Nanotechnology Tools for Antibacterial Materials

Loris Rizzello; Roberto Cingolani; Pier Paolo Pompa

Disclosures

Nanomedicine. 2013;8(5):807-821. 

In This Article

Effects of Randomly Organized Nanotopography on Bacterial Adhesion

Study of the relationship between surface roughness/porosity and bacterial adhesion traces back to the early 1980s, when it was mainly for dentistry and environmental applications.[35–39] Due to technological limitations, these studies were confined to examining antibacterial effects of poorly controlled random roughness. The biological investigations generally used colony-forming units assays, and direct counting under light or scanning electron microscopy to estimate the number of microorganisms attached to surfaces following incubation. Similar approaches were pursued into the 1990s, until nanotechnology was applied to study the bacteria–substrate interaction. In particular, Razatos and coworkers used the atomic force microscope (AFM) to analyze and quantify the initial events of bacterial adhesion with unprecedented resolution and precision.[40] They coated the AFM cantilever tip with a confluent layer of Escherichia coli, and then used the coated cantilevers to obtain force–distance curves describing the interaction forces between the bacteria and poly(methyl methacrylate)-functionalized surfaces (Figure 2). By combining the high sensitivity of AFM with isogenic bacterial strains differing in cell surface composition (e.g., mutants expressing progressively truncated lipopolysaccharides), the authors were able to discriminate the contribution of specific cell surface components in the adhesion forces.

Figure 2.

Atomic force microscope-based approach for probing the interaction forces between bacteria and abiotic surfaces.
Scanning electron microscope images of (A) bare silicon nitride tip and (B) silicon nitride tip coated with Escherichia coli. (C) Average force curves between silicon nitride tips coated with two different E. coli (D21 and D21f2, which differ in the length of core lipopolysaccharide molecules) and glass spin-coated with poly(methyl methacrylate). Magnification: (A) 1800× and (B) 1500×.
Reproduced with permission from [40].

In addition to providing new characterization methods, nanotechnology provided improved nanometer-resolution fabrication techniques to control the size and shape of randomly organized nanorough surfaces. These techniques included plasma etching (based on reactive-ion etching), acid-mediated etching, anodic oxidation and controlled polymer coatings.[41–43] Many of these methods have been applied to explore the influence of surface roughness on bacterial adhesion.

In this framework, Bakker et al. studied bacterial adhesion on polyurethane-coated glass plates of varying elastic moduli and surface roughness. The authors found a direct relationship between the mean surface roughness and the attachment of micron-sized marine bacteria (Marinobacter hydrocarbonoclasticus, Psychrobacter sp. SW5H and Halomonas pacifica) by using multiple linear regression analyses.[44] Nanoroughness-dependent bacterial adhesion was confirmed in later studies,[45–50] underscoring the importance of surface nanofeatures for adhesion. However, other works found the opposite trend, namely that a decrease in the topographical pattern (obtained by chemical etching) leads to an increase in the number of attached bacteria.[51,52] In addition, hydrophobic surfaces have been demonstrated to delay the adhesion of microorganisms when compared with hydrophilic substrates, which typically promote their attachment.[53–55] However, controversial results have also been reported.[56] Similar discrepancies also arose concerning the role played by surface energy on bacterial adhesion.[57]

Although many studies have tried to define a general correlation between surface nanotopography and bacterial adhesion,[58–62] a definitive explanation is still lacking. As recently suggested,[25] this is probably due to the relatively crude approaches that have often been used to characterize surface nanotopographies. Accurate physicochemical characterization of a substrate requires a combination of nanometer-resolution surface methods, such as AFM, nanoindentation and scanning electron microscopy, together with elemental analyses, for example, energy dispersive x-ray spectroscopy, time-of-flight secondary-ion mass spectrometry and x-ray photoelectron spectroscopy.[63,64] Furthermore, because of the intrinsically random organization of these surfaces, significant intra-sample and sample-to-sample variability may occur, thus requiring systematic investigations with higher statistical validity.

An additional limitation of past studies is the oversimplified description of microorganisms, which were treated as inert, perfectly smooth and geometrically regular colloidal microparticles, with surface properties and interaction dynamics estimated on purely theoretical grounds.[65] Although such assumptions were useful for examining the basic physicochemical interactions between abiotic surfaces and bacteria, they were inadequate for understanding the detailed aspects of the interaction. Additional important issues have to be taken into account. First, bacteria are rapidly evolving living systems that vary the protein composition of their cell envelope over time depending on environmental and physiological conditions,[66] secrete proteins by specific secretory pathways,[67] produce extracellular polymeric substance,[68] and extend external organelles such as flagella and adhesive fimbriae.[69] Second, microorganisms have heterogeneous shapes and sizes. For example, the Streptococcus species (S. pneumoniae, S. pyogenes and S. oralis) and Staphilococcus spcies (S. aureus, S. epidermidis and S. haemolyticus) have a spherical shape, but other microorganisms possess a bacillus (µ-rod) profile (e.g., Bacillus subtilis or Bacillus cereus), or even a more complex spiral shape (Spirochetes) (Figure 3). Third, the bacterial growth conditions, such as incubation time, characteristics of the growth medium (e.g., ionic strength), temperature and physical agitation during incubation, also affect the interaction at the interface. For these reasons, simplistic topological and physicochemical models for the bacteria and surfaces are inadequate to reliably predict the relationships between nanotopography and bacterial adhesion.

Figure 3.

The complex sizes, shapes and surface features characterizing microorganisms.
(A) The spherical shape of Staphylococcus epidermidis. (B) The rod-like structure of E. coli. (C) The spiral shape of leptospires. (D) The polysaccharide capsule of S. pneumoniae avoids bacterial clearance performed by host cells, since the bacterial antigens are hidden to both immunoglobulin proteins and phagocytic cell lines, such as macrophages. (E) The adhesive organelles' type 1 fimbriae allow bacteria to adhere to both host tissues and abiotic surfaces, thus starting the colonization processes, and biofilm formation and development.
E. coli: Escherichia coli; S. pneumoniae: Streptococcus pneumoniae.
(A) Image courtesy of the National Institute of Allergy and Infectious Diseases.
(C) Reproduced with permission from [201].
(D) Image courtesy of the CDC.
(E) Image courtesy of the Texas Department of State Health Services (TX, USA).

Another important issue in this research field is the biological characterization of the interaction events, since most experimental approaches are based on the observation of morphological changes (assessed by scanning electron microscopy, transmission electron microscopy and confocal/fluorescence microscopy), or on counting and viability assays. However, these approaches generally do not take into account the detailed molecular events accompanying the bacteria–substrate interactions. In this framework, we recently uncovered specific changes in the genomic and proteomic profile of E. coli adhering onto flat (control) and nanorough surfaces, which were produced by a spontaneous Galvanic displacement reaction[70–73] (Figure 4). We found that E. coli adhered to nanostructured surfaces undergo genetic phase variation of fimbrial subunit expression (the fimbrial operon was switched 'off', due to the overexpression of the LrhA repressor and upregulation of the FimE recombinase), as well as general stress conditions (suggested by the activation of the two-component system CpxP/R pathway). Furthermore, by proteomic analyses, we detected 15 proteins (involved in biosynthesis, peptide transport, metabolic pathways and DNA repair) that were differently regulated in bacteria growing on nanorough versus smooth surfaces.

Figure 4.

Molecular response of Escherichia coli adhering onto flat and nanorough gold surfaces.
Bacteria were grown on (A) flat and (B) nanorough gold substrates. The nanostructured surfaces led Escherichia coli cells to repress the expression of the adhesive organelles' type 1 fimbriae. (C) Expression levels of fimbrial gene subunits in the treated bacteria (growing onto nanorough surfaces) compared with control bacteria (growing on flat surfaces). (D–F) Global protein expression profile by means of 2D-difference gel electrophoresis proteomic assays. (D) Fluorescent staining of a 2D gel and (E) PC analyses for analyzing the statistical populations of up- and down-regulated proteins. (F) The electrophoresis proteomic assay shows a trend for down- (green) and up-regulation (red) of proteins for flat and nanorough samples.
PC: Principal component.
Reproduced with permission from [70].

In summary, despite the large body of literature on the topic, the question of whether antibacterial substrates should be designed with nanostructured or flat features remains unresolved, and will require more systematic investigations. Future efforts should be directed toward fine fabrication methods, high-resolution physicochemical characterization of surfaces, and deeper biochemical and molecular biology investigations of bacteria–surface interactions.

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