International Journal of Hygiene and Environmental Health 217 (2014) 219–225 Contents lists available at ScienceDirect International Journal of Hygiene and Environmental Health journa l h om epage: www.elsevier.com/locate/ijheh Preferential colonization and release of Legionella pneumophila from mature drinking water biofilms grown on copper versus unplasticized polyvinylchloride coupons Helen Y. Buse a,∗ , Jingrang Lu b , Ian T. Struewing a , Nicholas J. Ashbolt b a Dynamac Corporation c/o US Environmental Protection Agency, Cincinnati, OH, USA b National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, OH, USA a r t i c l e i n f o Article history: Received 14 December 2012 Received in revised form 8 April 2013 Accepted 22 April 2013 Keywords: CDC biofilm reactor Copper uPVC Drinking water HPC ATP a b s t r a c t Legionella occurrence in premise drinking water (DW) systems contributes to legionellosis outbreaks, especially in the presence of suitable protozoan hosts. This study examined L. pneumophila behavior within DW biofilms grown on copper (Cu) and unplasticized polyvinylchloride (uPVC) surfaces in the presence of Acanthamoeba polyphaga. One year-old DW biofilms were established within six CDC biofilm reactors: three each containing Cu or uPVC coupons. Biofilms were then inoculated with L. pneumophila (uPVC-Lp and Cu-Lp), or L. pneumophila and A. polyphaga (uPVC-Lp/Ap and Cu-Lp/Ap) and compared to sterile water inoculated controls (uPVC- and Cu-Control) over a 4 month period. L. pneumophila appeared more persistent by qPCR within Cu biofilms in the presence of A. polyphaga compared to uPVC biofilms with or without A. polyphaga, but maintained their cultivability in uPVC biofilms compared to Cu biofilms. Also, persistent shedding of L. pneumophila cells (assayed by qPCR) in the effluent water implied coloniza- tion of L. pneumophila within Cu-coupon reactors compared to no detection from uPVC-coupon reactor effluent 14 days after inoculation. Hence, L. pneumophila appeared to colonize Cu surfaces more effectively and may be shed from the biofilms at a greater frequency and duration compared to L. pneumophila col- onized uPVC surfaces with host amoebae playing a role in L. pneumophila persistence within Cu biofilms. Published by Elsevier GmbH. Introduction The presence of Legionella pneumophila within engineered water systems is a frequent health concern because of the significant cases of Legionella-associated disease outbreaks reported in the United States (Craun et al., 2010) let alone sporadic cases that go unreported (Dufresne et al., 2012). L. pneumophila is ubiquitous in the aquatic environment and is extremely persistent in drinking water systems (DWS) where free-living amoebae (FLA) are con- sidered to be primary hosts for their intracellular multiplication [reviewed in Lau and Ashbolt, 2009]. However, the intracellular growth of L. pneumophila within FLA is not always required for L. pneumophila amplification within mixed DWS biofilm communi- ties (Surman et al., 2002; Temmerman et al., 2006; Wadowsky and Yee, 1983; Yee and Wadowsky, 1982). Thus, FLA hosts as well as other drinking water microbes and surfaces may synergistically and ∗ Corresponding author at: 26 W Martin Luther King Drive, Cincinnati, OH 45268, USA. Tel.: +1 513 569 7930. E-mail address: buse.helen@epa.gov (H.Y. Buse). independently, play an important role in the growth and survival of L. pneumophila within drinking water environments. DWS biofilms are the preferred site for L. pneumophila growth (Armon et al., 1997; Rogers et al., 1994; Rogers and Keevil, 1992), and biofilm development may differ between in-premise plumbing materials, such as chlorinated-, plasticized-, and unplasticized- polyvinylchloride (cPVC, pPVC, uPVC, respectively) (Armon et al., 1997; Kuiper et al., 2004; Lehtola et al., 2007; Rogers et al., 1994), polybutylene (Rogers et al., 1994; Rogers and Keevil, 1992), cross- linked polyethylene (PE-X) (Moritz et al., 2010; Valster et al., 2010), copper (Cu) (Messi et al., 2011; Moritz et al., 2010; Rogers et al., 1994), and stainless steel (SS) (Murga et al., 2001). However, with a few exceptions, comparisons of L. pneumophila-associated biofilm growth between different surface materials are rare. Where com- parisons have been made, L. pneumophila appears to survive better on PVC rather than glass-associated laboratory grown biofilms (Armon et al., 1997), whereas Cu pipe biofilms may contain no culturable L. pneumophila compared to polybutylene and cPVC grown biofilms at 20 and 50 ◦ C (Rogers et al., 1994). L. pneumophila has been reported to persist for up to four weeks in biofilms, as determined by culture and fluorescent in situ hybridization (FISH) analysis, with L. pneumophila colonization highest within 1438-4639/$ – see front matter. Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.ijheh.2013.04.005