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

• Published in: Analyst (London) 2015-11, Vol.140 (22), p.7629-7636
• Main Authors: Alcaine, S D, Pacitto, D, Sela, D A, Nugen, S R
• Format: Article
• Language: English
• Subjects: Alkaline phosphatase; Alkaline Phosphatase - genetics; Alkaline Phosphatase - metabolism; ampicillin; antibiotic resistance; Antibiotics; Bacteria; Bacteriophage T7 - enzymology; Bacteriophage T7 - genetics; Bacteriophages; Biosensing Techniques - methods; capital; Chemiluminescence; Colorimetry; diagnostic techniques; Enzyme Assays - methods; Escherichia coli; Escherichia coli - genetics; Escherichia coli - isolation & purification; Escherichia coli - metabolism; Escherichia coli - virology; Escherichia coli Infections - diagnosis; Escherichia coli Infections - microbiology; genes; Genetic Engineering - methods; Humans; Laboratory equipment; Phage; Up-Regulation
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c5an01181g

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.