A Syringe-Based Biosensor to Rapidly Detect Low Levels of Escherichia Coli (ECOR13) in Drinking Water Using Engineered Bacteriophages

Abstract

A sanitized drinking water supply is an unconditional requirement for public health and the overall prosperity of humanity. Potential microbial and chemical contaminants of drinking water have been identified by a joint effort between the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF), who together establish guidelines that define, in part, that the presence of Escherichia coli (E. coli) in drinking water is an indication of inadequate sanitation and a significant health risk. As E. coli is a nearly ubiquitous resident of mammalian gastrointestinal tracts, no detectable counts in 100 mL of drinking water is the standard used worldwide as an indicator of sanitation. The currently accepted EPA method relies on filtration, followed by growth on selective media, and requires 24-48 h from sample to results. In response, we developed a rapid bacteriophage-based detection assay with detection limit capabilities comparable to traditional methods in less than a quarter of the time. We coupled membrane filtration with selective enrichment using genetically engineered bacteriophages to identify less than 20 colony forming units (CFU) E. coli in 100 mL drinking water within 5 h. The combination of membrane filtration with phage infection produced a novel assay that demonstrated a rapid, selective, and sensitive detection of an indicator organism in large volumes of drinking water as recommended by the leading world regulatory authorities.

Keywords: E.coli; bacteriophage; drinking water; rapid detection.

Figure 1

Assay format for the detection of E. coli in drinking water. (a) The water is filtered through a 0.22 µm cellulose filter in order to separate the bacteria. (b) The filter is then removed from the housing and placed on LB media in order to resuscitate the trapped bacteria. (c) Following the application of the engineered phages (grey), an infection cycle results in the expression and release of a reporter enzyme consisting of NanoLuc (blue) and a carbohydrate binding module (orange) with specificity to cellulose. (d) The fusion enzyme binds to the cellulose filter and the luminescent activity can then be determined.

Figure 2

Phages (NRGp5 or T7 Select) were added to individual cultures of E. coli (ECOR13) after 3 h of enrichment. The E. coli in samples without phages added continued to the exponential phase, while the application of phages resulted in a decline in optical density after about two hours.

Figure 3

Luminescence was measured from E. coli samples with and without the phages of T7 Select, which did not contain a gene for a reporter enzyme, NRGp4, but which contained a gene for NanoLuc-CBM, and NRGp5, which contained an optimized NanoLuc-CBM gene. It can be seen that the reporter genes resulted in luminescence with the optimized gene providing a higher signal. Error bars indicate the standard deviation of three biological triplicates and the limit of detection (lowest positive signal) was calculated as the negative control + 3x the standard deviation. The standard deviation for phages NRGp4 and NRGp5 was too low to produce visible error bars at the final time points of the assay.

Figure 4

NRGp5 was added at a high multiplicity of infection (MOI) to varying concentrations of E. coli ECOR13. The luminescence of the lysate resulting from the infections was measured following the addition of substrate. The results suggest relative relationship (R² = 0.9941) between the concentration of E. coli and the generation of the luminescent signal over several orders of magnitude.

Figure 5

To demonstrate the ability of the phage-based assay to distinguish between viable and non-viable E. coli ECOR13 cells, 10³ bacterial cells were exposed to either 70% ethanol or PBS, and washed. The cells were then either infected with NRGp5 or incubated without phages. The only cell treatment regime to display luminescence following incubation and substrate addition was the non-ethanol treated E. coli ECOR13 infected with NRGp5.

Figure 6

Performance of the phage-based syringe filter detection assay compared to standard plate counts. Water (100 mL) was processed through a syringe filter (0.22 um, regenerated cellulose) where bacteria (E. coli ECOR13) were enriched prior to the addition of phage NRGp5. Reporter enzymes were expressed, immobilized onto the cellulose filter, and luminescence was measured.