Thursday, 04 June 2015 22:49

Pyrosequencing analysis of roof-harvested rainwater and river water used for domestic purposes

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A significant number of households in rural South Africa rely on roof-harvested rainwater (RHRW) for domestic purposes. Although, there is a general public health perception that RHRW is safe to drink, the presence of potential pathogens has been reported in this water source. Generally, the microbiological methods used to evaluate water quality depend on conventional culturing methods, which may underestimate total pathogen content and diversity and, thus limit the extent to which one can fully understand potential infectious risks from RHRW use. However, the use of high-throughput next-generation sequencing, (pyrosequencing) offers an alternative, in which detailed community structure can be achieved in combination with a fairly high taxonomic resolution. Not only does high-throughput next-generation sequencing allow for the detection and identification of dominant bacteria phylotype profiles within a sample but the high sequence numbers produced allows for the detection of rare species including pathogens within bacterial communities.

In a bid to explore health risk implications from microbial communities in RHRW, scientists at the Department of Microbiology and plant pathology (University of Pretoria) used Pyrosequencing targeting the V1-V3 hypervariable of the 16S rDNA to investigate the bacterial diversity roof-harvested rainwater (RHRW) used for potable purposes by rural households in Luthengele village in the Eastern Cape Province of South Africa. The work was undertaken as part of a Water Research Commission (WRC) funded project (WRC Project No K5/2175, 2013). A total of nine water samples were collected in 2013 from seven randomly selected RHRW tanks and two individual samples from river water that is also used for potable purposes by the villagers and water that had been collected from a RHRW tank and stored in the kitchen prior to use (kitchen water).

















Analysis of our sequences using the SnoWMan pyrosequencing analysis pipeline and the greengenes taxonomic reference database showed diverse bacterial communities in RHRW, river and kitchen water samples. The phylum Proteobacteria dominated the data set and included the classes; Betaproteobacteria (50.4%), Alphaproteobacteria (16.2%), Verrucomicrobiae (6.6%), Planctomycetacia (5.7%) and Sphingobacteria (3%) in all the samples, although the class Verrucomicrobiae was most dominant in river water. Comparison of communities between groups showed that eleven genera were shared by all three while ten were shared between tank water and river water; five between tank water and kitchen water and no unique genera was shared between river water and kitchen water. The most abundant genera in both tank and kitchen water belonged to the phylum Proteobacteria whereas in river water it was genera of the phylum Planctomyces. In total, 60 genera were detected from the total amplicon sequences library and that tank water samples contained a greater variety of bacteria (50 genera) than the river water (38) genera) and kitchen water (22 genera) (with permission for reprint from the journal Environmental Monitoring and Assessment, 187(2), 1-17).

























Analysis of our sequences for the presence of potentially pathogenic bacteria showed Legionella signatures to be the most prevalent and were detected in six of the nine samples. Sequences of Acinetobacter and Pseudomonas were only detected in kitchen water, whereas Clostridia, Chromobacterium, Yersinia, Serratia and Legionella pneumophila were detected in Tank water (with permission for reprint from the journal Environmental Monitoring and Assessment, 187(2), 1-17).

Our findings demonstrated the potential of pyrosequencing as a method to define sequences of potentially pathogenic species. However, the limitations to this approach include, the potential uncertainty in accurate pathogen identification using the 16s rRNA gene and the need for greater sequencing depth to attain a level where primary pathogens in water samples are in abundance. This work provides guidance for prioritizing subsequent culturable and quantitative analysis, to ensure that potentially significant pathogens are not left out of risk estimations.

Journal Reference: Chidamba, L., & Korsten, L. (2015). Pyrosequencing analysis of roof-harvested rainwater and river water used for domestic purposes in Luthengele Village in the Eastern Cape Province of South Africa. Environmental Monitoring and Assessment, 187(2), 1-17.

Lise Korsten and Lizyben ChidambaAuthors: Lizyben Chidamba and This email address is being protected from spambots. You need JavaScript enabled to view it., Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0002, South Africa.

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