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

Abstract

The metabolic stages of bacterial development and viability under different stress conditions induced by disinfection, chemical treatments, temperature, or atmospheric changes have been thoroughly investigated. Here, we aim to evaluate early metabolic modifications in bacteria following induced stress, resulting in alterations to bacterial metabolism. A protocol was optimized for bacterial preparation using energy-dispersive X-ray (EDX) microanalysis coupled with scanning electron microscopy (SEM), followed by optimizing EDX data acquisition and analysis. We investigated different preparation methods aiming to detect modifications in the bacterial chemical composition at different states. We first investigated Escherichia coli, acquiring data from fresh bacteria, after heat shock, and after contact with 70% ethanol, in order to prove the feasibility of this new strategy. We then applied the new method to different bacterial species following 1 h of incubation with increasing doses of antibiotics used as a stress-inducing agent. Among the different materials tested aiming to avoiding interaction with bacterial metabolites, phosphorous-doped silicon wafers were selected for the slide preparation. The 15 kV acceleration voltage ensured all the chemical elements of interest were excited. A thick layer of bacterial culture was deposited on the silicon wafer providing information from multiple cells and intra-cellular composition. The EDX spectra of fresh, heat-killed, and alcohol-killed E. coli revealed important modifications in magnesium, potassium, and sodium. Those same alterations were detected when applying this strategy to bacteria exposed to antibiotics. Tests based on SEM-EDX acquisition systems would provide early predictions of the bacterial viability state in different conditions, yielding earlier results than culture.

Keywords: EDX; SEM; bacterial viability; chemical composition; energy dispersive X-ray; metabolic state; scanning electron microscopy.

Figure 1

(A) Processed EDX spectra of fresh and heat-killed Escherichia coli (Q5586). Each curve represents the average of three spectra taken from the same bacterial deposition. Spectra are offset by 3. (B) Evolution of the K/C, Mg/C, and Na/C ratios between the fresh and the heat-killed E. coli.

Figure 2

(A) Processed EDX spectra of E. coli (Q5586) at the fresh state and after exposure to 70% ethanol. Each curve represents the average of three spectra taken from the same bacterial deposition. Spectra are offset by 3. (B) Evolution of the K/C, Mg/C, and Na/C ratios between the fresh and the alcohol-killed E. coli.

Figure 3

EDX spectra and Mg/C ratio of Gram-negative bacilli incubated with imipenem. EDX spectra of (A, D, G) susceptible (Q5586, Q5580 and P9549) and (B, E, H) resistant (P1872, Q2247, and P9548) strains of E. coli, Klebsiella pneumoniae and Enterobacter cloacae, respectively. Each curve represents the average of three spectra taken from the same bacterial deposit. Spectra are offset by 5 (C, F, I) Mg/C ratio of tested strains calculated from the respective EDX spectra. Error bars: standard deviations. Blue and red triangles: MIC of tested strains. ^*p < 0.01; ^**p < 0.001; ^**p < 0.0001; (ns): not significant.

Figure 4

Comparison of Mg/C ratio, K/C ratio, and Na/C ratio of a Klebsiella pneumoniae susceptible strain incubated with various concentrations of imipenem. K/C ratio (A) and Na/C ratio (B) as a function of Mg/C ratio. Numbers in the figure represent the imipenem concentration. Dashed lines are linear fitting for all plots.