Phage-Based Fluorescent Biosensor Prototypes to Specifically Detect Enteric Bacteria Such as E. coli and Salmonella enterica Typhimurium

Abstract

Water safety is a major concern for public health and for natural environment preservation. We propose to use bacteriophages to develop biosensor tools able to detect human and animal pathogens present in water. For this purpose, we take advantage of the highly discriminating properties of the bacteriophages, which specifically infect their bacterial hosts. The challenge is to use a fluorescent reporter protein that will be synthesized, and thus detected, only once the specific recognition step between a genetically modified temperate bacteriophage and its bacterial host has occurred. To ensure the accuracy and the execution speed of our system, we developed a test that does not require bacterial growth, since a simple 1-hour infection step is required. To ensure a high sensitivity of our tool and in order to detect up to a single bacterium, fluorescence is measured using a portable flow cytometer, also allowing on-site detection. In this study, we have constructed and characterized several "phagosensor" prototypes using the HK620 bacteriophage and its host Escherichia coli TD2158 and we successfully adapted this method to Salmonella detection. We show that the method is fast, robust and sensitive, allowing the detection of as few as 10 bacteria per ml with no concentration nor enrichment step. Moreover, the test is functional in sea water and allows the detection of alive bacteria. Further development will aim to develop phagosensors adapted on demand to the detection of any human or animal pathogen that may be present in water.

New strategies for the enumeration of enteric pathogens in water

“…The multiplicity of the infection is a natural amplification of a signal and increases the sensitivity of the method. Unfortunately, this type of phage detection is not a viable quantification method when testing natural water sources because VBNC or injured cells will not replicate the phage dependably (Vinay et al ., ; Wang et al ., ).…”

Detection of Microbiological Content in Water as Indicator of Its Ecological State − Mini‐Review

“…For instance, light emission signal is received by whole cell biosensor where, in the presence of gene‐inducing environmental pollutants, cells react with production of fluorescent proteins . Biosensor prototype based on modified bacteriophage carrying gene of fluorescent reporter protein was successfully tested for the detection of E. coli in sea water samples .…”

Quantitative and synthetic biology approaches to combat bacterial pathogens

“…To facilitate detection and quantification, engineered phages have been equipped with a wide range of reporter genes, which code for fluorescence [57] or enzymatic activities [58-60]. These readouts are often more rapid to quantify and require less steps than plaque assays or immunoassays [61].…”