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1. Legionella pneumophila inactivation potency of silver nanoparticles and ionic silver and copper enhanced with microwave radiation

   Ayres, C.; Saleh, N. B.; Kirisits, M. J.; Lawler, D. F. (November 2019, 8th Sustainable Nanotechnology Organization Conference)

   Legionella pneumophila is a virulent bacterial pathogen that can cause a severe and deadly form of pneumonia called Legionnaires’ disease. Risk of infection increases when L. pneumophila are harbored inside free-living amoebae, which are resistant to traditional disinfection processes but lyse upon heat exposure. This project aims to develop a point-of-use technology based on microwave (MW) radiation and nanomaterial (e.g., silver, copper oxide, carbon nanotubes) exposure for L. pneumophila control. In this alternative technology, we hypothesize that amoebae will be lysed via localized interfacial heating, and the released L. pneumophila will be inactivated subsequently by heat, metal ions (from nanoparticle dissolution), and reactive oxygen species (ROS) produced in the process. The synergistic effect of microwaves and silver nanoparticles for enhanced, rapid inactivation has been demonstrated for Escherichia coli and planktonic L. pneumophila. Inactivation greater than 3-logs of each species has been achieved when subjected to silver nanoparticles (2-5 mg/L) and MW (2,450 MHz; 70 W) radiation. A mechanistic study using E. coli has determined the dominant interaction to be between released ions and MW radiation. Ultimately, the nanomaterials will be immobilized on a plaster of Paris or ceramic surface for flow through applications where both amoeba lysing and L. pneumophila inactivation will be achieved. 

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2. Synergy between microwave radiation and silver for inactivation of Legionella pneumophila

   Saleh, N. B.; Ayres, C.; Lawler, D. F.; Kirisits, M. J. (July 2020, 1st Virtual Canadian Symposium on Water Quality Research)

   Legionella pneumophila is an opportunistic human pathogen that can cause a severe and deadly form of pneumonia called Legionnaires’ disease. Over the past decade, the number of reported cases of Legionnaires’ disease has quadrupled in the U.S., with 8,000-18,000 hospitalizations per year at a yearly incidence rate of 1.7/100,000. Within the water sector, this public health risk is exacerbated by the proliferation of L. pneumophila in complex biological matrices such as biofilms and within free-living amoebae. Traditional disinfection technologies fail to effectively mitigate this emerging pathogen issue, necessitating development of point-of-use (POU) technologies with high inactivation efficacy. We aim to harness microwave (MW) radiation and take advantage of its synergy with ion-mediated toxicity to effectively inactivate L. pneumophila. In this study, planktonic L. pneumophila cells have been exposed to ionic and nano-particulate silver. While neither treatment alone is effective over a short exposure period, a combined treatment of silver with MW radiation successfully achieves 3-4 log removal within 6 min of irradiation, as shown in Figure 1. Enhanced toxicity was observed when L. pneumophila was pre-exposed to either treatment (i.e., MW heating or silver exposure) prior to exposure to the other; these results suggest that silver ion transport within the cells is facilitated by heat treatment. Data presented here serve as the proof-of-concept toward the development of a L pneumophila inactivation device that harnesses MW radiation and can potentially mitigate this public health risk, even if the cells are protected by amoebae or biofilms. 

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3. Interactive Effects of Copper Pipe, Stagnation, Corrosion Control, and Disinfectant Residual Influenced Reduction of Legionella pneumophila during Simulations of the Flint Water Crisis

   https://doi.org/10.3390/pathogens9090730  

   Martin, Rebekah L.; Strom, Owen R.; Pruden, Amy; Edwards, Marc A. (September 2020, Pathogens)

   null (Ed.)

   Flint, MI experienced two outbreaks of Legionnaires’ Disease (LD) during the summers of 2014 and 2015, coinciding with use of Flint River as a drinking water source without corrosion control. Using simulated distribution systems (SDSs) followed by stagnant simulated premise (i.e., building) plumbing reactors (SPPRs) containing cross-linked polyethylene (PEX) or copper pipe, we reproduced trends in water chemistry and Legionella proliferation observed in the field when Flint River versus Detroit water were used before, during, and after the outbreak. Specifically, due to high chlorine demand in the SDSs, SPPRs with treated Flint River water were chlorine deficient and had elevated L. pneumophila numbers in the PEX condition. SPPRs with Detroit water, which had lower chlorine demand and higher residual chlorine, lost all culturable L. pneumophila within two months. L. pneumophila also diminished more rapidly with time in Flint River SPPRs with copper pipe, presumably due to the bacteriostatic properties of elevated copper concentrations caused by lack of corrosion control and stagnation. This study confirms hypothesized mechanisms by which the switch in water chemistry, pipe materials, and different flow patterns in Flint premise plumbing may have contributed to observed LD outbreak patterns. 

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4. Exploitation of Phosphoinositides by the Intracellular Pathogen, Legionella pneumophila

   https://doi.org/10.5772/intechopen.89158  

   Pike, Colleen; Noll, Rebecca; Neunuebel, Ramona. (October 2019, Intech)

   Manipulation of host phosphoinositide lipids has emerged as a key survival strategy utilized by pathogenic bacteria to establish and maintain a replication-permissive compartment within eukaryotic host cells. The human pathogen, Legionella pneumophila, infects and proliferates within the lung’s innate immune cells causing severe pneumonia termed Legionnaires’ disease. This pathogen has evolved strategies to manipulate specific host components to construct its intracellular niche termed the Legionella-containing vacuole (LCV). Paramount to LCV biogenesis and maintenance is the spatiotemporal regulation of phosphoinositides, important eukaryotic lipids involved in cell signaling and membrane trafficking. Through a specialized secretion system, L. pneumophila translocates multiple proteins that target phosphoinositides in order to escape endolysosomal degradation. By specifically binding phosphoinositides, these proteins can anchor to the cytosolic surface of the LCV or onto specific host membrane compartments, to ultimately stimulate or inhibit encounters with host organelles. Here, we describe the bacterial proteins involved in binding and/or altering host phosphoinositide dynamics to support intracellular survival of L. pneumophila. 

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5. Effects of Copper on Legionella pneumophila Revealed via Viability Assays and Proteomics

   https://doi.org/10.3390/pathogens13070563  

   Song, Yang; Mena-Aguilar, Didier; Brown, Connor L; Rhoads, William J; Helm, Richard F; Pruden, Amy; Edwards, Marc A (July 2024, Pathogens)

   Cu is an antimicrobial that is commonly applied to premise (i.e., building) plumbing systems for Legionella control, but the precise mechanisms of inactivation are not well defined. Here, we applied a suite of viability assays and mass spectrometry-based proteomics to assess the mechanistic effects of Cu on L. pneumophila. Although a five- to six-log reduction in culturability was observed with 5 mg/L Cu2+ exposure, cell membrane integrity only indicated a <50% reduction. Whole-cell proteomic analysis revealed that AhpD, a protein related to oxidative stress, was elevated in Cu-exposed Legionella relative to culturable cells. Other proteins related to cell membrane synthesis and motility were also higher for the Cu-exposed cells relative to controls without Cu. While the proteins related to primary metabolism decreased for the Cu-exposed cells, no significant differences in the abundance of proteins related to virulence or infectivity were found, which was consistent with the ability of VBNC cells to cause infections. Whereas the cell-membrane integrity assay provided an upper-bound measurement of viability, an amoebae co-culture assay provided a lower-bound limit. The findings have important implications for assessing Legionella risk following its exposure to copper in engineered water systems. 

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