Atomic force microscopy: application to investigation of Escherichia coli morphology before and after exposure to cefodizime

Abstract

Atomic force microscopy (AFM) is a recently developed technique that allows for the investigation of the surface morphology of a biological specimen at an unprecedented level of resolution. The aim of the present study was to explore some of the new opportunities offered by AFM by studying the morphological and surface alterations induced in Escherichia coli by supra-MICs and sub-MICs of a beta-lactam antibiotic (cefodizime). The underlying principle of AFM is the scanning and sensing of the topography of a sample by means of near-field microscopy that makes it possible to obtain simultaneous digital measurements of the x, y, and z coordinates of any point on the bacterial surface with great resolution (x and y, approximately 20 A; z, approximately 1 A). Unlike scanning electron microscopy, performance of AFM does not require a vacuum, drying to the critical point, or the coating of the bacterial surface with a metal layer. The digital storage of the information makes it easy to rotate the image, observe the bacterial surface and induced structural alterations from different points of view, and obtain a cross-section at any desired point with precise, automatic measurement of the heights and sizes of normal versus damaged bacteria. Use of the new and outstanding technique of AFM will make it possible for researchers to investigate biological samples immersed in biological fluids and will also make it possible for them to study the morphological alterations of living bacteria exposed to antibiotics as they are taking place.

FIG. 1

Schematic diagram of an atomic force microscope.

FIG. 2-4

AFM images of E. coli under different conditions. FIG. 2. Common morphology of E. coli without exposure to cefodizime. Bars, 500 nm. FIG. 3 and 4. Example of bacterial damage after exposure to supra-MICs of cefodizime. Bars, 500 nm.

FIG. 5-7

AFM images of E. coli under different conditions. FIG. 5. Example of bacterial flattening and emptying after exposure to supra-MICs of cefodizime. Bars, 500 nm. FIG. 6. E. coli filamentation following exposure to sub-MICs of cefodizime. Bars, 500 nm. FIG. 7. Filamentation and bulge formation in E. coli after exposure to sub-MICs of cefodizime. Bars, 500 nm.

FIG. 8

Example of height and size measurements of selected cross-section in a damaged bacterium (f, fimbria).