Diagnosis of Infecting Pathogen Bacteria by Multiplex PCR and Evaluation of Inhibition Antibiofilm Using Zinc Oxide Nanoparticle abstract
Bacterial infections can occur in various body tissues, including the respiratory tract, urinary tract, gastrointestinal tract, and bloodstream. This study aimed to identify three important pathogenic species—Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa—using both phenotypic and genotypic methods. Bacterial isolates were initially identified through standard diagnostic tests and confirmed by multiplex PCR. Three randomly selected isolates corresponding to each pathogen were subjected to gene sequencing and compared with reference strains from NCBI. In addition, the
anti-biofilm activity of zinc oxide (ZnO) nanoparticles biosynthesized from Lactobacillus spp. extract was evaluated. The synthesized ZnO nanoparticles were characterized using FTIR, XRD, FE-SEM, and AFM. XRD analysis revealed distinct peaks indicative of a crystalline phase, while AFM and FE-SEM showed spherical nanoparticles with an average diameter of 58.30 nm. The study also assessed the ability of ZnO nanoparticles to inhibit biofilm formation. Results revealed no statistically significant association between the sample type (burn, wound, and urine) and the infecting pathogen (p=0.37).
Multiplex PCR amplification was successful in 28 isolates, with co-infections detected as follows: 57.15% of isolates showed triple infection (all three pathogens), while double infections were observed in 57.14% (E. coli and P. aeruginosa), 46.42% (E. coli and K. pneumoniae), and 42.86% (P. aeruginosa and K. pneumoniae) of isolates. Single infections were detected in 50.01% (E. coli), 28.58% (P. aeruginosa), and 17.86% (K. pneumoniae) of isolates. Sequencing analysis revealed 99% and 98% similarity with reference genes for E. coli, P. aeruginosa, and K. pneumoniae, respectively. A highly significant reduction in biofilm formation was observed after treatment with ZnO nanoparticles (p≤0.001). In conclusion, genotypic and phenotypic methods were effective for pathogen identification and ZnO nanoparticles demonstrated significant potential in inhibiting biofilm formation, offering a promising approach for combating bacterial infections.
