Optical photothermal infrared spectroscopy: A novel solution for rapid identification of antimicrobial resistance at the single-cell level via deuterium isotope labeling

The rise and extensive spread of antimicrobial resistance (AMR) has become a growing concern, and a threat to the environment and human health globally. The majority of current AMR identification methods used in clinical setting are based on traditional microbiology culture-dependent techniques which are time-consuming or expensive to be implemented, thus appropriate antibiotic stewardship is provided retrospectively which means the first line of treatment is to hope that a broad-spectrum antibiotic works. Hence, culture-independent and single-cell technologies are needed to allow for rapid detection and identification of antimicrobial-resistant bacteria and to support a more targeted and effective antibiotic therapy preventing further development and spread of AMR. In this study, for the first time, a non-destructive phenotyping method of optical photothermal infrared (O-PTIR) spectroscopy, coupled with deuterium isotope probing (DIP) and multivariate statistical analysis was employed as a metabolic fingerprinting approach to detect AMR in Uropathogenic Escherichia coli (UPEC) at both single-cell and population levels. Principal component-discriminant function analysis (PC-DFA) of FT-IR and O-PTIR spectral data showed clear clustering patterns as a result of distinctive spectral shifts (C–D signature peaks) originating from deuterium incorporation into bacterial cells, allowing for rapid detection and classification of sensitive and resistant isolates at the single-cell level. Furthermore, the single-frequency images obtained using the C–D signature peak at 2,163 cm⁻¹ clearly displayed the reduced ability of the trimethoprim-sensitive strain for incorporating deuterium when exposed to this antibiotic, compared to the untreated condition. Hence, the results of this study indicated that O-PTIR can be employed as an efficient tool for the rapid detection of AMR at the single-cell level.