Atomic Force Microscopy Investigation of the Morphology and Topography of Colistin-Heteroresistant Acinetobacter baumannii Strains as a Function of Growth Phase and in Response to Colistin Treatment

Abstract

The prevalence of infections caused by multidrug-resistant gram-negative Acinetobacter baumannii strains and the lack of novel antibiotics under development are posing a global dilemma, forcing a resurgence of the last-line antibiotic colistin. Our aim was to use atomic force microscopy (AFM) to investigate the morphology and topography of paired colistin-susceptible and -resistant cells from colistin-heteroresistant A. baumannii strains as a function of bacterial growth phase and colistin exposure. An optimal AFM bacterial sample preparation protocol was established and applied to examine three paired strains. Images revealed rod-shaped colistin-susceptible cells (1.65 ± 0.27 μm by 0.98 ± 0.07 μm) at mid-logarithmic phase, in contrast to spherical colistin-resistant cells (1.03 ± 0.09 μm); the latter were also more diverse in appearance and exhibited a rougher surface topography (7.05 ± 1.3 nm versus 11.4 ± 2.5 nm for susceptible versus resistant, respectively). Cellular elongation up to ∼18 μm at stationary phase was more commonly observed in susceptible strains, although these “worm-like” cells were also observed occasionally in the resistant population. The effects of colistin exposure on the cell surface of colistin-susceptible and -resistant cells were found to be similar; topographical changes were minor in response to 0.5 μg/ml colistin; however, at 4 μg/ml colistin, a significant degree of surface disruption was detected. At 32 μg/ml colistin, cellular clumping and surface smoothening were evident. Our study has demonstrated for the first time substantial morphological and topographical differences between colistin-susceptible and -resistant cells from heteroresistant A. baumannii strains. These results contribute to an understanding of colistin action and resistance in regard to this problematic pathogen.