Aims:  To investigate the effect of oxygen limitation, glucose‐starvation and temperature on the susceptibility of Escherichia coli towards the quaternary ammonium biocide benzalkonium chloride (BAC). Methods and Results:  The effect of BAC on planktonic and sessile cells were investigated using the gfp‐tagged E. coli K‐12 strain MG1655[pOX38Km]. Increasing temperature from 10°C to 30°C increased the bactericidal effect of BAC for both starved and nonstarved E. coli under aerobic and anaerobic conditions. The lowest minimum bactericidal concentration was observed for cells in anaerobic media at 30°C (30 mg l−1 BAC). Decreasing cell densities increased the decay rate for BAC‐exposed cells for both starved and nonstarved E. coli. Biofilms of E. coli exposed to BAC in anaerobic medium showed a greater percentage of membrane‐compromised cells than biofilms grown in aerobic medium. Image analyses of BAC‐exposed biofilms showed that membrane‐compromised cells were occasionally located in the interior structure of the biofilm microcolonies. Conclusions:  Increasing temperatures and the absence of oxygen, and energy substrates increased the antimicrobial effect of BAC towards E. coli. Significance and Impact of the Study:  The results are relevant for understanding the disinfection efficacy of quaternary ammonium compounds towards planktonic and sessile bacteria.

Traditional cultivation-dependent tests for coliform bacteria in food and drinking water take 18-24 h to complete. Bioluminescence-based enzyme assays can potentially reduce analysis time for indicator bacteria such as coliforms. In the present study, we developed a simple presence/absence (P/A) bioluminescence procedure for rapid detection of coliform bacteria in groundwater-based drinking water. The bioluminescence procedure targeting b-D-galactosidase activity in coliform bacteria was based on hydrolysis of 6-O-b-galactopyranosyl-luciferin. Bacteria immobilized on membrane filters were enriched for 6-8 h in selective media containing isopropyl-b-D-thiogalactopyranoside (IPTG) to induce b-D-galactosidase activity in coliform bacteria. The equivalent of approximately 300 E. coli cells was required for bioluminescence detection of b-D-galactosidase activity. In comparison, PCR based detection of E. coli in drinking water required approximately 30 target cells. Analysis of contaminated drinking water samples showed comparable results for coliform bacteria using traditional multiple-tube fermentation, Colilert-18, and the bioluminescence procedure. Aeromonas hydrophila or indigenous groundwater bacteria did not interfere with the procedure. The bioluminescence procedure can be combined with commercial substrates such as Fluorocult or Colilert-18, and will allow the detection of one coliform in 100 ml drinking water within one working day. The results suggest the bioluminescence assays targeting b-D-galactosidase activity may be used for or for early warning screening of drinking water and/or rapid identification of contaminated drinking water wells.

Land slugs are occasionally observed as contaminants in groundwater wells and drinking water treatment plants including storage tanks. Slugs may feed on carrion and feces, and they are potential vectors of pathogens such as Listeria monocytogenes. We isolated L. monocytogenes from the pest slug Arion vulgaris and examined persistence and survival of human and slug derived L. monocytogenes in groundwater-based drinking water and biofilms. L. monocytogenes survival was evaluated using cultivation and cultivation-independent techniques. L. monocytogenes remained culturable for 35–47 days in drinking water with first order decay rates between 0.314 and 0.457 h−1 (T99 ≥10 days). Attachment of L. monocytogenes to filter sand delayed washout from drinking water filters and increased persistence 2–3-fold. Indigenous biofilms stimulated initial surface attachment 10–100 fold but L. monocytogenes declined more rapidly in drinking water biofilms compared to virgin filters not colonized by microorganisms. Grazing by protozoa likely attenuated L. monocytogenes survival in some drinking water biofilms. A comparable survival pattern was observed for L. monocytogenes and the fecal indicator bacterium E. coli. The study suggests that live L. monocytogenes can persist for weeks as sessile organisms in groundwater-based drinking water supplies and may subsequently be released to the drinking water.