ABSTRACT
Wastewater-based epidemiology (WBE) is useful for the surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in communities, complementing clinical diagnostic testing of individuals. In this Review, we summarize recent progress and highlight remaining challenges in monitoring SARS-CoV-2 RNA in wastewater systems for community and environmental surveillance. Very low concentrations of viral particles and RNA present in the complicated wastewater and sewage sample matrix require efficient sample processing and sensitive detection. We discuss advantages and limitations of available methods for wastewater sample processing, including collection, separation, enrichment, RNA extraction, and purification. Efficient extraction of the viral RNA and removal of interfering sample matrices are critical to the subsequent reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for sensitive detection of SARS-CoV-2 in wastewater. We emphasize the importance of implementing appropriate controls and method validation, which include the use of surrogate viruses for assessing extraction efficiency and normalization against measurable chemical and biological components in wastewater. Critical analysis of the published studies reveals imperative research needs for the development, validation, and standardization of robust and sensitive methods for quantitative detection of viral RNA and proteins in wastewater for WBE.
ABSTRACT
We report the existence and resuscitation of viable but nonculturable (VBNC) Escherichia coli O157:H7 cells in drinking water induced by the common point-of-use disinfection treatments of boiling or microwaving. Tap water and saline samples containing E. coli O157:H7 culturable cells from a bovine isolate or two clinical isolates were boiled (1, 10, or 15 min) on a hot plate or microwaved (1.5 min) to reach boiling. No culturable E. coli O157:H7 cells were observed in the treated samples using conventional plating methods. In samples boiled for 1 or 10 min, two viability assays separately detected that 2-5.5% of the cells retained an intact membrane, while 28 to 87 cells out of the initial 108 cells retained both measurable intracellular esterase activity and membrane integrity. In samples boiled for 15 min, no viable cells were detected. The microwaved samples contained 6-10% of cells with an intact membrane, while 21 to 108 cells out of the initial 108 cells retained both membrane integrity and esterase activity. The number of viable cells retaining both metabolic activity and membrane integrity were consistent in all samples, supporting the survival of a small number of E. coli O157:H7 cells in the VBNC state after boiling for 1 or 10 min or microwaving. Furthermore, the VBNC E. coli O157:H7 cells regained growth at 37 °C in culture media containing autoinducers produced by common non-pathogenic E. coli, commonly present in the human intestine, and norepinephrine. The resuscitated cells were culturable on conventional plates and expressed mRNA encoding the E. coli O157 lipopolysaccharide gene (rfbE) and the H7 flagellin gene (fliC). This study highlights potential concerns for public health risk management of VBNC E. coli O157:H7 in drinking water disinfected by heat treatment at point-of-use. The public health significance of these concerns warrants further investigation.
