In situ monitoring of the shikimate pathway: a combinatorial approach of Raman reverse stable isotope probing and hyperspectral imaging

Abstract

Sensing and visualizing metabolites and metabolic pathways in situ for tracking their spatiotemporal dynamics in a non-destructive manner has significant requirement. The shikimate pathway is an important cellular mechanism which...

Sensing the Bactericidal and Bacteriostatic Antimicrobial Mode of Action Using Raman Deuterium Stable Isotope Probing (DSIP) in Escherichia coli

“…The next analysis was to examine whether the D 2 O incorporation also induces spectral changes in the biofingerprint region, which can be utilized as a metabolic marker to differentiate among different modes of antibiotic action. A weak peak observed at 989 cm –1 appears due to the deuteration of the microbial phenylalanine aromatic ring. ,, Another interpretation of origin of this band is due to the deuteration of scissoring CH 2 leading to the formation of CD 2 . As the deuterium concentration is at 40%, complete redshift of this band was not observed.…”

“…A weak peak observed at 989 cm −1 appears due to the deuteration of the microbial phenylalanine aromatic ring. 21,24,30 Another interpretation of origin of this band is due to the deuteration of scissoring CH 2 leading to the formation of CD 2 . 30 As the deuterium concentration is at 40%, complete redshift of this band was not observed.…”

“…The differences between bactericidal and bacteriostatic modes of action can be observed via the metabolic activity leading to a difference in C–D peak intensity. Raman spectroscopy is a nondestructive and noninvasive analytical technique with hidden potentials, which require further exploration by combining other methodologies such as stable isotope probing. , Recently, the Raman DSIP-based approach has been utilized to detect antimicrobial-resistant microbial species. Yang et al showed the integration of Raman DSIP with advanced multivariate analysis to study the spectral dynamics to classify sensitive, intrinsic tolerant, evolved tolerant, and resistant cells .…”

“…This also leads to the appearance of other unique biomolecular peaks. , The deuterium stable isotope probing methodology with heavy water as the probing source for the microbial study was introduced by Berry et al for identifying and sorting active microbes . The appearances of new deuterated peaks that are red-shifted arise due to the isotopic effect, i.e., the substitution of H by D leading to an increase in the reduced mass of the molecular bond owing to a decrease in wavenumber. ,, This C–H/D biomolecular substitution occurs due to the rapid intracellular biocatalytic exchange between D of D 2 O and H atom of nicotinamide adenine dinucleotide phosphate (NADPH) which is redox active. , Previously, Bernatová et al reported the use of Raman spectroscopy for investigating the action mechanism of bactericidal and bacteriostatic action based on DNA and protein signals in the Raman bio fingerprint region (600–-1800 cm –1 ) of Staphylococcus epidermis …”