Molecular insights into small RNA-mediated regulation of biofilm formation and multidrug resistance in Pseudomonas aeruginosa under zinc oxide nanoparticle exposure.

backgroundPseudomonas aeruginosa is an opportunistic pathogen with marked biofilm-forming capacity and increasing multidrug resistance, prompting the need for alternative antimicrobials. Zinc oxide (ZnO) nanomaterials exhibit antibacterial potential; however, their effects on small RNA-mediated regulation remain unclear.

objectiveTo assess the antimicrobial and antibiofilm activities of biosynthesized ZnO and determine its effects at subinhibitory concentrations on selected small regulatory RNAs and biofilm-associated genes in clinical P. aeruginosa isolates. MATERIALS AND

methodsFifty clinical isolates were identified and tested for antibiotic susceptibility and biofilm formation. The biosynthesized ZnO was characterized using UV-Vis, FTIR, EDX, FE-SEM, and AFM. The MIC and antibiofilm activity were evaluated using resazurin, broth microdilution, agar diffusion, and crystal violet assays. Three multidrug-resistant isolates underwent PCR and RT-qPCR analyses of ErsA, SrbA, amrZ, and algD after exposure to 12,500 and 25,000 μg/mL ZnO.

resultsAmong biofilm-forming isolates, 55% were strong, 33% moderate, and 11% weak producers; 88.8% of multidrug-resistant isolates showed strong or moderate biofilm formation. The ZnO nanoparticles had a mean diameter of 40.75 nm and MIC of 50,000 μg/mL. Significant biofilm inhibition occurred at 25,000 μg/mL (p = 0.039) and 50,000 μg/mL (p = 0.01) concentrations. The inhibition zones at 50,000 μg/mL were 16 ± 1.2 mm, 15 ± 1.0 mm, and 17 ± 1.1 mm for urine, burn, and wound isolates, respectively. Gene expression analysis revealed source-dependent transcriptional responses: urine isolates showed marked upregulation of SrbA (58.89-fold) and algD (43.71-fold), indicating pre-adaptation to environmental stressors, while wound isolates exhibited predominantly downregulation of biofilm-associated genes. Burn isolates displayed a biphasic response, with stress pathway activation at 1/4 MIC but gene suppression at 1/2 MIC.

conclusionBiosynthesized ZnO exerts concentration-dependent antibacterial and antibiofilm effects against clinical P. aeruginosa while differentially modulating sRNA-linked regulatory networks under sub-MIC exposure. The key findings demonstrate that ZnO nanoparticles effectively inhibit biofilm formation at concentrations ≥25,000 μg/mL, while sub-inhibitory exposure triggers source-specific adaptive transcriptional responses mediated through sRNA regulatory circuits. These findings conclusively support the potential of biosynthesized ZnO nanoparticles as adjunctive antimicrobial agents against MDR P. aeruginosa biofilms, with the critical caveat that therapeutic concentrations must be maintained above the MIC to prevent adaptive resistance enhancement through sRNA-mediated stress responses.