Engineered water systems (EWS, premise plumbing systems, cooling towers, air conditioning systems, etc.) are rich sources of biofilms and free-living amoebae that promote proliferation and survival of the opportunistic premise plumbing bacterial pathogens, such as L. pneumophila. L. pneumophila persistence in EWS poses a risk of legionellosis, a type of pneumonia that can often be fatal to immunocompromised or elderly individuals. Despite its global occurrence, many fundamental questions regarding L. pneumophila transportation and persistence in EWS remain unanswered.

This study describes the development of a novel, deoxyribonucleic acid (DNA)-loaded, biocompatible, and biodegradable surrogate with similar size, shape, surface charge, and hydrophobicity to L. pneumophila in order to mimic their mobility and persistence in EWS. This is the first study to utilise biopolymer surrogates with ‘customised’ surface properties, size, and shape to model L. pneumophila mobility and persistence in a simulated plumbing system.

In order to construct the surrogate, cell surface charge, hydrophobicity, and morphology of L. pneumophila were characterised in lag, exponential, and stationary growth phases. The effect of growth-dependent variations in L. pneumophila on their attachment ability was investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D) instrument. L. pneumophila cell surfaces were most hydrophobic in the stationary phase. Increased hydrophobicity of stationary phase L. pneumophila assisted the bacteria to form more rigid bonds with stainless steel surfaces, compared to lag and exponential phase cells in QCM-D attachment studies.

The surrogates were constructed using alginate and CaCO3 adapting a simple, one-pot co- precipitation technique and surface modified with amino acids to match the surface charge and hydrophobicity of stationary phase L. pneumophila. KH3 tracer DNA was loaded onto surrogates to enable their detection and quantification in simulated EWS studies using qPCR. Surrogate characterisation using scanning electron microscopy, nitrogen adsorption-desorption kinetics, qNano, and Fourier-transform infrared spectroscopy revealed that they consisted of mesoporous calcite nanostructures with high surface area self-assembled to form uniform, rod- shape structures of ~1.3 µm in length and 1 µm in width. The high surface area and porous structure of surrogates permitted efficient DNA loading.

Surrogate’s ability to mimic L. pneumophila was evaluated in A. polyphaga co-culture and biofilm attachment/detachment studies. The results of these studies indicated that surrogates successfully mimicked L. pneumophila mobility and persistence in a simulated plumbing system at 30 ºC. Both L. pneumophila and surrogates showed similar trends in A. polyphaga engulfment and biofilm attachment/detachment studies. With further validations, the surrogate developed in this study may be used as a new tool to predict L. pneumophila risk in regulated water systems.

Biopolymer surrogates developed in this study offer many advantages as opposed to the traditional E. coli faecal indicator surrogates for L. pneumophila. Biopolymer surrogates are more representative of L. pneumophila accounting for their ‘customised’ surface charge, hydrophobicity, size, and shape. The surrogate activity can be easily detected and quantified using qPCR. In addition, surrogates can be applied in a wide array of eco-sensitive aquatic environments owing to their biocompatible and biodegradable nature. The non-hazardous nature of these surrogates will facilitate pathogen studies in any non-physical containment-2 laboratory, without the requirement of specialised training or equipment. Therefore, the work described in this thesis provides new insights not only into the mobility and persistence of L. pneumophila in EWS but also into the fabrication of safe, eco-friendly, easily detectable, and more representative surrogates for pathogen mimics.