Rapid screening of bacteriostatic and bactericidal antimicrobial agents against Escherichia coli by combining machine learning (artificial intelligence) and UV-VIS spectroscopy.

Antibiotics are compounds that have a particular mode of action upon the microorganism they are targeting. However, discovering and developing new antibiotics is a challenging and timely process. Antibiotic development process can take up to 10-15 years and over $1billion to develop a single new therapeutic product. Rapid screening tools to understand the mode of action of the new antimicrobial agent are considered one of the main bottle necks in the antimicrobial agent development process. Classical approaches require multifarious microbiological methods and they do not capture important biochemical and organism therapeutic-interaction mechanisms. This work aims to provide a rapid antibiotic-antimicrobial biochemical diagnostic tool to reduce the timeframes of therapeutic development, while also generating new biochemical insight into an antimicrobial-therapeutic screening assay in a complex matrix. The work evaluates the effect of antimicrobial action through "traditional" microbiological analysis techniques with a high-throughput rapid analysis method using UV-VIS spectroscopy and chemometrics. Bacteriostatic activity from tetracycline and bactericidal activity from amoxicillin were evaluated on a system using non-resistant Escherichia coli O157:H7 by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and UV-VIS spectroscopy (high-throughput analysis). The data were analysed using principal component analysis (PCA) and support vector machine (SVM) classification. The rapid diagnostic technique could easily identify differences between bacteriostatic and bactericidal mechanisms and was considerably quicker than the "traditional" methods tested.

Bacterial attachment on sub-nanometrically smooth titanium substrata.

Despite the volume of work that has been conducted on the topic, the role of surface topography in mediating bacterial cell adhesion is not well understood. The primary reason for this lack of understanding is the relatively limited extent of topographical characterisation employed in many studies. In the present study, the topographies of three sub-nanometrically smooth titanium (Ti) surfaces were comprehensively characterised, using nine individual parameters that together describe the height, shape and distribution of their surface features. This topographical analysis was then correlated with the adhesion behaviour of the pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa, in an effort to understand the role played by each aspect of surface architecture in influencing bacterial attachment. While P. aeruginosa was largely unable to adhere to any of the three sub-nanometrically smooth Ti surfaces, the extent of S. aureus cell attachment was found to be greater on surfaces with higher average, RMS and maximum roughness and higher surface areas. The cells also attached in greater numbers to surfaces that had shorter autocorrelation lengths and skewness values that approached zero, indicating a preference for less ordered surfaces with peak heights and valley depths evenly distributed around the mean plane. Across the sub-nanometrically smooth range of surfaces tested, it was shown that S. aureus more easily attached to surfaces with larger features that were evenly distributed between peaks and valleys, with higher levels of randomness. This study demonstrated that the traditionally employed amplitudinal roughness parameters are not the only determinants of bacterial adhesion, and that spatial parameters can also be used to predict the extent of attachment.

Air-directed attachment of coccoid bacteria to the surface of superhydrophobic lotus-like titanium.

Superhydrophobic titanium surfaces fabricated by femtosecond laser ablation to mimic the structure of lotus leaves were assessed for their ability to retain coccoid bacteria. Staphylococcus aureus CIP 65.8T, S. aureus ATCC 25923, S. epidermidis ATCC 14990T and Planococcus maritimus KMM 3738 were retained by the surface, to varying degrees. However, each strain was found to preferentially attach to the crevices located between the microscale surface features. The upper regions of the microscale features remained essentially cell-free. It was hypothesised that air entrapped by the topographical features inhibited contact between the cells and the titanium substratum. Synchrotron SAXS revealed that even after immersion for 50 min, nano-sized air bubbles covered 45% of the titanium surface. After 1 h the number of cells of S. aureus CIP 65.8T attached to the lotus-like titanium increased to 1.27×10(5) mm(-2), coinciding with the replacement of trapped air by the incubation medium.

Nature inspired structured surfaces for biomedical applications.

Nature has created an array of superhydrophobic surfaces that possess water-repellent, self-cleaning and anti-icing properties. These surfaces have a number of potential applications in the biomedical industry, as they have the potential to control protein adsorption and cell adhesion. Natural superhydrophobic surfaces are typically composed of materials with a low intrinsic surface free-energy (e.g the cuticular waxes of lotus leaves and insect wings) with a hierarchical structural configuration. This hierarchical surface topography acts to decrease the contact area of water droplets in contact with the surface, thereby increasing the extent of the air/water interface, resulting in water contact angles greater than 150º. In order to employ these surfaces in biotechnological applications, fabrication techniques must be developed so that these multi-scale surface roughness characteristics can be reproduced. Additionally, these fabrication techniques must also be able to be applied to the material required for the intended application. An overview of some of the superhydrophobic surfaces that exist in nature is presented, together with an explanation of the theories of their wettability. Also included is a description of some of the biomedical applications of superhydrophobic surfaces and fabrication techniques that can be used to mimic superhydrophobic surfaces found in nature.

Bacterial attachment on optical fibre surfaces.

Optical fibres have received considerable attention as high-density sensor arrays suitable for both in vitro and in vivo measurements of biomolecules and biological processes in living organisms and/or nano-environments. The fibre surface was chemically modified by exposure to a selective etchant that preferentially erodes the fibre cores relative to the surrounding cladding material, thus producing a regular pattern of cylindrical wells of approximately 2.5 mum in diameter and 2.5 mum deep. The surface hydrophobicity of the etched and non-etched optical fibres was analysed using the sessile pico-drop method. The surface topography was characterised by atomic force microscopy (AFM), while the surface chemistry was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Six taxonomically different bacterial strains showed a consistent preference for attachment to the nano-scale smoother (R(q) = 273 nm), non-etched fibre surfaces (water contact angle, theta = 106 degrees +/- 4 degrees). In comparison, the surfaces of the etched optical fibres (water contact angle, theta = 96 degrees +/- 10 degrees) were not found to be amenable to bacterial attachment. Bacterial attachment on the non-etched optical fibre substrata varied among different strains.

Differences in colonisation of five marine bacteria on two types of glass surfaces.

The retention patterns of five taxonomically different marine bacteria after attachment on two types of glass surfaces, as-received and chemically etched, have been investigated. Contact angle measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), X-ray fluorescence spectroscopy (XRF) and X-ray photoelectron spectrometry (XPS) were employed to investigate the impact of nanometer scale surface roughness on bacterial attachment. Chemical modification of glass surfaces resulted in a approximately 1 nm decrease in the average surface roughness (R(a)) and the root-mean-squared roughness (R(q)) and in a approximately 8 nm decrease in the surface height and the peak-to-peak (R(max)) and the 10-point average roughness (R(z)). The study revealed amplified bacterial attachment on the chemically etched, nano-smoother glass surfaces. This was a consistent response, notwithstanding the taxonomic affiliation of the selected bacteria. Enhanced bacterial attachment was accompanied by elevated levels of secreted extracellular polymeric substances (EPS). An expected correlation between cell surface wettability and the density of the bacterial attachment on both types of glass surfaces was also reported, while no correlation could be established between cell surface charge and the bacterial retention pattern.