Next-Generation Diagnostics: Multiparametric Bacteriophage Reporters for RealTime Pathogen Detection and Phage Therapy Monitoring

Abstract

Bacteriophage-based diagnostic systems are promising alternatives to conventional bacterial detection methods because they offer host specificity, rapid signal generation, and the ability to distinguish between viable and non-viable bacterial cells. However, these systems often rely on substrates or include large DNA inserts into the phage genome. This study explored the feasibility of a peptide-based reporter phage strategy as a preliminary proof-of-concept model.

Bacteriophage EPIMAM01, a lytic Escherichia coli phage, was selected as the model phage based on its genomic characteristics and host range profile. The dispensable Soc gene was chosen as the intended target locus for future genome modification. Compatibility between EPIMAM01 and commercially available NEB 10-beta E. coli was first verified by plaque assay and efficiency-of-plating experiments. A vector plasmid was successfully recombined using a reporter gene insert with the native promoter through Golden Gate Assembly and transforming NEB bacteria were generated through Heat Shock. Primers amplifying the Soc region and insert sequence were validated for downstream applications through PCR amplification. Molecular validation confirming successful assembly of the recombinant construct was achieved through PCR and nanopore sequencing. Additionally, an ELISA-based detection system was optimized using three synthetic peptides for the detection of natural peptide in downstream applications.

On the other hand, the recombineering experiment, which aimed to introduce the recombinant plasmid into phage EPIMAM01 for homologous recombination and peptide expression, failed to produce a detectable signal via ELISA. This implied the lack of peptide production. Additionally, PCR amplification experiments with the phage post-NEB infection using insert-amplifying primers pointed to unsuccessful recombinant phage production. The results of these experiments suggested that homologous recombination most probably did not occur, or it occurred at a low frequency and could not be detected.

Overall, this study established several foundational components of a peptide-based reporter phage workflow, including host-phage compatibility assessment, target-region validation, recovery of candidate plasmid clones, and optimization of peptide immunodetection. However, it was incapable of detecting peptide production and generating recombinant phages. Future optimization will include using a CRISPR-Cas13 selection system and a different insert sequence with a stronger promoter to allow for better transcription.