Aspergillus oryzae attenuates quorum sensing -associated virulence factors and biofilm formation in Klebsiella pneumoniae extended-spectrum beta-lactamases

Abstract

Background: Klebsiella pneumoniae communicate between and among species using quorum sensing (QS). Biofilm formation and virulence factors are regulated by QS. This QS is indirectly responsible for K. pneumoniae pathogenicity. Inhibiting QS is a novel and highly effective method for controlling K. pneumoniae extended-spectrum beta-lactamases (KP-ESBL) infections. This study aimed to investigate how Aspergillus oryzae extracellular protein (AOEP) affected QS and KP-ESBL virulence factors. Methods: Methods used included minimal inhibitory concentration (MIC) through the microdilution method, biofilms with crystal violet staining, extracellular polysaccharides using the Congo Red assay, quantifying the expression of genes coding for capsular polysaccharide (wzI gene) and adhesion (mrkA gene) through quantitative reverse-transcription polymerase chain reaction (RT-qPCR), siderophore level measurement using Chrome Azurol sulphonate assay (CAS assay), biofilm morphology using a scanning electron microscope (SEM), and confirmation using the life span killing assay method on Caenorhabditis elegans (C. elegans). Results: In vitro studies revealed that AOEP inhibited biofilms and exopolysaccharides (EPS) in KP-ESBL at the sub-MIC level. In addition, AOEP inhibited the expression of the mrkA gene, which is involved in the adhesion process. Furthermore, an in vivo study revealed that AOEP levels of 75 and 150 µg/mL respectively increased C. elegans survival rates by 72.67% and 80.76% against K. pneumoniae infection. Conclusions: Our findings suggest that the extracellular protein of A. oryzae may be an effective QS inhibitor and a novel anti-virulence agent to control bacterial pathogens.

Secondary metabolite of Aspergillus oryzae impairs cell envelope integrity Klebsiella pneumoniae producing extended-spectrum beta-lactamase

“…In a 250 ml Erlenmeyer flask containing 2% glucose, 100 ml of potato dextrose broth media was inoculated with 8 mm (diameter) of fungal mycelium (Nacef et al, 2020;Rachma et al, 2022). Under static condition, the flask was incubated in a shaker incubator (Boxun BSD-TX270) at 27°C for 72 hours (OD 600 = 1.2).…”

“…Murein layer-(lpp-) deficient bacteria exhibit increased OM permeability, periplasmic component leakage, and increased outer membrane vesicle (OMV) release (Nevermann et al, 2019;Schwechheimer et al, 2015). Bacteria lacking pal exhibit decreased outer membrane integrity, increased susceptibility to antibiotics and detergents, increased periplasmic protein leakage, and increased OMV release (Graham et al, 2021;Schwechheimer et al, 2013). The collapse of the cell envelope integrity will affect the structures dependent on it, such as capsular polysaccharides (CPSs), fimbriae type 1, and siderophores.…”