Citation:

Kleier, K., J. Hoelle, M. Rodgers, AND H. Ryu. A comparison of assays measuring the viability of Legionella pneumophila after treatment with copper and silver ions. Presented at Water Quality Technology Conference, Indianapolis, Indiana, November 14 - 17, 2016.

Impact/Purpose:

This presentation will describe the research efforts that have been conducted at EPA ORD Cincinnati with respect to copper silver ionization. The data presented are bench scale studies that show water dosed with these ions and then the culturability of Legionella after exposure, along with other studies that measure viability such as qPCR treated with PMA or EMA.

Description:

Background: The relatively high prevalence of Legionella pneumophila in premise plumbing systems has been widely reported. Published reports indicate Legionella has a comparatively high resistance to chlorine and moreover has the ability to grow in phagocytic amoeba which could provide additional protection in chlorinated drinking water distribution systems. Copper-Silver (Cu-Ag) ionization treatment systems are commercially available for use in large building water systems to help control the risks from Legionella bacteria. The objectives of this study were to develop and optimize Legionella viability assays and use them to investigate the viability of Legionella bacteria after exposure to water treated with coppper and silver ions. Methods: Log phase L. pneumophila cells were used in all experiments and were generated by incubation at 35C for 48 hours in buffered yeast extract broth. Viability assays used included plating on buffered charcoal yeast extract agar to determine the number of culturable cells and treating cells with propidium monoazide (PMA) or ethidium monoazide (EMA) followed by quantitative PCR targeting mip gene of L. pneumophila. The qPCR viability assays were optimized using L. pneumophila inactivated by heat treatment at 65C for 60 min. The effectiveness of Cu-Ag ionization treatment was studied by inoculating L. pneumonia at 105 CFU/mL in water collected directly from a building water system that employed this technology and incubating for 3 and 24 hours at ambient temperature. Additionally, the effect on L. pneumophila viability by changing the pH and chloride concentration of copper and silver dosed water was studied. Results: It was determined that optimum spiking concentrations for PMA and EMA treatment experiments ranged from 104 to 105 CFU/mL, while greater than 106 CFU/mL showed significant false-positive qPCR results. Similarly, inactivation rates of L. pneumophila in Cu-Ag ionization with spiking concentrations of greater than 106 CFU/mL showed significant underestimation compared to lower spiking levels. The mip gene copy numbers in the bacteria were relatively consistent before and after heat treatment, whereas a more than 90% reduction in amplifiable copy number was observed in heat-killed bacteria treated with PMA or EMA. In contrast, the 24-hour exposure of L. pneumophila to Cu-Ag ionized water showed no significant variations of mip gene copy numbers determined by PMA- and EMA-qPCR, whereas culturable cell numbers decreased by more than 90% and 99.9% after 3- and 24-hour incubation periods, respectively. Water pH and chloride concentrations had no significant impact on the efficacy of Cu-Ag ionization, showing meaningful levels of inactivation rates even in the worst testing condition (i.e., at higher pH and greater chloride concentrations). Conclusions: Altogether, these results indicate that Cu-Ag ionization treatment impacts the viability of Legionella bacteria as measured by culture techniques, but not as measured by qPCR of the mip gene after treatment with PMA and EMA. Additionally these results have implications regarding the contributions to Legionella toxicity of the copper and silver ions. Acknowledgement: This work was funded by the EPA. Any opinions expressed in this abstract are those of the authors and do not necessarily reflect the views of the EPA.

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