Reporter Phage-Based Detection of Bacterial Pathogens: Design Guidelines and Recent Developments

Abstract

Fast and reliable detection of bacterial pathogens in clinical samples, contaminated food products, and water supplies can drastically improve clinical outcomes and reduce the socio-economic impact of disease. As natural predators of bacteria, bacteriophages (phages) have evolved to bind their hosts with unparalleled specificity and to rapidly deliver and replicate their viral genome. Not surprisingly, phages and phage-encoded proteins have been used to develop a vast repertoire of diagnostic assays, many of which outperform conventional culture-based and molecular detection methods. While intact phages or phage-encoded affinity proteins can be used to capture bacteria, most phage-inspired detection systems harness viral genome delivery and amplification: to this end, suitable phages are genetically reprogrammed to deliver heterologous reporter genes, whose activity is typically detected through enzymatic substrate conversion to indicate the presence of a viable host cell. Infection with such engineered reporter phages typically leads to a rapid burst of reporter protein production that enables highly sensitive detection. In this review, we highlight recent advances in infection-based detection methods, present guidelines for reporter phage construction, outline technical aspects of reporter phage engineering, and discuss some of the advantages and pitfalls of phage-based pathogen detection. Recent improvements in reporter phage construction and engineering further substantiate the potential of these highly evolved nanomachines as rapid and inexpensive detection systems to replace or complement traditional diagnostic approaches.

The Many Applications of Engineered Bacteriophages—An Overview

“…Bacteria are the simplest target as bacteriophages have evolved to bind to them. A number of detection systems simply rely on the reproduction of bacteriophages to indicate the presence of the corresponding host cells [ 116 , 117 ]. These methods are not especially rapid as it generally takes extended culture to detect sufficient bacteriophages either by plaque formation or increases in specific phage proteins.…”

The Age of Phage: Friend or Foe in the New Dawn of Therapeutic and Biocontrol Applications?

“…Lytic and lysogenic phages can be engineered to encode reporting elements to be used as markers, as is the case with luminescent, fluorescent, and colorimetric detection assays [284]. Upon host infection, the expression of reporter proteins such as fluorescent proteins, luciferases, and hydrolyzing enzymes aids in amplifying the detection signal with the addition of a substrate [285]. Reporter phages which have been engineered to exhibit bioluminescence upon infection of targeted bacterial hosts have been used for the detection of Staphylococcus aureus [286], Listeria monocytogenes [287], Salmonella spp.…”

The State of the Art in Biodefense Related Bacterial Pathogen Detection Using Bacteriophages: How It Started and How It’s Going

“…There are many more examples of how phages are used for pathogen detection for non-biothreat organisms that are well reviewed [2][3][4][6][7][8]. This is in part due to the greater interest in bacteria that cause food borne illness and other common bacterial pathogens that are easier to work with in the laboratory and do not have biosecurity concerns.…”

“…From the Center for Disease Control and Prevention's (CDC) gold standard for "phage typing" bacterial isolates to the development of the phage display system for discovery of binding peptides and antibodies, researchers have found creative ways to exploit phages for novel pathogen detection platforms. There are many reviews on the use of phages for pathogen detection using different assays, platforms and biosensors [1][2][3][4][5][6][7][8][9]. This review will focus on how phages have been exploited for detection of those pathogens that are of most concern due to their history and potential for use as biowarfare and bioterrorism agents.…”