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

Abstract

The mode of action of antibiotics can be broadly classified as bacteriostatic and bactericidal. The bacteriostatic mode leads to the arrested growth of the cells, while the bacteriocidal mode causes cell death. In this work, we report the applicability of deuterium stable isotope probing (DSIP) in combination with Raman spectroscopy (Raman DSIP) for discriminating the mode of action of antibiotics at the community level. Escherichia coli, a well-known model microbe, was used as an organism for the study. We optimized the concentration of deuterium oxide required for metabolic activity monitoring without compromising the microbial growth. Our findings suggest that changes in the intensity of the C-D band in the high-wavenumber region could serve as a quantifiable marker for determining the antibiotic mode of action. This can be used for early identification of the antibiotic's mode of action. Our results explore the new perspective that supports the utility of deuterium-based vibrational tags in the field of clinical spectroscopy. Understanding the antibiotic's mode of action on bacterial cells in a short and objective manner can significantly enhance the clinical management abilities of infectious diseases and may also help in personalized antimicrobial therapy.

Figure 1

Identifying optimal deuterium concentration on E. coli cells. (A) Growth profile at different concentrations of D₂O in culture medium with incubation time points. (B) Average Raman spectra of E. coli cells cultured in different D₂O abundance. (C) Plot showing relation between ratiometric CD/CD+CH intensity percentage with D₂O abundance in the culture medium (shaded area shows the standard deviation).

Figure 2

Discrmination of Raman spectra of deuterated E. coli (biofingerprint region) cells growing in different D₂O abundance using principal component analysis (PCA); (A) score plot between principal component 1 and principal component 2; (B) scree plot showing variance contributions by first 10 PCs; (C) loading profile of PC1 and PC2 in the biofingerprint region.

Figure 3

Growth profile of E. coli in culture medium with antibiotic treatment at 6 h; (A) bacteriostatic antibiotic activity (BS; chloramphenicol and tetracycline); (B) bactericidal antibiotics activity (BC; norfloxacin and ciprofloxacin). [* – Treatment time point].

Figure 4

Raman deuterium stable isotope probing for differentiating the antimicrobial mode of action; qualitative analysis using Raman spectra of microbial cells in Raman silent reigion at different incubation time points. (A) Control, (B) chloramphenicol treated, (C) tetracycline treated, (D) norfloxacin treated, (E) ciprofloxacin treated; quasi-quantitative analysis using relative ratiometric intensity (CD/CD+CH) with incubation time; (F) control cells versus bacteriostatic effected cells; (G) control versus bactericidal effected cells (shaded area shows the standard deviation).

Figure 5

Deuterated band at 989 cm^–1 in biofingerprint region as possible qualitative spectral marker band for differentiating the antimicrobial mode of action; qualitative analysis using average Raman spectra of microbial cells at biofingerprint region at different incubation time points. (A) Control (B) chloramphenicol treated, (C) tetracycline treated, (D) norfloxacin treated, (E) ciprofloxacin treated (shaded area shows the standard deviation).