SUMMARY
In this study, the Lactobacillus reuteri bacterium was isolated from probiotic powder (containing different Lactobacillus strains and Saccharomyces), in addition to producing of extracellular extract and intracellular extract from this isolate. The isolate was cultured on Man Rogosa Sharpe (MRS) agar after growth and then identified by microscopic examination, biochemical tests and molecular assay. Biologically, the extracellular extract and intracellular extract from Lactobacillus reuteri JCM1112 were used to novel synthesize silver nanoparticles to the best of our knowledge, this is the first. Two methods were used to prepare silver nanoparticles, the first method was to use the extracellular extract by adding 1 g of silver nitrate to 10 ml of the extracellular extract from Lactobacillus reuteri to synthesize the nanoparticles, and the second method was to use the intracellular extract of the same bacterium by adding 1 g of silver nitrate to 10 mL of intracellular extract for nanoparticle synthesis.
Pseudomonas aeruginosa bacteria were isolated and identified from 100 clinical samples were collected from October 2021to February 2022, from patients in Erbil hospitals including Rozhawa Hospital and Emergency Hospital. Among these samples, 19 (19%) P.aeruginosa were diagnosed depending on the morphological features on culture media, biochemical tests,Vitek-2 compact while six (6) isolates of P. aeruginosa were confirmed by molecular assay. The concentration of DNA ranges from 18.9 to 21.7 ng/ μl.
In addition to comparing results with reference strain ATCC 50126. The results showed that 19 tested isolates appeared Gram negative P. aeruginosa bacteria, red rods, non-spore forming, non-capsulated cell, motile, positive β hemolysis. positive oxidase and catalase tests.
Antibiotic susceptibility testing revealed multidrug resistance patterns in both P. aeruginosa isolates.
The extracellular and Intracellular extraction of Lactobacillus reuteri were done by using culture fermentation broth media , ultrasonic techniques and using to biosynthesis of Extracellular and Intracellular Lactobacillus reuteri / silver nanoparticles were characterized by different techniques (UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), Zeta potential, Scanning Electron Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX), Transmission Electron Microscopy (TEM) and X-ray Diffraction (XRD) were exhibited near-spherical morphology with an average size of 86.54 nm, slightly rough surface and -30.16 mV and electrophoretic mobility of -2.36 cm²/Vs and stabilizing biomolecules that prevent nanoparticle aggregation in solution. The XRD analysis revealed a crystallinity of 76.89% with an average crystal size of 8.35 nm and an interplanar distance of 0.233 nm. Molecular docking revealed strong interactions between an optimized Ag3 cluster and key amino acid residues (Arg277, Gln279, Trp280, Asp273, and Val276) in P. aeruginosa protein. These interactions, with binding energies ranging from -1.89 to -3.68 kcal/ mol, suggested a potential mechanism for the observed antibacterial activity.
All biofilm-forming isolates were tested to determine the Minimum Inhibitory Concentration (MIC) of commercial nanoparticles (NPs) and biosynthesized NPs, by Resazurin MTP method the range of MICs was from 1-1024 μg/ml. The results showed that MICs of extracellular synthesis, intracellular synthesis and commercial NPs against P. aeruginosa isolates were 16, 64 and 32 μg /ml respectively.
The effect of MIC for AgNPs and commercial AgNPs against biofilm-forming- isolates was used to evaluate the antibacterial and antibiofilm activity tests. The results of antibiofilm activity by using MTP assay showed that both NPs can prevent biofilm formation in all 15 isolates (100%).
The effectiveness of silver nanoparticles as anti-virulence agents was tested against Pseudomonas aeruginosa producing virulence factors such as catalase, hemolysin, and by using the sub minimal inhibitory concentrations for both types of silver nanoparticles, the results showed the loss or decrease of the ability of these isolates to produce these factors when treated with nanoparticles, and the results of light microscopy and the scanning electron microscope for those treated isolates showed the morphological changes occur, as the cells appeared in irregular shapes and the presence of abnormalities in the cell membrane was noticed and the cell contents were released to the outside. The results showed that both types of nanoparticles were devoid of any cytotoxicity on human red blood cells through the absence of any hemolysis of red blood cells and by using the concentration of 100% of the nanoparticles.
Antimicrobial activity of silver nanoparticles was evaluated for their combined effects with antibiotics against five (5) isolates of Pseudomonas aeruginosa. The antibacterial activities increased in the presence of biologically synthesized silver nanoparticles.