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.
Highlights
The guidelines for monitoring bacterial contamination in a public supply of water, or in a supply of food, are stringent to mitigate threats to public health
Analytical methods approved by the Environmental Protection Agency (EPA) must ensure that a public water supply maintains fewer than one colony-forming unit (CFU) of coliform bacteria in 100 mL of water,[1] and the Food and Drug Administration (FDA) has a “zero tolerance” policy for the presence of bacterial species such as E. coli O157:H7, Salmonella sp., and Listeria monocytogenes in foodstuffs.[2,3]
Microbiological cultures determine the number of live bacteria in a sample, but require incubation periods ranging from several hours to several days at temperatures between 30 - 45 oC;[7,8] the “rapid” test for coliform bacteria approved by the EPA requires a 16-hour incubation at 35 oC
Summary
The guidelines for monitoring bacterial contamination in a public supply of water, or in a supply of food, are stringent to mitigate threats to public health. Several commercial products are available to detect coliform bacteria in a sample of water, at the point of access, but these products are culture-based, and require incubation periods of 12 hours or longer.
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