HEPES-Stabilized Encapsulation of Salmonella typhimurium

Abstract

Most bacteria, planktonic and sessile, are encapsulated inside loosely bound extracellular polymeric substance (EPS) in their physiological environment. Imaging a bacterium with its capsule requires lengthy sample preparation to enhance the capsular contrast. In this study, Salmonella typhimurium was investigated using atomic force microscopy for a practical means of imaging an encapsulated bacterium in air. The investigation further aimed to determine the relation between the buffers used for preparing the bacterium and the preservation of the capsular material surrounding it. It was observed that rinsing bacteria with HEPES buffer could stabilize and promote capsule formation, while rinsing with PBS, Tris, or glycine removes most of the capsular EPS. For bacteria rinsed with HEPES and air-dried, the height images showed only the contour of the capsular material, while the phase and amplitude images presented the detailed structures of the bacterial surface, including the flagella encapsulated inside the capsular EPS. The encapsulation was attributed to the cross-linking of the acidic exopolysaccharides mediated by the piperazine moiety of HEPES through electrostatic attraction. This explanation is supported by encapsulated bacteria observed for samples rinsed with N,N'-bis(2-hydroxyethyl)-piperazine solution and by the presence of entrapped HEPES within the dry capsular EPS suggested by micro-Raman spectroscopy.

Are the Soft, Liquid-Like Structures Detected around Bacteria by Ambient Dynamic Atomic Force Microscopy Capsules?

“…This kind of substance, imaged by ambient AFM, especially when it appears around single bacteria (as in our center-intermediate zones), is virtually identical to what has been associated with bacterial capsules in the some other investigations (30,31). When detected in larger quantities (as in our peripheral areas), they have been attributed to EPS produced by the bacteria (25).…”

“…and/or stability, experiments with nonbiological particles (Al 2 O 3 powder) were performed. As the suspending liquid we used KPi buffer, since it produced the liquid-like substances around our S. epidermidis cells, and a HEPES solution, as this buffer has been shown to result in the formation of similar liquid-like substances in other work (31). With KPi as the suspending liquid, both isolated and aggregated alumina particles were found at the center of the spot, with no evidence of any kind of liquid-like substances in this area (Fig.…”

“…Bacterial extracellular polysaccharides often form very thin, hydrated, and soft capsules around bacteria, which are considered virulence factors, contributing to their attachment and evasion of host defenses (31) and protecting them from desiccation. For example, pneumonia-causing Streptococcus pneumococcal bacteria have noncapsulated and capsulated strains.…”

“…Indeed, AFM has proved capable of scanning the surface of liquids, and this is being currently explored in areas such as the study of wetting properties at ultrasmall scales (19). In this sense, several works have recently reported the observation of capsules using ambient AFM (25,27,30,31) in the form of tiny amounts of a liquidlike substance around bacteria. Theoretical models are being currently developed for fluid microbial capsules (3).…”

Use of Atomic Force Microscopy and Transmission Electron Microscopy for Correlative Studies of Bacterial Capsules

“…This result indicates that the presence of HEPES buffer, even in very small concentrations (2 mM in our case), significantly affects the mechanical properties of the capsules. The higher phase contrast observed for bacterial capsules exposed to HEPES buffer is consistent with AFM phase imaging of Salmonella enterica serovar Typhimurium, for which it was observed that rinsing the bacteria with HEPES buffer could stabilize and promote capsule formation (35). It is likely that molecules from the HEPES buffer are trapped in the capsular matrix and may not be completely removed during rinsing with MilliQ water.…”