 31.6-49.4% within a small, conserved core region of 79 bp. However, BLASTn analysis showed 99% identity with known sequences for the entire gene length (493 bp). Antibiotic resistance was highest with Aztreonam (35%) and Imipenem (28%). Spearman correlation indicated that the presence of rhlR and fliC positively correlated with antibiotic resistance severity (rho = 0.525, p = 0.0085). Conclusion: rhlR and fliC are present in a considerable number of pediatric strains, where the fliC gene is highly variable. Moreover, the positive correlation between gene burden and resistance severity supports the development of new quorum-sensing-based therapies.