Abstract
Quantitative PCR (qPCR) and next generation sequencing (NGS) are nucleic acid based microbiology techniques that provide new insights into drinking water quality, but considerable uncertainty remains around their correct interpretation. We noticed the presence of bacterial DNA from various putative pathogens, including from faecal indicator bacteria (FIB), in disinfected water, when culturable FIB were absent. To understand these observations better we studied the effect of chlorination on conventional and DNA based microbial water quality assessments. Surface water chlorination reduced plate counts for various FIB by up to >6 log units, intact cell counts by flow cytometry by 3.3 log units, and 16S rRNA gene copies by qPCR by 1.5 and 1.6 log units for total bacteria and total coliforms, respectively. Nanopore sequencing of 16S rRNA amplicons with the portable MinION device revealed the DNA from several families containing putative pathogens appeared to be more resistant than that of other bacteria to degradation by chlorine disinfection. For instance, 16S rRNA genes assigned to the Enterobacteriaceae family, members of which are mostly the target of coliform tests, increased in relative abundance from 0.001 ± 0.0002% to 0.0036 ± 0.003% after chlorine treatment. Hence, metagenomic drinking water data needs to be interpreted with caution. Plate counts and flow cytometry in combination with DNA based analysis provide more robust insight than NGS or qPCR alone.
Highlights
We there fore sought to address the following research questions; 1) How do data obtained with a range of microbial water quality assessment methods compare when assessing water before and after disinfection with chlorine? 2) What is the impact of chlorination on the water microbial community structure derived from DNA-based molecular methods such as 16S rRNA next generation sequencing (NGS)? 3) What strategies need to be adopted to more robustly interpret molecular data obtained from disinfected systems? To answer these questions, we first analysed and compared real-world drinking water samples collected from various parts of the globe using both traditional and molecular microbial methods
None of the drinking water samples showed any evidence of cul turable faecal indicator bacteria, which included coliform, faecal streptococci and heterotrophic bacteria (Table S1)
The implication is that some of the water sources used for processing or manufacturing the drinking water were likely contaminated with faecal bacteria, but water was rendered fit for consumption by disinfection
Summary
In low-income countries, a significant proportion of the population uses a wide range of drinking water sources, including water supplied through drinking water distribution systems (DWDS), commercially available bottled or jar water, and locally accessible water resources such as roof-collected rainwater, springs, wells and boreholes (Sobsey et al, 2003; Wright et al, 2004; Arnold and Colford Jr, 2007; Mohapatra et al, 2014; Kostyla et al, 2015; Acharya et al, 2019; Kumar et al, 2020). POU chlorination plays a major role in providing safe drinking water in many rural areas, and helps to significantly inactive majority of waterborne pathogens and reduces the mortality due to diarrhoea (Mohamed et al, 2015) In these settings, the quality of water is typically not routinely monitored, and insufficient information is available for the sound protection of public health (Wright et al, 2004). We there fore sought to address the following research questions; 1) How do data obtained with a range of microbial water quality assessment methods (culture-based, flow cytometry, qPCR and NGS) compare when assessing water before and after disinfection with chlorine? 2) What is the impact of chlorination on the water microbial community structure derived from DNA-based molecular methods such as 16S rRNA NGS? To further enhance our understanding, we studied experimentally the impacts of chlorination on the detection of FIB and putative pathogens in surface water, as many people in devel oping countries use local surface water as a source of their drinking water
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