Combined impact of biosynthesized selenium nanoparticles and imipenem against carbapenem-resistant Pseudomonas aeruginosa and their associated virulence factors.

backgroundCarbapenem-resistant P. aeruginosa (CRPA) is a significant nosocomial pathogen characterized by extensive antibiotic resistance, representing a serious public health concern. It is regarded as a high-priority target for antibacterial research. This study aimed to isolate and identify CRPA isolates and the biosynthesis of selenium nanoparticles (Se-NPs) as a novel therapeutic approach for combating CRPA strains and their capacity to form biofilms, alone or in combination with imipenem.

methodsCRPA isolates were isolated from different clinical samples, identified, and subjected to antibiotic profiling using Vitek-2 method. The detection of biofilm was performed using Congo red agar (CRA), Microdilution broth assay (MBA), and qRT-PCR detection of Bap and ompA genes. Biosynthesis of Se-NPs with a cell-free filter (CFF) of Streptomyces sp. was done and characterized with various techniques, including UV-Vis, XRD, TEM, FTIR, and Zeta potential measurement. The antibacterial efficacy and minimum inhibitory concentrations (MICs) were determined using disc diffusion and microdilution techniques. The checkerboard assay was used to formulate various combinations of imipenem and Se-NPs, alongside time-kill assays to assess their antimicrobial efficacy. Furthermore, the cytotoxic effects and hemolytic activity of Se-NPs, imipenem and their combination were assessed.

resultsThe identification process and antibiotic susceptibility testing confirmed that the bacterial isolates were found to be CRPA. Phenotypic analysis revealed that the CRPA produced biofilm, and qRT-PCR demonstrated that all CRPA strains under study have the Bap and ompA genes. The CFF of Streptomyces sp. was able to biosynthesize Se-NPs which presented UV-Visible spectrometric profile with sharp peak at 290 nm. Se-NPs appeared to be a spherical shape, with particle sizes ranging from 20 to 100 nm under TEM and have zeta potential value of -40 mV. The MICs of Se-NPs and imipenem ranged from 6 to 14 and 12 to 14 µg/ml, respectively. The fractional inhibitory concentration index (FICI) values ranged from 0.37 to 0.50 against tested CRPA strain with a significant reduction in the concentrations of Se-NPs and imipenem. QRT-PCR showed that Se-NPs alone or combination of Se-NPs and imipenem led to a reduction of Bap and ompA gene expression compared to control (p ≤ 0.0001). The study showed a significant difference in cell viability was observed across normal or cancer cell lines at high concentrations. However, the combination of Se-NPs and imipenem demonstrated enhanced selectivity toward cancer cells, with HepG-2 cells showing significantly lower viability compared to normal HFP-4 cells across all tested concentrations. Se-NPs alone showed moderate hemolysis percentages of 1.9% at 12 h and 2.3% at 24 h while the hemolytic activity Se-NPs and imipenem combination was reduced to 1.4% and 1.7% at 12 and 24 h, representing approximately 26% and 26% reductions in haemolysis compared to Se-NPs alone at the respective time points.

conclusionThis study confirms that the biosynthesized Se-NPs exhibit potent synergistic effects with imipenem against CRPA, significantly reducing biofilm formation and the expression of virulence genes Bap and ompA.