Rapid Detection of Imipenem Resistance in Gram-Negative Bacteria Using Tabletop Scanning Electron Microscopy: A Preliminary Evaluation

Abstract

Background: Enabling faster Antimicrobial Susceptibility Testing (AST) is critical, especially to detect antibiotic resistance, to provide rapid and appropriate therapy and to improve clinical outcomes. Although several standard and automated culture-based methods are available and widely used, these techniques take between 18 and 24 h to provide robust results. Faster techniques are needed to reduce the delay between test and results.Methods: Here we present a high throughput AST method using a new generation of tabletop scanning electron microscope, to evaluate bacterial ultra-structural modifications associated with susceptibilities to imipenem as a proof of concept. A total of 71 reference and clinical strains of Gram-negative bacteria were used to evaluate susceptibility toward imipenem after 30, 60, and 90 min of incubation. The length, width and electron density of bacteria were measured and compared between imipenem susceptible and resistant strains.Results: We correlated the presence of these morphological changes to the bacterial susceptibility and their absence to the bacterial resistance (e.g., Pseudomonas aeruginosa length without [2.24 ± 0.61 μm] and with [2.50 ± 0.68 μm] imipenem after 30 min [p = 3.032E-15]; Escherichia coli width without [0.92 ± 0.07 μm] and with [1.28 ± 0.19 μm] imipenem after 60 min [p = 1.242E-103]). We validated our method by a blind test on a series of 58 clinical isolates where all strains were correctly classified as susceptible or resistant toward imipenem.Conclusion: This method could be a potential tool for rapidly identifying carbapenem-resistance in Enterobacterales in clinical microbiology laboratories in <2 h, allowing the empirical treatment of patients to be rapidly adjusted.

Antimicrobial susceptibility testing for Gram positive cocci towards vancomycin using scanning electron microscopy

“…The advantages of the tabletop SEM used in this study relies in its ease of use and user-friendly interface, the rapid acquisition of micrographs, and being an affordable tool (€ 50k) that can be installed in clinical labs. A similar strategy for clinically relevant Gram-negative bacteria against imipenem was investigated by our team, showing morphological changes after one hour of incubation ( Haddad et al., 2021b ). Previously, we also developed an SEM strategy for bacterial identification from blood cultures ( Haddad et al., 2021a ).…”

“…The samples were prepared as described in our recent publication, for AST of Gram-negative bacteria ( Haddad et al., 2021b ). A patent application related to this strategy and methodology was also deposited (WO2020/183,072 A1).…”

“…Micrographs were acquired on the TM4000 Plus tabletop SEM (Hitachi, Tokyo, Japan) at each timepoint using Zigzag software that allows for a rapid and automated acquisition. Low magnification micrographs were recorded at 10 kV accelerating voltage with a 500X magnification, whereas higher magnifications (5000X and 7000X) were recorded with an accelerating voltage of 15 kV, as previously described ( Haddad et al., 2021b ).…”

A preliminary investigation into bacterial viability using scanning electron microscopy–energy-dispersive X-ray analysis: The case of antibiotics

“…Monitoring and understanding bacterial metabolic alterations imply comparing bacteria under stress conditions induced by temperature change, disinfection, and biochemical treatments. Our choice of antibiotics to study the metabolic changes in bacteria reveals details with progressive changes at increasing concentrations ( Hanberger et al, 1991 ; Cushnie et al, 2016 ; Haddad et al, 2021b ). When the cell wall is damaged, pores are created, leading to diffusive ion movement, resulting in a decrease of K + and Mg 2+ and an increase in Na + ( Lu et al, 2017 ).…”

“…Fluorescence detection by flow cytometry or imaging has also been extensively researched and widely used for the detection and assessment of viable potential foodborne pathogens in various ecosystems ( Diaper and Edwards, 1994 ; Boulos et al, 1999 ; Adams et al, 2003 ; Delgado-Viscogliosi et al, 2005 ; Brauge et al, 2019 ). Recently, scanning electron microscopy (SEM) has been re-evaluated for applications in clinical microbiology ( Cushnie et al, 2016 ; Hannachi et al, 2020 ; Haddad et al, 2021a , b ). In addition to high-resolution images, SEM can provide information on the chemical elements present in a specimen when coupled with energy-dispersive X-ray spectroscopy (EDX).…”