Chlorination effects on DNA based characterization of water microbiomes and implications for the interpretation of data from disinfected systems.

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.

Attenuation of bacterial hazard indicators in the subsurface of an informal settlement and their application in quantitative microbial risk assessment.

Pit latrines provide essential onsite sanitation services to over a billion people, but there are concerns about their role in infectious disease transmission, and impacts on groundwater resources. We conducted fieldwork in an informal settlement in Dar es Salaam, where cholera is endemic. We combined plate counting with portable MinION sequencing and quantitative polymerase chain reaction (qPCR) methods for characterization of bacteria in pit latrine sludge, leachate, shallow and deep groundwater resources. Pit latrine sludge was characterized by log10 marker gene concentrations per 100 mL of 11.2 ± 0.2, 9.9 ± 0.9, 6.0 ± 0.3, and 4.4 ± 0.8, for total bacteria (16S rRNA), E. coli (rodA), human-host-associated Bacteroides (HF183), and Vibrio cholerae (ompW), respectively. The ompW gene observations suggested 5 % asymptomatic Vibrio cholerae carriers amongst pit latrine users. Pit leachate percolation through one-meter-thick sand beds attenuated bacterial hazard indicators by 1 to 4 log10 units. But first-order removal rates derived from these data substantially overestimated the longer-range hazard attenuation in the sand aquifers. Cooccurrence of human sewage marker gene HF183 in all shallow groundwater samples testing positive for ompW genes demonstrated the human origin of Vibrio cholerae hazards in the subsurface. All borehole water samples tested negative for ompW and HF183 genes, but 16S rRNA gene sequencing data suggested ingress of faecal pollution into boreholes at the peak of the "long rainy season". Quantitative microbial risk assessment (QMRA) predicted a gastrointestinal disease burden of 0.05 DALY per person per year for the community, well above WHO targets of 10-4-10-6 DALY for disease related to drinking water.