Despite numerous legionellosis outbreaks attributed to contaminated engineered water systems (EWSs), no suitable surrogate has been established for studying Legionella pneumophila in the EWSs. In this study, we developed a novel biopolymer surrogate by using DNA-encapsulated, amino acid-modified alginate−calcium carbonate microparticles. The surrogate mimicked L. pneumophila's size, rod shape, surface charge, and hydrophobicity. Preliminary proof-of-concept validations were conducted with the surrogate and L. pneumophila in laboratory flow-through biofilm bioreactor experiments with and without chlorine. The 2 entities displayed similar attachment/detachment magnitudes and kinetics to/from Pseudomonas f luorescens biofilms grown on stainless steel piping material. Their relative concentrations and peak attachment values were within the same orders of magnitude, and both exhibited slower attachment than detachment processes. Compared with the experiments without chlorine, surrogate and L. pneumophila biofilm attachment was significantly reduced in the presence of chlorine. P. f luorescens biofilm formation was not affected by exposure to the surrogate microparticles that were made of biocompatible materials. The promising results from our study suggest that further validations of this new surrogate are warranted. It may create new pathways for investigating L. pneumophila mobility and persistence in EWSs, which will facilitate better control of legionellosis risks.

Nonbacterial microflora in wastewater remain understudied compared to their bacterial counterparts despite their importance in the wastewater treatment process. In this study, we report the temporal and spatial distributions of DNA viruses, archaea, protozoa, and fungi in raw wastewater influent, effluent, oxidation pond water, and oxidation pond sediments by using shotgun metagenomic sequencing.

Significance and Impact of the Study: The role of growth phase-dependent variations in cell surface hydrophobicity and charge on adhesion of the opportunistic pathogen Legionella pneumophila to plumbing materials remains poorly described. In this study, we characterized the effect of L. pneumophila cell surface properties on adhesion to stainless steel, an increasingly used plumbing material in potable water systems. These results help us understand the persistence of L. pneumophila in engineered water systems. To our knowledge, this is the first report describing the role of growthdependent variations of L. pneumophila cell surface properties in adhesion to a plumbing material surface.

Biopolymer microparticles have been developed for applications that require biocompatibility and biodegradability, such as drug delivery. In this study, we assessed the production of microparticles using carnauba wax, κ-carrageenan, alginate, and poly (lactic-co-glycolic acid) (PLGA) with the aim of developing a novel, DNA-tracer-loaded, biopolymer surrogate with a size, shape, surface charge, and relative hydrophobicity similar to stationary-phase Legionella pneumophila to mimic the bacteria’s mobility and persistence in engineered water systems. We found that the type and concentration of biopolymer, reaction conditions, and synthesis methods affected the morphology, surface charge, relative hydrophobicity, and DNA tracer loading efficiency of the biopolymer microparticles produced. Carnauba wax, κ-carrageenan, and alginate (Protanal®, and low and medium viscosity) produced highly polydisperse microspheres. In contrast, PLGA and alginate-CaCO3 produced uniform microspheres and rod-shaped microparticles, respectively, with high DNA tracer loading efficiencies (PLGA 70% and alginate-CaCO3 95.2 ± 5.7%) and high reproducibilities. Their synthesis reproducibility was relatively high. The relative hydrophobicity of PLGA microspheres closely matched the cell surface hydrophobicity of L. pneumophila but not the bacterial morphology, whereas the polyelectrolyte layer-by-layer assembly was required to enhance the relative hydrophobicity of alginate-CaCO3 microparticles. Following this surface modification, alginate-CaCO3 microparticles represented the best match to L. pneumophila in size, morphology, surface charge, and relative hydrophobicity. This new biopolymer surrogate has the potential to be used as a mimic to study the mobility and persistence of L. pneumophila in water systems where the use of the pathogen is impractical and unsafe.

The internalization of L. pneumophila within amoebae is a critical component of their life cycle in EWS, as it protects the bacteria from commonly used water disinfectants and provides a niche for their replication. Intracellularly replicated forms of L. pneumophila are also more virulent and resistant to sanitizers.

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