Morphological and nanostructural surface changes in Escherichia coli over time, monitored by atomic force microscopy

Abstract

The present study aims at evaluating intrinsic changes in Escherichia coli (E. coli) surface over time, by Atomic Force Microscopy (AFM). For that purpose, bacteria were immobilized on mica or on mica previously functionalized by the deposition of a polyelectrolyte multilayer cushion. AFM images reveal that E. coli population goes through different stages. Firstly, after a week, the number of healthy bacteria decreases resulting in a release of cellular components which likely become, in turn, a nutrition source for increasing the healthy population after around two weeks. Finally, after one month, most of the bacteria is dead. Our study shows a transition of a healthy rod-shaped bacterium to a dead collapsed one. Most importantly, along with the morphological evolution of bacteria, are the structure changes and the mechanical properties of their outer membrane, emphasized by AFM phase images with very high resolution. Indeed, the surface of healthy bacteria is characterized by a phase separation pattern, thereafter mentioned as "ripples". Bacterial ageing goes along with the loss of this organized structure, turning into circular areas with irregular boundaries. These changes are likely caused by a re-organization, due to external stress, of mainly lipopolysaccharides (LPS) present in the outer membrane of E. coli.

Localized incorporation of outer membrane components in the pathogen Brucella abortus

“…Our single cell analyses using fluorescence microscopy revealed a high heterogeneity between single cells with R-LPS labeling ranging from absence to patchy and even full labeling (Figs 1A and EV1). Furthermore, in AFM analyses, the areas enriched in R-LPS correlated with a high roughness (R a ) value (Fig 2), suggesting that irregular surface structures, already reported for E. coli (Amro et al, 2000;Gammoudi et al, 2016) and S. meliloti (Greif et al, 2010), correspond to mixes of long and short LPS molecules. To our knowledge, this is the first study making a correlation between the presence of R-LPS clusters localized by fluorescence microscopy and the physical surface structure of the bacterial surface investigated by AFM.…”

Probing the threshold of membrane damage and cytotoxicity effects induced by silica nanoparticles in Escherichia coli bacteria

“…Firstly, it emphasized the particular ultrastruc ture present on healthy E.coli's surface. This "brain like" organization, here called ripples, is attributed to the conformation of LPS molecules in the OM [56]. Then, AFM observations confirmed, at the cellular level, the existence of a NPs size threshold, 50 nm < Φ c < 80 nm.…”

“…Such invaginations were reported in antibiotic (penicillin and amoxicillin) treatment of E. coli, interfering with enzymes involved in the peptidoglycan synthesis [58]. In addition, SiO 2 NPs − smaller than Φ c induce a reorganization in E. coli's OM structure from a ripples pattern to more compact and rounded aggregates, typical of weakened bacteria [56]. This transition might arise from (i) the spatial reorganization of the LPS molecules from a ripples structure to an aggregated one or (ii) from the transition in their molecular conformation from an extended to a more condensed one.…”

“…Hereafter, samples were observed, in tapping mode, in the day following their preparation to prevent any interference, like ageing of E. coli biofilm [56], with the potential damage caused by SiO 2 NPs. Different areas were systematically scanned and images shown there after are representative of the cells in a given set of colonies and of all the tested samples.…”

Analytical Protocols for Separation and Electron Microscopy of Nanoparticles Interacting with Bacterial Cells

“…A commonly used biological assay, for example, tests the antimicrobial effect of NPs by evaluating the bacterial viability after exposure to NPs. 15,16 Physical aspects of NP− bacteria interactions tend to be analyzed by high-resolution imaging techniques, such as electron, 8,9,11,12,14 atomic force, 17 or Raman 13 microscopies, which can provide direct and quantitative information about NPs and cells (such as proximity, penetration, aggregation) but typically not directly in solution environments where NPs and bacteria interact. Artifacts introduced during preparation for imaging, therefore, can obscure evidence of NP−bacteria interactions.…”