The limitation of 16S rRNA gene sequencing (DNA-based) for microbial community analyses in water is the inability to differentiate live (dormant cells as well as growing or non-growing metabolically active cells) and dead cells, which can lead to false positive results in the absence of live microbes. Propidium-monoazide (PMA) has been used to selectively remove DNA from dead cells during downstream sequencing process. In comparison, 16S rRNA sequencing (RNA-based) can target live microbial cells in water as both dormant and metabolically active cells produce rRNA. The objective of this study was to compare the efficiency and sensitivity of DNA-based, PMA-based and RNA-based 16S rRNA Illumina sequencing methodologies for live bacteria detection in water samples experimentally spiked with different combination of bacteria (2 gram-negative and 2 gram-positive/acid fast species either all live, all dead, or combinations of live and dead species) or obtained from different sources (First Nation community drinking water; city of Winnipeg tap water; water from Red River, Manitoba, Canada). The RNA-based method, while was superior for detection of live bacterial cells still identified a number of 16S rRNA targets in samples spiked with dead cells. In environmental water samples, the DNA- and PMA-based approaches perhaps overestimated the richness of microbial community compared to RNA-based method. Our results suggest that the RNA-based sequencing was superior to DNA- and PMA-based methods in detecting live bacterial cells in water.

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Approximately 20% of the 600 First Nations reserves across Canada are under a drinking water advisory, often due to unacceptable levels of bacteria. In this study, we detected fecal bacteria at an alarmingly high frequency in drinking water sources in a fly-in First Nations community, most notably in buckets/drums of homes without running water where Escherichia coli levels ranged from 20 to 62,000 CFU/100mL. The water leaving the water treatment plant was free of E. coli and its free residual chlorine concentration (0.67 mg/L) was within the range typically observed for treated water in Canada. Water samples from taps in homes served by cisterns, and those sampled from the water truck and community standpipe, always showed unacceptable levels of E. coli (1 to 2,100 CFU/100mL) and free residual chlorine concentrations below the 0.2 mg/L required to prevent bacterial regrowth. Samples from taps in homes served by piped water had lower levels of E. coli (0 to 2 CFU/100mL). DNA-and RNA-based 16S rRNA Illumina sequencing demonstrated that piped and cisterns water distribution systems showed an abundance of viable cells of Alphaproteobacteria indicative of biofilm formation in pipes and cisterns. The alpha diversity, based on observed OTUs and three other indices, was lowest in water truck samples that supplied water to the cistern and the low free residual chlorine concentration (0.07 mg/L) and predominance of Betaproteobacteria (63% of viable cells) that were immediately detected after the truck had filled up at the water treatment plant was indicative of contamination by particulate matter. Given these findings, First Nation residents living without running water and relying on inadequate water distribution systems are at higher risk of contracting water-born illnesses. We urge all governments in Canada to expand their investments in supporting and sustaining water as a human right in Canada's First Nations communities. 3 GRAPHICAL ABSTRACT