Classification of Clinical Isolates of Klebsiella pneumoniae Based on Their in vitro Biofilm Forming Capabilities and Elucidation of the Biofilm Matrix Chemistry With Special Reference to the Protein Content

Abstract

Klebsiella pneumoniae is a human pathogen, capable of forming biofilms on abiotic and biotic surfaces. The limitations of the therapeutic options against Klebsiella pneumoniae is actually due to its innate capabilities to form biofilm and harboring determinants of multidrug resistance. We utilized a newer approach for classification of biofilm producing Klebsiella pneumoniae isolates and subsequently we evaluated the chemistry of its slime, more accurately its biofilm. We extracted and determined the amount of polysaccharides and proteins from representative bacterial biofilms. The spatial distribution of sugars and proteins were then investigated in the biofilm matrix using confocal laser scanning microscopy (CLSM). Thereafter, the extracted matrix components were subjected to sophisticated analysis incorporating Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, one-dimensional gel-based electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), and MALDI MS/MS analysis. Besides, the quantification of its total proteins, total sugars, uronates, total acetyl content was also done. Results suggest sugars are not the only/major constituent of its biofilms. The proteins were harvested and subjected to SDS-PAGE which revealed various common and unique protein bands. The common band was excised and analyzed by HPLC. MALDI MS/MS results of this common protein band indicated the presence of different proteins within the biofilm. The 55 different proteins were identified including both cytosolic and membrane proteins. About 22 proteins were related to protein synthesis and processing while 15 proteins were identified related to virulence. Similarly, proteins related to energy and metabolism were 8 and those related to capsule and cell wall synthesis were 4. These results will improve our understanding of Klebsiella biofilm composition and will further help us design better strategies for controlling its biofilm such as techniques focused on weakening/targeting certain portions of the slime which is the most common building block of the biofilm matrix.

Proteomic and Transcriptomic Analyses Indicate Reduced Biofilm-Forming Abilities in Cefiderocol-Resistant Klebsiella pneumoniae

“…Biofilms are aggregates of single or multiple bacterial species encased in a protective extracellular matrix, which is typically composed of secreted exopolysaccharides (EPS), environmental DNA (eDNA), proteins, surfactants, lipids and water ( Stewart, 2002 ; Artini et al, 2013 ; Tan et al, 2015 ). The process of biofilm formation includes surface attachment and movement, formation of microcolonies, maturation and eventual diffusion ( Parrino et al, 2019 ; Singh et al, 2019 ). This ability is widely distributed among microbes and contributes to a broad spectrum of defense mechanisms ( Hall-Stoodley et al, 2004 ; Piperaki et al, 2017 ).…”

Galactose-Clicked Curcumin-Mediated Reversal of Meropenem Resistance among Klebsiella pneumoniae by Targeting Its Carbapenemases and the AcrAB-TolC Efflux System

“…In the present study, 157 nonrepetitive, consecutive K. pneumoniae isolates from intensive care unit patients were collected from the routine bacteriology laboratory of the Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University Varanasi, India, from March 2019 to December 2019. Definitive identification of collected isolates was performed using established biochemical tests as described earlier [ 44 ]. After identification, the K. pneumoniae isolates were stocked in brain heart infusion broth (BHI) (HiMedia, India) with 10% glycerol (Merck, Germany) at –80 °C.…”

“…S is the degree of synergy that establishes a synergistic interaction for its positive values and an antagonistic interaction for its negative values. At various time points, growth rates were ascertained by calculating the slope of the growth curve being analysed for 4.5 h, as described previously [ 44 ].…”

Making of water soluble curcumin to potentiate conventional antimicrobials by inducing apoptosis-like phenomena among drug-resistant bacteria

“…Confocal laser scanning microscopic evaluation of the synergistic antibacterial activity of soluble curcumin and vancomycin. For confocal analysis of the synergistic effects of sCur, we grew K. pneumoniae in chambered slides as described previously with modifications 42 . Briefly, we grew MRSA overnight and then diluted it 1:500 in fresh BHI broth to adjust its absorbance to 0.2 at λ max 600 nm.…”

“…We spectrophotometrically monitored the effect of sCur on growth rates of the respective bacterial isolates both in the presence and absence of the drugs as described previously 19 , 42 . Briefly, overnight grown bacterial cells were allowed to grow in the fresh BHI broth to their early exponential phase.…”