A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water

Troy C Hinkley,Sam R Nugen,Joey N Talbert,Spencer Garing,Anne-Laure M Le Ny,Joseph E Peters,Kevin P Nichols,Sangita Singh

doi:10.1038/s41598-018-33097-4

Abstract

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100 mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specific enumeration of E. coli in contaminated drinking water is critical to mitigate significant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane filtration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifically engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specific to cellulose. These constructs were then inserted into the E. coli-specific phage T7 which were used to infect E. coli trapped on a cellulose filter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose filter in proximity to the lysed cells. Following substrate addition, the location and quantification of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed.

Highlights

Clean drinking water has been declared a fundamental human right, yet millions still lack access to consistently clean sources of potable water[1]
Detection of bacterial pathogens or their indicators in larger samples of water is critical to ensuring safe drinking water in developing countries
Given the zero tolerance regulatory requirements for drinking water of 0 CFU/100 mL, sample sizes below 100 mL risk the possibility of false negative results

Summary

Introduction

Clean drinking water has been declared a fundamental human right, yet millions still lack access to consistently clean sources of potable water[1]. The United States EPA and FDA have set a limit of zero CFU generic E. coli in 100 mL for drinking water and postharvest produce rinse water, respectively. Untreated agricultural water, such as that used for irrigation, has a maximum geometric mean (GM) of 126 CFU or less with a statistical threshold value (STV) of 410 or less of generic E. coli in a 100 mL sample[11]. The tubes are assessed for bacterial growth and the highest dilution numbers for each replicate are used to statistically estimate the MPN of the original sample While both EPA 1604 and MPN can detect a single E. coli CFU in 100 mL of water, the prolonged incubation periods necessary for visual identification make them less practical for time sensitive applications. There is a significant need to rapidly detect E. coli in water samples while maintaining high sensitivity and quantification

Methods

Results

Conclusion