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

Abstract

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.

Keywords: Uropathogenic Escherichia coli; antimicrobial resistance; infrared spectroscopy; microbiology; single-cell; stable-isotope probing.

Figure 1

PC-DFA scores plots of pre-processed FT-IR spectral data (4,000–2,000 cm⁻¹) of the UPEC isolates grown in LB broth containing 80% D₂O, with (10 mg/l TMP) and without TMP at 4 different time points of 1 h (A), 2 h (B), 3 h (C) and 4 h (D). TMP-susceptible isolates are shown in blue, while TMP-resistant isolates are shown in red. The black outline represents the TMP-treated (T) condition, while those without it represent the untreated (U) condition (control group). UPEC isolates investigated in this study are represented by different numbers and symbols. Blue arrows indicate the trend detected for the treated TMP-susceptible isolates.

Figure 2

FT-IR spectra of susceptible UPEC isolates investigated in this study. The green arrows highlight the fatty acids’ CH₂ (at 2,925 cm⁻¹ and 2,854 cm⁻¹) and C–D stretching vibrations (at 2,471 cm⁻¹ and 2,418 cm⁻¹) (A), ‘DF1 loadings plot of FT-IR spectral data at 3h timepoint illustrating the most significant vibrational peaks (green strips) contributing to the discrimination patterns (B). All spectra were normalized using extended multiplicative signal correction (EMSC). Each spectrum represents the average of four technical replicates. Different colored lines and numbers represent the different isolates, dotted lines represent treated groups, while untreated groups are represented as normal lines.

Figure 3

Single-cell O-PTIR spectral data of UPEC isolates at 3h timepoint (A). Red arrows show the C–D stretching vibrational peaks at 2,159 cm⁻¹ and 2,195 cm⁻¹. Different colored lines and numbers represent the different isolates and are also indicated in the figure. The letters (T) and (U) indicate TMP-treated and untreated conditions. 3D PCA scores plot of O-PTIR spectral data at 3 h timepoint (B). The blue colored stars represent susceptible isolates and the red colored diamonds represent resistant isolates. The black outline represents TMP-treated groups, while those without it represent untreated groups. Different symbols and numbers represent the different UPEC isolates in this study, the letters (T) and (U) indicate TMP-treated and untreated conditions. The percent total explained variance (TEV) is shown on the PC axes.

Figure 4

Box whisker plot of O-PTIR spectral data indicating the peak intensity ratio of the selected C–D stretching vibration (2,159 cm⁻¹) to amide I (1,654 cm⁻¹) at the 4 h timepoint. Blue and red box colors represent TMP-treated (T) and untreated (U) groups of bacterial cells, respectively. The numbers in the figure represent the UPEC isolates. (R) and (S) characterize the resistant and susceptible strains, respectively.

Figure 5

Optical images of untreated (A), and TMP-treated (D) E. coli cells (isolate 147, TMP-sensitive). The corresponding single-frequency O-PTIR images were obtained using the amide I vibration at 1,655 cm⁻¹ for the untreated (B), and TMP-treated (E) cells, and the C–D vibration at 2,163 cm⁻¹ for the untreated (C), and TMP-treated (F) cells.