Phage & phosphatase: a novel phage-based probe for rapid, multi-platform detection of bacteria

Abstract

Genetic engineering of bacteriophages allows for the development of rapid, highly specific, and easily manufactured probes for the detection of bacterial pathogens. A challenge for novel probes is the ease of their adoption in real world laboratories. We have engineered the bacteriophage T7, which targets Escherichia coli, to carry the alkaline phosphatase gene, phoA. This inclusion results in phoA overexpression following phage infection of E. coli. Alkaline phosphatase is commonly used in a wide range of diagnostics, and thus a signal produced by our phage-based probe could be detected using common laboratory equipment. Our work demonstrates the successful: (i) modification of T7 phage to carry phoA; (ii) overexpression of alkaline phosphatase in E. coli; and (iii) detection of this T7-induced alkaline phosphatase activity using commercially available colorimetric and chemilumiscent methods. Furthermore, we demonstrate the application of our phage-based probe to rapidly detect low levels of bacteria and discern the antibiotic resistance of E. coli isolates. Using our bioengineered phage-based probe we were able to detect 10(3) CFU per mL of E. coli in 6 hours using a chemiluminescent substrate and 10(4) CFU per mL within 7.5 hours using a colorimetric substrate. We also show the application of this phage-based probe for antibiotic resistance testing. We were able to determine whether an E. coli isolate was resistant to ampicillin within 4.5 hours using chemiluminescent substrate and within 6 hours using a colorimetric substrate. This phage-based scheme could be readily adopted in labs without significant capital investments and can be translated to other phage-bacteria pairs for further detection.

Methods for detection of viable foodborne pathogens: current state-of-art and future prospects

“…Examples of intracellular markers are adenosine-5 triphosphate or ATP (Griffiths 1996;Blasco et al 1998) and β-galactosidase (Neufeld et al 2003;Burnham et al 2014;Chen et al 2015). More recently, use of engineered lysogenic bacteriophages has also permitted the development of other prototype phage-based tests using a wider range of enzymatic reactions, including luciferase-based (Zhang et al 2016;Franche et al 2017), protease-based (Alcaine et al 2015a), and alkaline phosphatase-based (Alcaine et al 2015b) phage detection methods. Finally, as released intracellular content is highly conductive, changes of conductivity in the surrounding environment can be used as a signal of viable bacteria present in the sample.…”

Genetically modified bacteriophages in applied microbiology

“…One of the possible applications of these systems could be monitoring of antibiotic susceptibility of pathogens, as the luminescence signal response is dependent on host cell fitness (Alcaine et al . ).…”

Integrating recognition elements with nanomaterials for bacteria sensing

“…35 In addition, T7 phages were engineered to carry alkaline phosphatase or tobacco etch virus protease gene, generating phage-based platform for bacteria detection. 36–37 …”