Measuring Drug-Induced Changes in Metabolite Populations of Live Bacteria: Real Time Analysis by Raman Spectroscopy

Abstract

Raman difference spectroscopy is shown to provide a wealth of molecular detail on changes within bacterial cells caused by infusion of antibiotics or hydrogen peroxide. Escherichia coli strains paired with chloramphenicol, dihydrofolate reductase propargyl-based inhibitors, meropenem, or hydrogen peroxide provide details of the depletion of protein and nucleic acid populations in real time. Additionally, other reproducible Raman features appear and are attributed to changes in cell metabolite populations. An initial candidate for one of the metabolites involves population increases of citrate, an intermediate within the tricarboxyclic acid cycle. This is supported by the observation that a strain of E. coli without the ability to synthesize citrate, gltA, lacks an intense feature in the Raman difference spectrum that has been ascribed to citrate. The methodology for obtaining the Raman data involves infusing the drug into live cells, then washing, freezing, and finally lyophilizing the cells. The freeze-dried cells are then examined under a Raman microscope. The difference spectra [cells treated with drug] - [cells without treatment] are time-dependent and can yield population kinetics for intracellular species in vivo. There is a strong resemblance between the Raman difference spectra of E. coli cells treated with meropenem and those treated with hydrogen peroxide.

Antimicrobial Resistance Studies Using Raman Spectroscopy on Clinically Relevant Bacterial Strains

“…), respectively. This is well in agreement with the mechanism of action of ciprofloxacin, which binds to DNA gyrase, producing ciprofloxacin–gyrase DNA ternary complexes. ,,,,− These complexes can inhibit DNA replication and RNA transcription, causing massive DNA fragmentation and preventing cell growth, leading to bacterial cell death. Fluoroquinolones increase reactive oxygen spp.…”

Biofilm Eradication Using Biogenic Silver Nanoparticles

“…Changes in cellular composition can be monitored using changes in Raman band profiles, which can be associated with the morphological changes of microbial cells, providing an image of the antimicrobial effect [24]. Recent reports have shown that Raman difference spectroscopy is able to provide molecular details on changes within E. coli cells caused by antibiotics, hydrogen peroxide [20,25] or graphene oxide [19]. In this work, the interaction of PchNPs with E. coli caused a decrease in the intensity of most Raman bands, while other remain the same ( Table 1).…”

Raman spectroscopy and multivariate analysis as potential tool to follow Alzheimer’s disease progression

“…Raman spectroscopy and its derivatives, coupled with multivariate analysis, have been applied mainly to cancer diagnosis [ 12 – 19 ] on a range of sample formats, including fixed cells and tissues, as well as non-invasive biofluid measurements [ 20 ] has been used also in diagnosis and study of other diseases due to viral or bacterial infections [ 21 ], even to study biofluid profile after COVID-19 infection [ 22 ], until it has been used in neurodegenerative disease diagnosis [ 23 ]. Raman spectroscopy has been also applied to study stem cells [ 24 ], cell lines in vitro [ 25 ] and metabolomics [ 26 ].…”