Biosynthesized Chitosan Nanoparticles Mediated by Saccharomyces cerevisiae: A Comparative Study on the Gene Expression of ESBL-Encoding and Virulence-Associated Genes in Klebsiella pneumonia

Abstract

     Hypervirulent Klebsiella pneumoniae (HvKp) causes numerous severe diseases worldwide because it has many virulence factors encoded by numerous genes located on a large, important plasmid called the pLVPK plasmid. The present study aims to define the Saccharomyces cerevisiae aqueous extract (ScE) and its biosynthesized chitosan-coated nanoparticles' (ScNPs) impact on the expression of resistance genes (blaTEM, blaCTX-M, blaSHV) and virulence genes (iucA, rmpA, Prg344) in HvKp. Thirty bacterial isolates obtained from various clinical sources in hospitals in Baghdad, Iraq. The isolates were morphologically identified, and the identifications were confirmed using the Vitek-2 compact system. The 30 isolates were screened for extended spectrum beta-lactamase enzymes (ESBL) production using double disk synergy testing (DDST) and virulence tests, including the string test, siderophore production assay, and tellurite resistance assay, and verified the presence of resistance and virulence genes by polymerase chain reaction technique (PCR). Chitosan nanoparticles mediated by S. cerevisiae (ScNPs) were synthesized and characterized using UV-visible absorption spectroscopy (UV-VIS), scanning electron microscopy (SEM) analysis, atomic force microscopy (AFM), and X-ray diffractometer (XRD) techniques. The ScE and ScNPs were used to determine antibacterial activity using the 96-well microtiter plate method (MTP) and to study the effects on resistance and virulence genes in HvKp. The result revealed that all 30 bacterial isolates were confirmed as K. pneumoniae, and only five isolates displayed highly virulent and resistant abilities, classifying them as hypervirulent K. pneumoniae isolates. These isolates were selected for further experiments in this study. The results of the ScNPs syntheses revealed the presence of chitosan-Tripolyphosphate (TPP) as a crosslinking agent, and the process was visually confirmed by color changes and alterations in solubility. The characterization results showed new absorbance peaks in UV-Vis compared to the crude extract, and crystalline patterns detected by XRD, SEM, and AFM revealed the formation of nanoscale particles ranging from 26 to 48 nm, with discrete aggregates exhibiting both two and three-dimensional features and rough surfaces. The ScNPs exhibited substantially greater activity than the crude aqueous extract, with minimum inhibitory concentration (MIC) and sub-MIC values of 10.737 μg/ml and 5.368 μg/ml, respectively, compared to 125,000 μg/ml (MIC) and 62,500 μg/ml (sub-MIC) for ScE. Furthermore, the results showed that ScNPs and ScE have a significant impact on reducing gene expression below the 1.0 ± 0.0 ratio across five isolates. In conclusion, the ScNPs exhibit direct antibacterial activity and effectively down regulate resistance and virulence gene expression. Hence, it subsequently reduces the pathogenicity caused by HvKp.