Public health implications of Acanthamoeba and multiple potential opportunistic pathogens in roof-harvested rainwater tanks K.A. Hamilton a,b , W. Ahmed a,n , A. Palmer a , J.P.S. Sidhu a , L. Hodgers a , S. Toze a , C.N. Haas b a CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, Qld 4102, Australia b Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, USA article info Article history: Received 29 April 2016 Received in revised form 7 June 2016 Accepted 8 June 2016 Keywords: Roof-harvested rainwater Opportunistic pathogens Fecal indicator bacteria Quantitative PCR Health risks Rainwater survey abstract A study of six potential opportunistic pathogens (Acanthamoeba spp., Legionella spp., Legionella long- beachae, Pseudomonas aeruginosa, Mycobacterium avium and Mycobacterium intracellulare) and an acci- dental human pathogen (Legionella pneumophila) in 134 roof-harvested rainwater (RHRW) tank samples was conducted using quantitative PCR (qPCR). All five opportunistic pathogens and accidental pathogen L. pneumophila were detected in rainwater tanks except Legionella longbeachae. Concentrations ranged up to 3.1 Â 10 6 gene copies per L rainwater for Legionella spp., 9.6 Â 10 5 gene copies per L for P. aeruginosa, 6.8 Â 10 5 gene copies per L for M. intracellulare, 6.6 Â 10 5 gene copies per L for Acanthamoeba spp., 1.1 Â 10 5 gene copies per L for M. avium, and 9.8 Â 10 3 gene copies per L for L. pneumophila. Among the organisms tested, Legionella spp. (99% tanks) were the most prevalent followed by M. intracellulare (78%). A survey of tank-owners provided data on rainwater end-uses. Fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp. were enumerated using culture-based methods, and assessed for correlations with opportunistic pathogens and L. pneumophila tested in this study. Opportunistic pathogens did not correlate well with FIB except E. coli vs. Legionella spp. (tau ¼0.151, P ¼0.009) and E. coli vs. M. in- tracellulare (tau ¼0.14, P ¼0.015). However, M. avium weakly correlated with both L. pneumophila (Ken- dall’s tau ¼0.017, P ¼0.006) and M. intracellulare (tau ¼0.088, P ¼0.027), and Legionella spp. also weakly correlated with M. intracellulare (tau ¼0.128, P ¼0.028). The presence of these potential opportunistic pathogens in tank water may present health risks from both the potable and non-potable uses docu- mented from the current survey data. Crown Copyright & 2016 Published by Elsevier Inc. All rights reserved. 1. Introduction Increasing water scarcity has led to a greater reliance on al- ternative and decentralized potable and non-potable water re- sources in recent decades (Hanjra et al., 2012). Australia is the driest inhabited continent on Earth and suffered from a severe “millennium” drought from 2001 to 2009 (van Dijk et al., 2013). As a result of water scarcity in this region, the use of roof-harvested rainwater (RHRW) for domestic purposes is a widely accepted practice. This is beneficial for simultaneously conserving water and reducing stormwater runoff. Pathogens could be introduced to tanks via roof runoff con- taining fecal matter from birds, insects, bats, possums and reptiles. The microbiological quality of RHRW stored in tanks is generally assessed by monitoring Escherichia coli (E. coli) and Enterococcus spp., which are commonly found in the gut of warm-blooded an- imals (Albrechtsen, 2002; Lee et al., 2010). The presence of E. coli in tank water generally indicates fecal contamination and the presence of potential pathogens. Drinking water guidelines have been used to assess the microbial quality of the tank water. For most guidelines, this entails the non-detection of E. coli in 100 mL of water (NHMRC-NRMMC, 2004; WHO, 2004). Fecal indicator bacteria should be able to predict human health outcomes. From a public health perspective, the relationship between fecal indicator bacteria and pathogens is critical. Although case-control studies have established associations between untreated rainwater consumption and gastroenteritis (Brodribb et al., 1995; Merritt et al., 1999), epidemiological studies have not supported a strong linkage (Heyworth et al., 2006; Ro- drigo et al., 2011). The presence of multiple non-gastroenteritis- associated microbial pathogens (opportunistic in nature) in rain- water tanks have been reported (Tuffley and Holbeche, 1980; Dobrowsky et al., 2014; Chidamba and Korsten, 2015), supporting the need to assess potential health risks. Only a few studies Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/envres Environmental Research http://dx.doi.org/10.1016/j.envres.2016.06.017 0013-9351/Crown Copyright & 2016 Published by Elsevier Inc. All rights reserved. n Correspondence to: CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, Brisbane 4102, Australia. E-mail address: Warish.Ahmed@csiro.au (W. Ahmed). Environmental Research 150 (2016) 320–327