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

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.

Validating the utility of heavy water (Deuterium Oxide) as a potential Raman spectroscopic probe for identification of antibiotic resistance.

The impact of microbial infections is increasing over time, and it is one of the major reasons for death in both developed and developing countries. colistin is considered as the antibiotic of last choice for infections brought by major multidrug-resistant (MDR), gram-negative bacteria such as Enterobacter species, Acinetobacter species, and Pseudomonas aeruginosa. Existing approaches to diagnose these resistant species are relatively slow and take up to 2 to 3 days. In this work, we propose a novel interdisciplinary method based on Raman spectroscopy and heavy water to identify colistin-resistant microbes. Our hypothesis is based on the fact that resistant bacteria will be metabolically active in the culture medium containing antibiotics and heavy water, and these bacteria will take up deuterium instead of hydrogen to newly synthesized lipids and proteins. This effect will generate a 'C - D' bond-specific Raman spectral marker. Successful identification of this band in the spectral profile can confirm the presence of colistin-resistant bacteria. We have validated the efficacy of this approach in identifying colistin-resistant bacteria spiked in artificial urine and have compared sensitivity at different bacterial concentrations. Overall findings suggest that heavy water can potentially serve as a suitable Raman probe for identifying metabolically active colistin-resistant bacteria via urine under clinically implementable time and can be used in clinical settings after validation.