Abstract:
The ability of Klebsiella pneumoniae to form biofilm renders the pathogen recalcitrant to
various antibiotics. The difficulty in managing K. pneumoniae related chronic infections is due to its
biofilm-forming ability and associated virulence factors, necessitating the development of efficient
strategies to control virulence factors. This study aimed at evaluating the inhibitory potential of
selected phytochemical compounds on biofilm-associated virulence factors in K. pneumoniae, as
well as authenticating their antibiofilm activity. Five phytochemical compounds (alpha-terpinene,
camphene, fisetin, glycitein and phytol) were evaluated for their antibacterial and anti-biofilmassociated virulence factors such as exopolysaccharides, curli fibers, and hypermucoviscosity against
carbapenem-resistant and extended-spectrum beta-lactamase-positive K. pneumoniae strains. The
antibiofilm potential of these compounds was evaluated at initial cell attachment, microcolony
formation and mature biofilm formation, then validated by in situ visualization using scanning
electron microscopy (SEM). Exopolysaccharide surface topography was characterized using atomic
force microscopy (AFM). The antibacterial activity of the compounds confirmed fisetin as the best
anti-carbapenem-resistant K. pneumoniae, demonstrating a minimum inhibitory concentration (MIC)
value of 0.0625 mg/mL. Phytol, glycitein and α-terpinene showed MIC values of 0.125 mg/mL
for both strains. The assessment of the compounds for anti-virulence activity (exopolysaccharide
reduction) revealed an up to 65.91% reduction in phytol and camphene. Atomic force microscopy
detected marked differences between the topographies of untreated and treated (camphene and
phytol) exopolysaccharides. Curli expression was inhibited at both 0.5 and 1.0 mg/mL by phytol,
glycitein, fisetin and quercetin. The hypermucoviscosity was reduced by phytol, glycitein, and fisetin
to the shortest mucoid string (1 mm) at 1 mg/mL. Phytol showed the highest antiadhesion activity
against carbapenem-resistant and extended-spectrum beta-lactamase-positive K. pneumoniae (54.71%
and 50.05%), respectively. Scanning electron microscopy correlated the in vitro findings, with phytol
significantly altering the biofilm architecture. Phytol has antibiofilm and antivirulence potential
against the highly virulent K. pneumoniae strains, revealing it as a potential lead compound for the
management of K. pneumoniae-associated infections.
