Molecular detection of Acanthamoeba spp., Naegleria fowleri and Vermamoeba (Hartmannella) vermiformis as vectors for Legionella spp. in untreated and solar pasteurized harvested rainwater

Abstract

Background Legionella spp. employ multiple strategies to adapt to stressful environments including the proliferation in protective biofilms and the ability to form associations with free-living amoeba (FLA). The aim of the current study was to identify Legionella spp., Acanthamoeba spp., Vermamoeba (Hartmannella) vermiformis and Naegleria fowleri that persist in a harvested rainwater and solar pasteurization treatment system.MethodsPasteurized (45 °C, 65 °C, 68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples were screened for Legionella spp. and the heterotrophic plate count was enumerated. Additionally, ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) was utilized for the quantification of viable Legionella spp., Acanthamoeba spp., V. vermiformis and N. fowleri in pasteurized (68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples, respectively.ResultsOf the 82 Legionella spp. isolated from unpasteurized tank water samples, Legionella longbeachae (35 %) was the most frequently isolated, followed by Legionella norrlandica (27 %) and Legionella rowbothamii (4 %). Additionally, a positive correlation was recorded between the heterotrophic plate count vs. the number of Legionella spp. detected (ρ = 0.710, P = 0.048) and the heterotrophic plate count vs. the number of Legionella spp. isolated (ρ = 0.779, P = 0.0028) from the tank water samples collected. Solar pasteurization was effective in reducing the gene copies of viable V. vermiformis (3-log) and N. fowleri (5-log) to below the lower limit of detection at temperatures of 68–93 °C and 74–93 °C, respectively. Conversely, while the gene copies of viable Legionella and Acanthamoeba were significantly reduced by 2-logs (P = 0.0024) and 1-log (P = 0.0015) overall, respectively, both organisms were still detected after pasteurization at 93 °C.ConclusionsResults from this study indicate that Acanthamoeba spp. primarily acts as the vector and aids in the survival of Legionella spp. in the solar pasteurized rainwater as both organisms were detected and were viable at high temperatures (68–93 °C).

Legionella pneumophila and Protozoan Hosts: Implications for the Control of Hospital and Potable Water Systems

“…Numerous studies have demonstrated the presence of L. pneumophila in disinfected water supplies [60,61]. An important factor enabling L. pneumophila survival in the built environment is its interaction with a protozoan host [62][63][64] (as mentioned in Table 3). Thermal treatment is one of the most common methods used to disinfect hospitals and building water supplies.…”

“…Rhoads et al [64] reported that L. pneumophila associated with V. vermiformis can tolerate thermal (58 • C) treatment, and this disinfection protocol is unable to reduce microbial load in water. Published evidence suggests Legionella associated with Acanthamoeba are more thermos-tolerant and can survive at even higher temperatures ranging from 68-93 • C [63]. According to Steinert et al [38] members of L. pneumophila SG1 are more thermo-tolerant than SG2.…”

Unravelling the importance of the eukaryotic and bacterial communities and their relationship with Legionella spp. ecology in cooling towers: a complex network

“…Different species or strain of Legionella can infect different host species, depending on the type of effector protein present in the genome [24]. In addition, host cells provide a safe means of transportation and protection from harsh chemicals, such as disinfectants found in the cooling tower environment, as some protozoa host can produce cysts [25][26][27]. The number of known host species is quite expansive, spanning several distant phylogenetic groups.…”

Water Stagnation and Flow Obstruction Reduces the Quality of Potable Water and Increases the Risk of Legionelloses

“…Cargill et al (1992) reported that in contrast to free living cells, L. pneumophila existing in complex biofilms can tolerate high doses of disinfectant. Specifically, the amoeba Acanthamoeba and Vermamoeba provide additional protection from prolonged and persistent water treatment processes (Kilvington and Price, 1990;Dupuy et al, 2011;Cervero-Arago et al, 2014;Dobrowsky et al, 2016). Donlan et al (2005) observed that for L. pneumophila-amoebae complex biofilms, monochloramine (0.5 mg/L) was a more effective disinfectant compared to free chlorine (0.5 mg/L).…”