Filter-Based Assay for Escherichia coli in Aqueous Samples Using Bacteriophage-Based Amplification

Abstract

This paper describes a method to detect the presence of bacteria in aqueous samples, based on the capture of bacteria on a syringe filter, and the infection of targeted bacterial species with a bacteriophage (phage). The use of phage as a reagent provides two opportunities for signal amplification: (i) the replication of phage inside a live bacterial host and (ii) the delivery and expression of the complementing gene that turns on enzymatic activity and produces a colored or fluorescent product. Here we demonstrate a phage-based amplification scheme with an M13KE phage that delivers a small peptide motif to an F(+), α-complementing strain of Escherichia coli K12, which expresses the ω-domain of β-galactosidase (β-gal). The result of this complementation-an active form of β-gal-was detected colorimetrically, and the high level of expression of the ω-domain of β-gal in the model K12 strains allowed us to detect, on average, five colony-forming units (CFUs) of this strain in 1 L of water with an overnight culture-based assay. We also detected 50 CFUs of the model K12 strain in 1 L of water (or 10 mL of orange juice, or 10 mL of skim milk) in less than 4 h with a solution-based assay with visual readout. The solution-based assay does not require specialized equipment or access to a laboratory, and is more rapid than existing tests that are suitable for use at the point of access. This method could potentially be extended to detect many different bacteria with bacteriophages that deliver genes encoding a full-length enzyme that is not natively expressed in the target bacteria.

Detection of Escherichia coli in Drinking Water Using T7 Bacteriophage-Conjugated Magnetic Probe

“…Second, they allow for the separation of the bacteria from matrices like: milk, orange juice, turbid water and so on, that would otherwise interfere with colorimetric assays. 7,9 Smartphone-based technology has become increasingly more popularly for use in analytical diagnostics. Erickson and his co-workers developed a smartphone platform to quantify 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 vitamin D levels and detect pH change in saliva and sweat.…”

Colorimetric Detection of Escherichia coli Based on the Enzyme-Induced Metallization of Gold Nanorods

“…We next used our enzyme‐induced metallization colorimetric assay to detect E. coli cells. β‐gal is an essential enzyme in the inducible lactose transport and metabolism system of E. coli , and can be used as an indicator for their presence . Because phages can distinguish between live and inactive bacteria, their use as detection probes reduces the possibility of false‐positive results .…”

Effects of Environmental and Clinical Interferents on the Host Capture Efficiency of Immobilized Bacteriophages

“…5 Furthermore, phagefunctionalized surfaces can be used in biosensor designs using platforms such as surface plasmon resonance, 6-7 quartz crystal microbalance, 8 or dip-stick assays. 9 In an earlier study, we demonstrated the efficacy of a phage-functionalized surface for the capture and deactivation of bacteria. 10 The findings emphasized how the orientation of the immobilized phage, specifically the position of its host binding proteins on the surface, played a significant role in the capture of host bacteria.…”