High-Throughput Antimicrobial Susceptibility Testing of Escherichia coli by Wide-Field Mid-Infrared Photothermal Imaging of Protein Synthesis

Abstract

Antimicrobial resistance poses great threats to global health and economics. Current gold-standard antimicrobial susceptibility testing (AST) requires extensive culture time (36−72 h) to determine susceptibility. There is an urgent need for rapid AST methods to slow down antimicrobial resistance. Here, we present a rapid AST method based on wide-field mid-infrared photothermal imaging of protein synthesis from 13 C-glucose in Escherichia coli. Our wide-field approach achieved metabolic imaging for hundreds of bacteria at the single-cell resolution within seconds. The perturbed microbial protein synthesis can be probed within 1 h after antibiotic treatment in E. coli cells. The susceptibility of antibiotics with various mechanisms of action has been probed through monitoring protein synthesis, which promises great potential of the proposed platform toward clinical translation.

Video-rate mid-infrared photothermal imaging by single-pulse photothermal detection per pixel

“…Because of these two reasons, developing wide-field approaches requires a camera with a large well depth capacity and a mid-IR excitation source with much higher pulse energy, such as an infrared optical parametric oscillator, to achieve high-throughput measurements. Along these efforts, a rapid antimicrobial susceptibility testing platform is reported using an infrared optical parametric oscillator and a camera with 2 million well-depth capacity ( 38 ).…”

Mid-infrared Photothermal Imaging: Instrument and Life Science Applications

“…MIP is harnessed as a novel tool for analyzing the chemical content, metabolism, and physiology at the single cell or single virus level. Examples include the protein dynamic under drug stress, , fungal cell wall analysis, and single virus analysis. , As discussed below, these studies were pursued through either a label-free manner or use of isotope labeling.…”

“…The Goodacre group evaluated the incorporation of 13 C-glucose and 15 N-ammonium chloride by a single Escherichia coli (E. coli). , As shown in Figure d, SIP MIP imaging could detect spectral changes in phenotypic rapid antimicrobial susceptibility testing (AST) . The Cheng group used SIP MIP for high throughput rapid AST by monitoring 13 C-incorporated protein synthesis in single bacterial cells.…”

“…43,72 As shown in Figure 3d, SIP MIP imaging could detect spectral changes in phenotypic rapid antimicrobial susceptibility testing (AST). 68 The Cheng group used SIP MIP for high throughput rapid AST by monitoring 13 C-incorporated protein synthesis in single bacterial cells. E. coli was selected as the testbed, and 13 C-glucose was utilized for isotopic replacement in proteins, resulting in a peak shift of the amide I band from 1656 to 1612 cm −1 compared to the original 12 C-incorporation protein amide I.…”

“…The wide-field MIP microscope employs wide-field illumination of both the IR pump and visible probe beams onto a sample. − By exploring the difference in the transmission/reflection images between the IR-on and IR-off status, the transient photothermal effects in a whole field-of-view can be captured by a camera. Wide-field MIP significantly improves MIP imaging speed up to 1250 frames per second …”