P. H. Dobrowsky

Distribution of Indigenous Bacterial Pathogens and Potential Pathogens Associated with Roof-Harvested Rainwater

ABSTRACT The harvesting of rainwater is gaining acceptance among many governmental authorities in countries such as Australia, Germany, and South Africa, among others. However, conflicting reports on the microbial quality of harvested rainwater have been published. To monitor the presence of potential pathogenic bacteria during high-rainfall periods, rainwater from 29 rainwater tanks was sampled on four occasions (during June and August 2012) in a sustainable housing project in Kleinmond, South Africa. This resulted in the collection of 116 harvested rainwater samples in total throughout the sampling period. The identities of the dominant, indigenous, presumptive pathogenic isolates obtained from the rainwater samples throughout the sampling period were confirmed through universal 16S rRNA PCR, and the results revealed that Pseudomonas (19% of samples) was the dominant genus isolated, followed by Aeromonas (16%), Klebsiella (11%), and Enterobacter (9%). PCR assays employing genus-specific primers also confirmed the presence of Aeromonas spp. (16%), Klebsiella spp. (47%), Legionella spp. (73%), Pseudomonas spp. (13%), Salmonella spp. (6%), Shigella spp. (27%), and Yersinia spp. (28%) in the harvested rainwater samples. In addition, on one sampling occasion, Giardia spp. were detected in 25% of the eight tank water samples analyzed. This study highlights the diverse array of pathogenic bacteria that persist in harvested rainwater during high-rainfall periods. The consumption of untreated harvested rainwater could thus pose a potential significant health threat to consumers, especially children and immunocompromised individuals, and it is recommended that harvested rainwater be treated for safe usage as an alternative water source.

Domestic Rainwater Harvesting: Microbial and Chemical Water Quality and Point-of-Use Treatment Systems

Quality of the essential commodity, water, is being compromised by contaminants originating from anthropogenic sources, industrial activities, agriculture, etc. Water scarcity and severe droughts in many regions of the world also represent a significant challenge to availability of this resource. Domestic rainwater harvesting, which involves collection and storage of water from rooftops and diverse surfaces, is successfully implemented worldwide as a sustainable water supplement. This review focuses on chemical and microbial qualities of domestic rainwater harvesting, with a particular focus on sources of chemical pollution and major pathogens associated with the water source. Incidences of disease linked to consumption and utilization of harvested rainwater are also discussed. In addition, various procedures and methods used for disinfection and treatment of harvested rainwater, such as implementation of filter systems (activated carbon, slow sand filtration, etc.), heat treatment, and chlorination, among others, are also presented.

Prevalence of virulence genes associated with pathogenic Escherichia coli strains isolated from domestically harvested rainwater during low and high rainfall periods

ABSTRACT The possible health risks associated with the consumption of harvested rainwater remains one of the major obstacles hampering its large-scale implementation in water limited countries such as South Africa. Rainwater tank samples collected on eight occasions during the low- and high-rainfall periods (March to August 2012) in Kleinmond, South Africa, were monitored for the presence of virulence genes associated with Escherichia coli. The identity of presumptive E. coli isolates in rainwater samples collected from 10 domestic rainwater harvesting (DRWH) tanks throughout the sampling period was confirmed through universal 16S rRNA PCR with subsequent sequencing and phylogenetic analysis. Species-specific primers were also used to routinely screen for the virulent genes, aggR, stx, eae, and ipaH found in enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli, respectively, in the rainwater samples. Of the 92 E. coli strains isolated from the rainwater using culture based techniques, 6% were presumptively positively identified as E. coli O157:H7 using 16S rRNA. Furthermore, virulent pathogenic E. coli genes were detected in 3% (EPEC and EHEC) and 16% (EAEC) of the 80 rainwater samples collected during the sampling period from the 10 DRWH tanks. This study thus contributes valuable information to the limited data available regarding the ongoing prevalence of virulent pathotypes of E. coli in harvested rainwater during a longitudinal study in a high-population-density, periurban setting.

EMA-qPCR to monitor the efficiency of a closed-coupled solar pasteurization system in reducing Legionella contamination of roof-harvested rainwater.

Solar pasteurization is effective in reducing the level of indicator organisms in stored rainwater to within drinking water standards. However, Legionella spp. were detected at temperatures exceeding the recommended pasteurization temperatures using polymerase chain reaction assays. The aim of the current study was thus to apply EMA quantitative polymerase chain reaction (EMA-qPCR) to determine whether the Legionella spp. detected were intact cells and therefore possibly viable at pasteurization temperatures >70°C. The BacTiter-Glo™ Microbial Cell Viability Assay was also used to detect the presence of ATP in the tested samples, as ATP indicates the presence of metabolically active cells. Chemical analysis also indicated that all anions and cations were within the respective drinking water guidelines, with the exception of iron (mean: 186.76 μg/L) and aluminium (mean: 188.13 μg/L), which were detected in the pasteurized tank water samples at levels exceeding recommended guidelines. The BacTiter-Glo™ Microbial Cell Viability Assay indicated the presence of viable cells for all pasteurized temperatures tested, with the percentage of ATP (in the form of relative light units) decreasing with increasing temperature [70-79°C (96.7%); 80- 89°C (99.2%); 90-95°C (99.7%)]. EMA-qPCR then indicated that while solar pasteurization significantly reduced (p<0.05) the genomic copy numbers of intact Legionella cells in the pasteurized tank water (~99%), no significant difference (p>0.05) in the mean copy numbers was detected with an increase in the pasteurization temperature, with 6 × 10(3) genomic copies/mL DNA sample obtained at 95°C. As intact Legionella cells were detected in the pasteurized tank water samples, quantitative microbial risk assessment studies need to be conducted to determine the potential health risk associated with using the water for domestic purposes.

Microbial and Physico-chemical Characteristics Associated with the Incidence of Legionella spp. and Acanthamoeba spp. in Rainwater Harvested from Different Roofing Materials

The incidence of Legionella and Acanthamoeba spp. was correlated to microbial indicator analysis and physico-chemical characteristics of rainwater harvested from catchment areas constructed from galvanized zinc, Chromadek®, and asbestos, respectively. Quantitative PCR (qPCR) analysis indicated that no significant difference (p > 0.05) in copy numbers of Legionella spp. and Acanthamoeba spp. was recorded in tank water samples collected from the respective roofing materials. However, significant positive Spearman (ρ) correlations were recorded between the occurrences of Legionella spp. gene copies vs. nitrites and nitrates (p = 0.05) in all tank water samples. Significant positive correlations were also established between Acanthamoeba spp. vs. barium (p = 0.03), magnesium (p = 0.02), sodium (p = 0.02), silicon (p = 0.05), arsenic (p = 0.03), and phosphate (p = 0.01), respectively. Additionally, while no significant correlations were observed between Legionella spp. vs. the indicator bacteria (p > 0.05), positive correlations were observed between Acanthamoeba spp. vs. total coliforms (p = 0.01) and Acanthamoeba spp. vs. Escherichia coli (p = 0.02), respectively. Results obtained in the current study thus indicate that the incidence of Acanthamoeba and Legionella spp. in harvested rainwater was not influenced by the roofing material utilized. Moreover, it is essential that the microbial quality of rainwater be assessed before this water source is implemented for potable and domestic uses as untreated harvested rainwater may lead to legionellosis and amoebae infections.

Resistance of Legionella and Acanthamoeba mauritaniensis to heat treatment as determined by relative and quantitative polymerase chain reactions

Abstract Legionella and Acanthamoeba spp. persist in harvested rainwater pasteurized at high temperatures (> 72 °C) and the interaction mechanisms exhibited between these organisms need to be elucidated. The resistance of two Legionella reference strains (Legionella pneumophila ATCC 33152 and Legionella longbeachae ATCC 33462), three environmental strains [Legionella longbeachae (env.), Legionella norrlandica (env.) and Legionella rowbothamii (env.)] and Acanthamoeba mauritaniensis ATCC 50676 to heat treatment (50–90 °C) was determined by monitoring culturability and viability [ethidium monoazide quantitative polymerase chain reaction (EMA‐qPCR)]. The expression of metabolic and virulence genes of L. pneumophila ATCC 33152 (lolA, sidF, csrA) and L. longbeachae (env.) (lolA) in co‐culture with A. mauritaniensis ATCC 50676 during heat treatment (50–90 °C) was monitored using relative qPCR. While the culturability (CFU/mL) and viability (gene copies/mL) of the Legionella strains reduced significantly (p < 0.05) following heat treatment (60–90 °C), L. longbeachae (env.) and L. pneumophila ATCC 33152 were culturable following heat treatment at 50–60 °C. Metabolically active trophozoites and dormant cysts of A. mauritaniensis ATCC 50676 were detected at 50 °C and 60–90 °C, respectively. For L. pneumophila ATCC 33152, lolA expression remained constant, sidF expression increased and the expression of csrA decreased during co‐culture with A. mauritaniensis ATCC 50676. For L. longbeachae (env.), while lolA was up‐regulated at 50–70 °C, expression was not detected at 80–90 °C and in co‐culture. In conclusion, while heat treatment may reduce the number of viable Legionella spp. in monoculture, results indicate that the presence of A. mauritaniensis increases the virulence of L. pneumophila during heat treatment. The virulence of Legionella spp. in co‐culture with Acanthamoeba spp. should thus be monitored in water distribution systems where temperature (heat) is utilized for treatment. HighlightsResistance of Legionella spp. and A. mauritaniensis to heat treatment (50–90 °C).Following treatment (50–60 °C), L. longbeachae (env.) and L. pneumophila culturable.Cysts/trophozoites of A. mauritaniensis observed in samples treated at 50–90 °C.Viability (EMA‐qPCR) of all Legionella and Acanthamoeba spp. confirmed at 90 °C.Expression of sidF (increase) and csrA (decrease) in L. pneumophila during co‐culture.