In-situ fluorescence spectroscopy is a more rapid and resilient indicator of faecal contamination risk in drinking water than faecal indicator organisms.

Faecal indicator organisms (FIOs) are limited in their ability to protect public health from the microbial contamination of drinking water because of their transience and time required to deliver a result. We evaluated alternative rapid, and potentially more resilient, approaches against a benchmark FIO of thermotolerant coliforms (TTCs) to characterise faecal contamination over 14 months at 40 groundwater sources in a Ugandan town. Rapid approaches included: in-situ tryptophan-like fluorescence (TLF), humic-like fluorescence (HLF), turbidity; sanitary inspections; and total bacterial cells by flow cytometry. TTCs varied widely in six sampling visits: a third of sources tested both positive and negative, 50% of sources had a range of at least 720 cfu/100 mL, and a two-day heavy rainfall event increased median TTCs five-fold. Using source medians, TLF was the best predictor in logistic regression models of TTCs ≥10 cfu/100 mL (AUC 0.88) and best correlated to TTC enumeration (ρs 0.81), with HLF performing similarly. Relationships between TLF or HLF and TTCs were stronger in the wet season than the dry season, when TLF and HLF were instead more associated with total bacterial cells. Source rank-order between sampling rounds was considerably more consistent, according to cross-correlations, using TLF or HLF (min ρs 0.81) than TTCs (min ρs 0.34). Furthermore, dry season TLF and HLF cross-correlated more strongly (ρs 0.68) than dry season TTCs (ρs 0.50) with wet season TTCs, when TTCs were elevated. In-situ TLF or HLF are more rapid and resilient indicators of faecal contamination risk than TTCs.

Tryptophan-like and humic-like fluorophores are extracellular in groundwater: implications as real-time faecal indicators.

Fluorescent natural organic matter at tryptophan-like (TLF) and humic-like fluorescence (HLF) peaks is associated with the presence and enumeration of faecal indicator bacteria in groundwater. We hypothesise, however, that it is predominantly extracellular material that fluoresces at these wavelengths, not bacterial cells. We quantified total (unfiltered) and extracellular (filtered at < 0.22 µm) TLF and HLF in 140 groundwater sources across a range of urban population densities in Kenya, Malawi, Senegal, and Uganda. Where changes in fluorescence occurred following filtration they were correlated with potential controlling variables. A significant reduction in TLF following filtration (ΔTLF) was observed across the entire dataset, although the majority of the signal remained and thus considered extracellular (median 96.9%). ΔTLF was only significant in more urbanised study areas where TLF was greatest. Beneath Dakar, Senegal, ΔTLF was significantly correlated to total bacterial cells (ρs 0.51). No significant change in HLF following filtration across all data indicates these fluorophores are extracellular. Our results suggest that TLF and HLF are more mobile than faecal indicator bacteria and larger pathogens in groundwater, as the predominantly extracellular fluorophores are less prone to straining. Consequently, TLF/HLF are more precautionary indicators of microbial risks than faecal indicator bacteria in groundwater-derived drinking water.