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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Inactivation of Legionella Pneumophila-Harbored by Amoebae Using a Nano-Enabled Alternative Technology
Legionella pneumophila is a virulent bacterial pathogen that can cause a severe and deadly form of pneumonia called Legionnaires’ disease. Documented cases of Legionnaires’ disease have been rising since 2000. Risk of infection increases when L. pneumophila are harbored inside free-living amoebae, which are resistant to traditional disinfection processes. The ability of amoebae to phagocytose L. pneumophila allows amoebae to act as ‘Trojan horses’ for pathogen transport. This project aims to extract an unintended benefit from low-intensity microwave (MW) radiation (already found in many homes across economic cross-sections) by employing nanomaterials (e.g., silver, copper oxide, and carbon nanotubes) that are capable of harnessing such radiation and localizing the otherwise dissipated energy. 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. Traditionally, inactivation of up to 3-logs of planktonic L. pneumophila with dissolved silver requires hours of contact time. This study reports rapid inactivation (in minutes) of 3-log or higher when the planktonic L. pneumophila is subjected to AgNPs (5 mg/L) and MW radiation (2,450 MHz; 70 W). Ensuing phases of this project will incorporate copper oxide nanoparticles – which are anticipated to increase toxicity akin to copper-silver ionization systems currently employed in hospitals for L. pneumophila control – and enhance inactivation potency with potentially lower microwave radiation input and/or a lower concentration of nanoparticles. Ultimately, the nanomaterials will be immobilized on a plaster of Paris or ceramic surface for flow-through applications for lysing amoebae and inactivating L. pneumophila.
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- Award ID(s):
- 1805958
- NSF-PAR ID:
- 10174722
- Date Published:
- Journal Name:
- 2019 ASA, CSSA, and SSSA Annual Meeting
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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.more » « less
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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.more » « less
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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.more » « less
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Abstract Unregulated private wells are understudied potential sources of community-acquired Legionnaires’ disease. Here we conducted a comprehensive survey of 44 homes supplied by private wells in Wake County, North Carolina, quantifying Legionella spp. DNA, Legionella pneumophila DNA, and total bacterial 16S rRNA genes via real-time polymerase chain reaction in hot and cold drinking water samples, along with culturable L. pneumophila via IDEXX Legiolert in cold drinking water samples. Legionella spp. DNA, L. pneumophila DNA and culturable L. pneumophila were detected in 100, 65·5 and 15·9% of the 44 homes, respectively, and culturable levels were comparable to some municipal surveys applying the same methods. Total coliforms and Escherichia coli were monitored as representative faecal indicators and were found in 20·4 and 0·0% of homes. Within certain sample types, Legionella spp. and L. pneumophila gene copy numbers were positively associated with total bacteria (i.e. total 16S rRNA genes) and water softener use, but were not associated with faecal indicator bacteria, inorganic water parameters or other well characteristics. These findings confirm that occurrence of Legionella and L. pneumophila is highly variable in private wells.
Significance and Impact of the Study Legionella is the leading identified cause of waterborne disease outbreaks associated with US municipal water systems. While Legionella is known to occur naturally in groundwater, prior efforts to characterize its occurrence in unregulated private wells are limited to sampling at the wellhead and not in the home plumbing where Legionella can thrive. This work documents much higher levels of Legionella in home plumbing versus water directly from private wells and examines factors associated with higher Legionella occurrence.
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