Phenotypic Profiling of Antibiotic Response Signatures in Escherichia coli Using Raman Spectroscopy

Abstract

bIdentifying the mechanism of action of new potential antibiotics is a necessary but time-consuming and costly process. Phenotypic profiling has been utilized effectively to facilitate the discovery of the mechanism of action and molecular targets of uncharacterized drugs. In this research, Raman spectroscopy was used to profile the phenotypic response of Escherichia coli to applied antibiotics. The use of Raman spectroscopy is advantageous because it is noninvasive, label free, and prone to automation, and its results can be obtained in real time. In this research, E. coli cultures were subjected to three times the MICs of 15 different antibiotics (representing five functional antibiotic classes) with known mechanisms of action for 30 min before being analyzed by Raman spectroscopy (using a 532-nm excitation wavelength). The resulting Raman spectra contained sufficient biochemical information to distinguish between profiles induced by individual antibiotics belonging to the same class. The collected spectral data were used to build a discriminant analysis model that identified the effects of unknown antibiotic compounds on the phenotype of E. coli cultures. Chemometric analysis showed the ability of Raman spectroscopy to predict the functional class of an unknown antibiotic and to identify individual antibiotics that elicit similar phenotypic responses. Results of this research demonstrate the power of Raman spectroscopy as a cellular phenotypic profiling methodology and its potential impact on antibiotic drug development research.

Near-Real-Time Analysis of the Phenotypic Responses of Escherichia coli to 1-Butanol Exposure Using Raman Spectroscopy

“…Measurements were carried out using laser excitation of 532 nm (20 mW) for 25 s with spectral resolution of 9 to 15 cm Ϫ1 . A similar method has been described elsewhere (47). A minimum of 50 individual spectra was acquired per sample prior to data analysis.…”

Characterizing the Phenotypic Responses of Escherichia coli to Multiple 4-Carbon Alcohols with Raman Spectroscopy

“…Raman spectroscopy was performed as published previously [22,25], except that the analysis required 50 µL of washed cells. In short, cells were dried at room temperature on an aluminum surface and analyzed using a Bruker Senterra dispersive Raman spectrometer, which was attached to a confocal microscope (100ˆmagnification) and equipped with OPUS software (Bruker Optics, Billerica, MA, USA).…”

“…PCA was then used to reduce the dataset for DA. DA was then performed using the first 10 principal components, as described previously [25], in order to separate and cluster samples based on the different phenotypic responses to 4-carbon alcohol exposure.…”

“…The major advantages of the approach are that (i) results are returned in near real-time; (ii) it is non-destructive to the sample; and (iii) minimal sample preparation is required (chemical label-free). Raman spectroscopy has also been applied in other biological research for purposes of (among many others) (i) species characterization [23]; (ii) studying cell differentiation [24]; (iii) identifying the mechanism of action of antimicrobial compounds [25], and fermentation monitoring [26][27][28].…”

Predictive modeling inClostridium acetobutylicumfermentations employing Raman spectroscopy and multivariate data analysis for real-time culture monitoring

“…Several articles on use of Raman spectroscopy with cells are presented in the literature. For example, cell phenotypes of E. coli cultures have been studied via Raman spectroscopy, following exposure to 1-butanol 20 , different 4-carbon alcohols 21 , and antibiotics 22 . In a more recent publication by Freedman et al, Raman spectroscopy was used to differentiate between phenotypes of E. coli cultures enriched for 1-butanol tolerance 23 .…”