Rapid detection of Escherichia coli using bacteriophage-induced lysis and image analysis

Abstract

Rapid detection of bacterial pathogens is a critical unmet need for both food and environmental samples such as irrigation water. As a part of the Food safety Modernization Act (FSMA), The Produce Safety rule has established several requirements for testing for the presence of generic Escherichia coli in water, but the current method available for testing (EPA M1603) demands specified multiple colony verification and highly trained personnel to perform these tests. The purpose of the study was to assess a phage induced bacterial lysis using quantitative image analysis to achieve rapid detection of E. coli at low concentrations within 8 hours. This study aimed to develop a simple yet highly sensitive and specific approach to detect target bacteria in complex matrices. In the study, E. coli cells were first enriched in tryptic soy broth (TSB), followed by T7 phage induced lysis, concentration, staining and fluorescent imaging. Image analysis was conducted including image pre-processing, image segmentation and quantitatively analysis of cellular morphological features (area, eccentricity and full width at half maximum). Challenge experiments using realistic matrices, including simulated fresh produce wash water, coconut water and spinach wash water, demonstrated the method can be applied for use in situations that occur in food processing facilities. The results indicated E. coli cells that are lysed by T7 phages demonstrated significantly (P < 0.05) higher extracellular DNA release, altered cellular shape (from rod to circular) and diffused fluorescent signal intensity. Using this biosensing strategy, a sensitivity to detect Escherichia coli at 10 CFU/ml within 8 hours was achieved, both in laboratory medium and in complex matrices. The proposed phage based biosensing strategy enables rapid detection of bacteria and is applicable to analysis of food systems. Furthermore, the steps involved in this assay can be automated to enable detection of target bacteria in food facilities without extensive resources.

Application of Engineered Bacteriophage T7 in the Detection of Bacteria in Food Matrices

“…Moreover, using soluble substrates requires the release of enzyme from cells to the aqueous phase, which may not be efficient as alkaline phosphatase have high molecular weight of 94 kDa. In our prior research, we have observed a significant retention of cellular content including DNA and cell membrane after bacteriophage lysis of cells (Yang et al, 2020). Thus, an extended time of incubation may result in more efficient release of enzymes from residual cellular content after lysis as well as multiple repeat cycles of phage infection and the expression of exogenous enzyme can increase the effective enzyme concentration.…”

An Engineered Reporter Phage for the Fluorometric Detection of Escherichia coli in Ground Beef

“…The fact that T7 bacteriophage genome is relatively simple has allowed multiple genetic modifications by inserting different reporter genes. A number of phage-based bacteria detection approaches have been discussed and improved for higher bacteria detection sensitivity [ 14 , 17 , 29 , 30 ]. To extend the applicability of this system, our study offers a straightforward and rapid strategy using engineered T7 phages to detect E. coli in ground beef.…”

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

“…in fluid from orthopedic artificial joints [53]. Other methods have visualized the change in redox reactions by Salmonella enterica Typhi and Paratyphi after phage infection [81], or measured the release of eDNA from phage infected E. coli [82]. Table 1 provides a detailed overview of phage amplification-based detection assays for bacterial pathogens.…”