Natural freeze-thaw cycles may increase the risk associated with Salmonella contamination in surface and groundwater environments

Abstract

Groundwater contamination by bacteria poses a serious threat to our drinking water supplies. In cold climate regions, microorganisms introduced to upper soil layers by spreading of animal manure are subject to low temperatures and multiple cycles of freezing and thawing at the beginning of winter and during spring melt. We investigated the influence of temperature fluctuations around the freezing point, known as freeze-thaw (FT), on the inactivation rates, growth, and biofilm formation of a manure-isolated strain of Salmonella typhimurium. Moreover, the effects of FT on the transport characteristics of S. typhimurium in quartz sand were monitored in model porewater solutions of two different ionic strengths (IS: 10 and 100 mM KCl) and two different humic acid (HA) concentrations (1 and 5 mg/L). Increasing numbers of FT cycles were found to decrease the deposition of S. typhimurium onto quartz sand and increase the percentage of detached cells in sand-packed column experiments. Based on the calculated bacterial attachment efficiencies, the predicted minimum setback distances between the location of water supply wells and manure spreading activities are higher when the effects of FT are taken into consideration. While FT treatment significantly affected cell viability (in the presence of HA), most cells were in a viable but non-culturable (VBNC) state with compromised ability to form biofilm. This investigation demonstrates the effects of spring temperature variations in upper soil layers on S. typhimurium properties and the potential increased risk of bacterial contamination in representative aquifer environments in cold climate regions.

Keywords: Bacteria transport; Climate change; Freeze-thaw; Groundwater contamination; Public health risk; Salmonella.

Graphical abstract

Fig. 1

Breakthrough curves (BTCs) for transport of S. typhimurium in columns packed with clean quartz sand at 10 °C in (a) 10 mM KCl, (b) 100 mM KCl, (c) 10 mM KCl + 1 mg/L humic acid (HA) and, (d) 10 mM KCl + 5 mg/L HA, before FT treatment (-□-), after 4 FT (-○-), and after 10 FT (-Δ-). BTCs were identical for two replicate experiments.

Fig. 2

Detachment curves obtained by injecting a lower IS solution after BTCs of S. typhimurium through clean quartz sand at 10 °C in (a) 10 mM KCl (b) 100 mM KCl (c) 10 mM KCl + 1 mg/L HA and (d) 10 mM KCl + 5 mg/L HA before FT treatment (-□-), after 4 FT (-○-), and after 10 FT (-Δ-). Detachment curves were identical for two replicate experiments.

Fig. 3

Effects of freeze-thaw (FT) on S. typhimurium culturability (columns) on LB agar plates. The viability (cell membrane integrity) (-●-) of S. typhimurium cells in the column influent and effluent at different time points was measured using Live/Dead assay at 10 °C in (a) 10 mM KCl (b) 100 mM KCl (c) 10 mM KCl + 1 mg/L humic acid (HA) and (d) 10 mM KCl + 5 mg/L HA before FT treatment, after 4 FT, and after 10 FT. Viability results represent mean values ± SD for two replicate experiments and CFU/mL results represent mean values ± SD for three independent experiments. Asterisks indicate a statistically significant difference of results when compared to Before FT and control conditions (128 hr and 320 hr in KCl or LB) which was determined using Student's t-test (*: p < 0.05).

Fig. 4

Characterization of the effects of freeze-thaw (FT) on (a) S. typhimurium biofilm formation and (b) S. typhimurium growth rates in microtitre plate wells (at 37 °C) before FT treatment (-□-), after 4 FT (–+–), after 10 FT (--), after 128 hr (Ctrl) in 10 mM KCl (-○-), after 128 hr (Ctrl) in LB (-Δ-), after 320 hr (Ctrl) in KCl (–◊–) and after 320 hr (Ctrl) in LB (-□-). Normalized ODa = [OD – ODblank]/([ODCtrl – ODblank ]* [ODplanktonic – ODblank ]) and Normalized ODb = [OD – ODblank]/([ODCtrl – ODblank ]). Biofilm formation results represent mean values ± SD for three independent experiments and planktonic growth results represent mean values for two replicate experiments. Asterisks indicate a statistically significant difference of results when compared to Before FT and control conditions (128 hr and 320 hr in KCl or LB) which was determined using Student's t-test (*: p < 0.05).