Correlative ex situ and Liquid-Cell TEM Observation of Bacterial Cell Membrane Damage Induced by Rough Surface Topology

Abstract

Background: Nanoscale surface roughness has been suggested to have antibacterial and antifouling properties. Several existing models have attempted to explain the antibacterial mechanism of nanoscale rough surfaces without direct observation. Here, conventional and liquid-cell TEM are implemented to observe nanoscale bacteria/surface roughness interaction. The visualization of such interactions enables the inference of possible antibacterial mechanisms.

Methods and results: Nanotextures are synthesized on biocompatible polymer microparticles (MPs) via plasma etching. Both conventional and liquid-phase transmission electron microscopy observations suggest that these MPs may cause cell lysis via bacterial binding to a single protrusion of the nanotexture. The bacterium/protrusion interaction locally compromises the cell wall, thus causing bacterial death. This study suggests that local mechanical damage and leakage of the cytosol kill the bacteria first, with subsequent degradation of the cell envelope.

Conclusion: Nanoscale surface roughness may act via a penetrative bactericidal mechanism. This insight suggests that future research may focus on optimizing bacterial binding to individual nanoscale projections in addition to stretching bacteria between nanopillars. Further, antibacterial nanotextures may find use in novel applications employing particles in addition to nanotextures on fibers or films.

Keywords: antibacterial microparticles; antibacterial nanopatterns; antibacterial surface topology; graphene liquid cell; liquid TEM.

Figure 1

Schematic of plasma etching of PLGA MPs (A-C), SEM imaging of the control PLGA MPs and the plasma-etched PLGA MPs (D-G) and viability graph of E. coli control and in the presence of antibacterial agents (H). In (A) oxygen flows in low concentration (200 ppm volume) through a charged environment to create free radicals (B) which etch the surface, leaving behind rough MPs (C). In (D) unetched particles show smooth surface morphology. In (E) the PLGA MPs show mild dimpling of the surface after 1 min of plasma etching. In (F) the PLGA MPs etched for 2 mins show more pronounced, sharp patterns as opposed to the PLGA MPs shown in (D) and (E). Finally, in (G) 5 mins of etching severely damaged the MPs, reducing the PLGA to primarily amorphous PLGA aggregations. The bactericidal efficacy of PLGA MPs and AgNPs are compared in (H) where control and bacteria treated with 3 μg/mL AgNPs do not show decreased CFU/mL, while the 100 μg/mL AgNPs samples show decreased viability. The error bars in (H) represent the standard error. The unetched PLGA does not exhibit a statistically significant bactericidal effect, nor does the PLGA etched for 5 mins. However, the PLGA etched for 2 mins, which features rough MP surfaces, does exhibit a statistically significant bactericidal effect. The scale bar is 1 µm in (D), and 500 nm in (E), (F), and (G).

Figure 2

Bacterial death is induced by rough MP topography as observed in a cross-sectional image obtained via conventional TEM. In (A) and (B) low to high magnifications of surface interaction between an E. coli and a rough PLGA particle etched for 2 mins is shown. A sharp peak on the PLGA particle appears to have penetrated the E. coli bacterium cell envelope. The area bracketed in (A) is shown at higher magnification in (B). The scale bars in (A) and (B) are 200nm.

Figure 3

GLC encapsulation of E. coli and PLGA MPs shows localized damage to the cell envelope of the bacterium. In (A), a lower-magnification image shows an overview of the E. coli and PLGA MP. In (B), a higher magnification shows the PLGA MP and bacterium in close proximity. The cell wall proximal to the PLGA MP shows damage with a shape similar to the adjacent MP. A high contrast liquid indicates the cytosol of the bacterium, which clearly identifies the death of the bacterium. The cytosol has a distinct contrast from the PBS medium due to the proteins, glycans, and other bacterial components within it. Other areas of the E. coli cell wall are smooth and show no damage or degradation. The scale bars in (A) and (B) are 200 nm.

Figure 4

Schematic illustration of bacterial death mechanism. The bacterium is shown in blue on the left side of the image (i), whereas the cell envelope is indicated as a dark blue layer on the outer edge of the bacterium (ii). The PLGA particle is shown in grey on the right-hand side (iii). In (A) and (B) the particle has come into contact and deforms the cell envelope, before breaking it in (C) and( D). This damage then causes degradation of the cell envelope in (E) and (F), before the cell wall disintegrates in (G) and (H).