Bacterial and Fungal Cell Behaviour on Surfaces with Defined Topography

The fabrication of surface topographies on materials for antibacterial activity are mostly relatable to the naturally occurring surface features found on animal and plant surfaces such as the shark skin [1] or the lotus leaves [2]. Shark skin type topography includes a combination of 4, 8, 12 and 16 μm long vertical rod shaped lines with a width of 2 μm which was used to prevent Staphylococcus aureus biofilm formation as well as reduced the settlement of Ulva larvae zoospores on surfaces subjected to prolonged exposure to marine environment [3][1]. Lotus leaf topography on the other hand promoted bacterial colonisation only on the super-hydrophobic micro scaled interfacial air trapped structural crevices, thereby preventing bacterial growth directly onto the nano patterned surfaces [4]. Few other examples of nature inspired topography to prevent bacterial growth includes silica nanopollens, where the nanostructure is fully capable of hanging on to the bacterial surface to deliver enzymes that can cause bacterial membrane disruption and ultimately cell death [5]. The other example is the nanopillar pattern present on the Cicada (or tree bugs) wing surface, that are naturally evolved to prevent bacterial growth [6]. Dragonfly wings also have similar but finer surface patterns when compared with Cicada wings, comprising of hexagonal arrays of nanopillars spanning throughout the wing structure and these Dragonfly wing patterns can be fabricated on to titanium implants for exhibiting antibacterial surface activity [7]. These bioinspired patterns have often interested scientists to look for specific features existing naturally or design patterns for possible surface activity on cells.

The mechanism of interaction of these nanostructures with cells are of utmost interest in terms of exhibiting anti-bacterial/fungal surface activity and one of the possible mechanism is brought about by membrane disruption. The introduction of shear stress on the bacterial (Pseudomonas aeruginosa) membrane by a nanopillar protrusion (70 nm in width) into a much thinner (~10 nm in width) bacterial membrane disrupts the membrane [8]. However in surfaces where the roughness is of a magnitude much smaller than the bacteria itself, a study reported an increased bacterial growth and the bacteria grown on these surfaces had bigger size with an increased amount of exopolysaccharide (EPS) synthesis [9][10]. Surface roughness can be interestingly manipulated using highly flexible or fluid-like nanostructures capable of bending and swaying along with the weight of bacteria, as a result the bacteria fails to anchor onto its surface and avoiding overall bacterial adhesion [11][12][13]. Bacterial shape itself can be utilised to design surfaces that can prevent its colonisation on surfaces. A recent study took into account the aggregative behaviour of bacteria adhering on to surfaces and used a monolayer of polystyrene microsphere measuring 1500 nm in diameter to create a wall in between individual rod shaped bacterium (Pseudomonas aeruginosa) thereby preventing cell-cell communication and hence prevented biofilm formation on surfaces [14].  However in the case of a coccoid or an irregularly shaped bacterium, the monolayer arrangement of polystyrene colloid crystals might not favour biofilm prevention, which makes it important to study the effect of an elaborate range of topography in order to understand the relationship of surface topography with microbial cells. A study on a similar note showed that bacterial cells (Escherichia coli, Staphylococcus epidermidis and Bacillus subtilis) even with unambiguous morphology and structural attributes, preferred to survive within the cavities or ridges in between patterns rather than occupy elevated areas of nano-scaled fabricated surfaces with distinct topographies [15].  Another study tried to elucidate the adhesion preference of bacterial cells using a variety of different topographies on silica and alumina substrates and concluded that while alumina substrates exhibited non-specific adhesion preference, the silica substrates on the other hand induced substrate adhesion in the following order: E.coli ATCC 25922 > L.innocua _~ P.fluorescens > E.coli O157:H7 [16]. Diaz et al. studied the behaviour of Pseudomonas fluorescens on engineered metal surfaces that had a trench shaped topography and reported that bacterial confinement within engineered structures brings about different morphological changes in the bacteria which could be exploited to limit bacterial proliferation [17][18]. She studied Pseudomonas fluorescens organisation on topographically patterned surfaces and compared the arrangement of Pseudomonas fluorescens after modifying the material chemistry in the topography and found out that surface topography itself plays an integral role in determining the arrangement of bacterial cells over surfaces [19]. Although from literature, there are studies that utilise both surface topography and surface functionalisation as a means to activate surfaces for biological activity, it is often ambiguous that whether the achieved surface activity is because of surface topography itself or due to additional surface chemistry employed using wet chemistry or plasma polymerisation methods [20], [21].

Nanofabricated surfaces with increased tip sharpness within their topography resulted in disintegrating the bacterial membrane altogether, like in the case of highly protrusive black silicon surfaces that has applications in developing biomedical devices [22]. In another study by the same group bacterial adhesion on to black silicon surfaces seemed to vary with the topographical geometry of black silicon surfaces and the authors found out that the nano-pillars with height below 300 nm, 60 nm tip diameter and 600 nm pitch proved to be the most biocidal [23]. To prove the importance of structural geometry and topography towards microbial response, a study conducted with silicon nanowire arrays with heights of 3 μm, 300 nm in diameter and 10 – 15 μm in spacing that showed positive interaction with Shewanella oneidensis and Escherichia coli, with their swimming motility confined to mostly around the nanowire posts these bacteria flourished on this type of topography [24]. Hence, it will be correct to reason that the organisation of each individual units in a well-defined surface topography determines the type of microbial response elicited by a particular topography. A recent study utilised a vertically aligned micron scaled pillar arrays, made with polyurethane based resin using soft scale lithographic technique to modulate bacterial signalling within a co-culture of Pseudomonas aeruginosa and Escherichia coli biofilms [25]. Micro-topography of poly dimethylsiloxane (PDMS) as square shaped and hexagonal shaped 10 μm high patterns that has side lengths of 50 and 15 μm, respectively and inter-pattern distances of 15 μm for the squared ones and 2 μm for the hexagonal ones, showed an enhanced rate of conjugation [26]. Promoting bacterial conjugation by using surface topography brings about the possibility of improving the delivery efficacy of genetic elements into bacterial cells which gives the ability to create/delete mutations in the bacterial genome. There are very few literature explaining the activity of surface topography on fungal cells, however a study on fungal cells explains the “mechano-sensitive” sensors present on the surface of the cell membrane that can sense surface irregularities to bypass obstacles, this phenomenon is termed as thigmotropism [27]. Candida albicans uses another way of sensing a change in external pressure, that is by detecting a change within the closely balanced cytoskeletal force inwards and a turgor force outwards which helps keeps the cell membrane intact [27]. A review on the hyphal growth of Candida albicans elaborates on the signal transduction mechanisms including the formation of complex pathways under stressful environment [28].

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