Sensitive Detection of Deliquescent Bacterial Capsules through Nanomechanical Analysis.

Encapsulated bacteria usually exhibit strong resistance to a wide range of sterilization methods, and are often virulent. Early detection of encapsulation can be crucial in microbial pathology. This work demonstrates a fast and sensitive method for the detection of encapsulated bacterial cells. Nanoindentation force measurements were used to confirm the presence of deliquescent bacterial capsules surrounding bacterial cells. Force/distance approach curves contained characteristic linear-nonlinear-linear domains, indicating cocompression of the capsular layer and cell, indentation of the capsule, and compression of the cell alone. This is a sensitive method for the detection and verification of the encapsulation status of bacterial cells. Given that this method was successful in detecting the nanomechanical properties of two different layers of cell material, i.e. distinguishing between the capsule and the remainder of the cell, further development may potentially lead to the ability to analyze even thinner cellular layers, e.g. lipid bilayers.

Ecophysiological diversity of a novel member of the genus Alteromonas, and description of Alteromonas mediterranea sp. nov.

Nine non-pigmented, motile, Gram-negative bacteria originally designated as Alteromonas macleodii deep-sea ecotypes, were isolated from seawater samples collected from four separate locations; two deep-sea sites in the Mediterranean Sea and surface water of the Aegean Sea and English Channel. The six strains studied in vitro were found to tolerate up to 20 % NaCl. The DNA-DNA relatedness between the deep-sea ecotype strains was found to be between 75 and 89 %, whilst relatedness with the validly named Alteromonas species was found to be between 31 and 69 %. The average nucleotide identity (ANI) amongst the deep-sea ecotype strains was found to be 98-100 %; the in silico genome-to-genome distance (GGD), 85-100 %; the average amino acid identity (AAI) of all conserved protein-coding genes, 95-100 %; and the strains possessed 30-32 of the Karlin's genomic signature dissimilarity. The ANI between the deep-sea ecotype strains and A. macleodii ATCC 27126(T) and Alteromonas australica H 17(T) was found to be 80.6 and 74.6 %, respectively. A significant correlation was observed between the phenotypic data obtained in vitro and data retrieved in silico from whole genome sequences. The results of a phylogenetic study that incorporated a 16S rRNA gene sequence analysis, multilocus phylogenetic analysis (MLPA) and genomic analysis, together with the physiological, biochemical and chemotaxonomic data, clearly indicated that the group of deep-sea ecotype strains represents a distinct species within the genus Alteromonas. Based on these data, a new species, Alteromonas mediterranea, is proposed. The type strain is DE(T) ( = CIP 110805(T) = LMG 28347(T) = DSM 17117(T)).

Bacterial patterning at the three-phase line of contact with microtextured alkanes.

Aliphatic crystallites, characteristic of the eicosane and docosane components of naturally occurring lipids, were found to form microtextures that were structured by specific interactions with ordered graphite (HOPG) used as the underlying substratum, as confirmed by scanning electron microscopy (SEM) and fast Fourier transform (FFT) analysis. Confocal scanning laser microscopy (CLSM) showed highly directed bacterial alignment for two bacterial species (spherical and rod-shaped), reflecting the preferential orientation of the crystallite-air-water interfaces to give linear and triangular bacterial patterning. The mechanisms of bacterial attachment are demonstrated in terms of the balance between effective radial adhesional forces and the capillary forces resulting from the water contact angle of the bacteria at the three-phase line (TPL) of the lipid surface. It is suggested that these microtextured surfaces, which exhibit the ability to limit bacterial adhesion to a precise patterning at the lipid TPL, could be used as a means of controlling bacterial colonization.

Natural insect and plant micro-/nanostructsured surfaces: an excellent selection of valuable templates with superhydrophobic and self-cleaning properties.

Insects and plants are two types of organisms that are widely separated on the evolutionary tree; for example, plants are mostly phototrophic organisms whilst insects are heterotrophic organisms. In order to cope with environmental stresses, their surfaces have developed cuticular layers that consist of highly sophisticated structures. These structures serve a number of purposes, and impart useful properties to these surfaces. These two groups of organisms are the only ones identified thus far that possess truly superhydrophobic and self-cleaning properties. These properties result from their micro- and nano-scale structures, comprised of three-dimensional wax formations. This review analyzes the surface topologies and surface chemistry of insects and plants in order to identify the features common to both organisms, with particular reference to their superhydrophobic and self-cleaning properties. This information will be valuable when determining the potential application of these surfaces in the design and manufacture of superhydrophobic and self-cleaning devices, including those that can be used in the manufacture of biomedical implants.

Subtle Variations in Surface Properties of Black Silicon Surfaces Influence the Degree of Bactericidal Efficiency.

One of the major challenges faced by the biomedical industry is the development of robust synthetic surfaces that can resist bacterial colonization. Much inspiration has been drawn recently from naturally occurring mechano-bactericidal surfaces such as the wings of cicada (Psaltoda claripennis) and dragonfly (Diplacodes bipunctata) species in fabricating their synthetic analogs. However, the bactericidal activity of nanostructured surfaces is observed in a particular range of parameters reflecting the geometry of nanostructures and surface wettability. Here, several of the nanometer-scale characteristics of black silicon (bSi) surfaces including the density and height of the nanopillars that have the potential to influence the bactericidal efficiency of these nanostructured surfaces have been investigated. The results provide important evidence that minor variations in the nanoarchitecture of substrata can substantially alter their performance as bactericidal surfaces.

Bactericidal activity of self-assembled palmitic and stearic fatty acid crystals on highly ordered pyrolytic graphite.

The wings of insects such as cicadas and dragonflies have been found to possess nanostructure arrays that are assembled from fatty acids. These arrays can physically interact with the bacterial cell membranes, leading to the death of the cell. Such mechanobactericidal surfaces are of significant interest, as they can kill bacteria without the need for antibacterial chemicals. Here, we report on the bactericidal effect of two of the main lipid components of the insect wing epicuticle, palmitic (C16) and stearic (C18) fatty acids. Films of these fatty acids were re-crystallised on the surface of highly ordered pyrolytic graphite. It appeared that the presence of two additional CH2 groups in the alkyl chain resulted in the formation of different surface structures. Scanning electron microscopy and atomic force microscopy showed that the palmitic acid microcrystallites were more asymmetric than those of the stearic acid, where the palmitic acid microcrystallites were observed to be an angular abutment in the scanning electron micrographs. The principal differences between the two types of long-chain saturated fatty acid crystallites were the larger density of peaks in the upper contact plane of the palmitic acid crystallites, as well as their greater proportion of asymmetrical shapes, in comparison to that of the stearic acid film. These two parameters might contribute to higher bactericidal activity on surfaces derived from palmitic acid. Both the palmitic and stearic acid crystallite surfaces displayed activity against Gram-negative, rod-shaped Pseudomonas aeruginosa and Gram-positive, spherical Staphylococcus aureus cells. These microcrystallite interfaces might be a useful tool in the fabrication of effective bactericidal nanocoatings. STATEMENT OF SIGNIFICANCE: Nanostructured cicada and dragonfly wing surfaces have been discovered to be able physically kill bacterial cells. Here, we report on the successful fabrication of bactericidal three-dimensional structures of two main lipid components of the epicuticle of insect wings, palmitic (C16) and stearic (C18) acids. After crystallisation onto highly ordered pyrolytic graphite, both the palmitic and stearic acid films displayed bactericidal activity against both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus cells. The simplicity of the production of these microcrystallite interfaces suggests that a fabrication technique, based on solution deposition, could be an effective technique for the application of bactericidal nanocoatings.

The nature of inherent bactericidal activity: insights from the nanotopology of three species of dragonfly.

While insect wings are widely recognised as multi-functional, recent work showed that this extends to extensive bactericidal activity brought about by cell deformation and lysis on the wing nanotopology. We now quantitatively show that subtle changes to this topography result in substantial changes in bactericidal activity that are able to span an order of magnitude. Notably, the chemical composition of the lipid nanopillars was seen by XPS and synchrotron FTIR microspectroscopy to be similar across these activity differences. Modelling the interaction between bacterial cells and the wing surface lipids of 3 species of dragonflies, that inhabit similar environments, but with distinctly different behavioural repertoires, provided the relationship between surface structure and antibacterial functionality. In doing so, these principal behavioural patterns correlated with the demands for antimicrobial efficiency dictated by differences in their foraging strategies. This work now reveals a new feature in the design elegance of natural multi-functional surfaces as well providing insights into the bactericidal mechanism underlying inherently antimicrobial materials, while suggesting that nanotopology is related to the evolutionary development of a species through the demands of its behavioural repertoire. The underlying relationship between the processes of wetting, adhesion and capillarity of the lipid nanopillars and bactericidal efficiency suggests new prospects for purely mechano-responsive antibacterial surfaces.

The Bioeffects Resulting from Prokaryotic Cells and Yeast Being Exposed to an 18 GHz Electromagnetic Field.

The mechanisms by which various biological effects are triggered by exposure to an electromagnetic field are not fully understood and have been the subject of debate. Here, the effects of exposing typical representatives of the major microbial taxa to an 18 GHz microwave electromagnetic field (EMF)were studied. It appeared that the EMF exposure induced cell permeabilisation in all of the bacteria and yeast studied, while the cells remained viable (94% throughout the exposure), independent of the differences in cell membrane fatty acid and phospholipid composition. The resulting cell permeabilisation was confirmed by detection of the uptake of propidium iodine and 23 nm fluorescent silica nanospheres using transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Upon EMF exposure, the bacterial cell membranes are believed to become permeable through quasi-endocytosis processes. The dosimetry analysis revealed that the EMF threshold level required to induce the uptake of the large (46 nm) nanopsheres was between three and six EMF doses, with a specific absorption rate (SAR) of 3 kW/kg and 5 kW/kg per exposure, respectively, depending on the bacterial taxa being studied. It is suggested that the taxonomic affiliation and lipid composition (e.g. the presence of phosphatidyl-glycerol and/or pentadecanoic fatty acid) may affect the extent of uptake of the large nanospheres (46 nm). Multiple 18 GHz EMF exposures over a one-hour period induced periodic anomalous increases in the cell growth behavior of two Staphylococcus aureus strains, namely ATCC 25923 and CIP 65.8T.

Dual role of outer epicuticular lipids in determining the wettability of dragonfly wings.

Numerous natural surfaces possess superhydrophobicity and self-cleaning properties that would be extremely beneficial when applied in industry. Dragonfly wings are one example of such surfaces, and while their general surface structure is known, their precise chemical composition is not. Here, the epicuticular lipids of dragonfly wing membranes were characterized to investigate their significance in contributing to self-cleaning and superhydrophobic properties. After just 10s of lipid extraction using chloroform, the water contact angles exhibited by the wings decreased below the accepted threshold for superhydrophobicity (150°). Infrared spectra collected at the Australian Synchrotron contained characteristic absorption bands of amide, ester and aliphatic hydrocarbons moieties on the wing surfaces, the latter of which was decreased post-extraction with chloroform. GC-MS data analysis revealed that the epicuticular wax components were dominated by n-alkanes with even-numbered carbons, especially n-hexacosane, and palmitic acid. SEM and AFM data analysis conducted on the untreated and chloroform-extracted wing surfaces demonstrated that surface topography changed after extraction; the surface nanostructure was progressively lost with extended extraction times. The data presented here indicate that epicuticular lipids contribute not only to self-cleaning and superhydrophobic properties through their inherent hydrophobic nature, but also by forming the physical structure of the wing surface. This knowledge will be extremely valuable for reconstruction of dragonfly wing structures as a biomimetic template.

Molecular organization of the nanoscale surface structures of the dragonfly Hemianax papuensis wing epicuticle.

The molecular organization of the epicuticle (the outermost layer) of insect wings is vital in the formation of the nanoscale surface patterns that are responsible for bestowing remarkable functional properties. Using a combination of spectroscopic and chromatographic techniques, including Synchrotron-sourced Fourier-transform infrared microspectroscopy (FTIR), x-ray photoelectron spectroscopy (XPS) depth profiling and gas chromatography-mass spectrometry (GCMS), we have identified the chemical components that constitute the nanoscale structures on the surface of the wings of the dragonfly, Hemianax papuensis. The major components were identified to be fatty acids, predominantly hexadecanoic acid and octadecanoic acid, and n-alkanes with even numbered carbon chains ranging from C14 to C30. The data obtained from XPS depth profiling, in conjunction with that obtained from GCMS analyses, enabled the location of particular classes of compounds to different regions within the epicuticle. Hexadecanoic acid was found to be a major component of the outer region of the epicuticle, which forms the surface nanostructures, and was also detected in deeper layers along with octadecanoic acid. Aliphatic compounds were detected throughout the epicuticle, and these appeared to form a third discrete layer that was separate from both the inner and outer epicuticles, which has never previously been reported.