P. aeruginosa is a bacterial pathogen responsible for a wide range of infections. As a result, the
World Health Organization identified it as one of the top priority pathogens that urgently calls for
the development of novel treatments. Bacteriophages have emerged as a promising therapeutic
approach and their properties can be enhanced by phage-engineering. This opens an extensive
variety of possibilities, allowing to assemble chimeric phages with new functions. Considering the
slow turnover of conventional diagnostic methods and the problems associated with the molecular
and immunogenic methods, this study aimed at assembling a bioluminescence-based reporter
phage for the fast and sensitive detection of P. aeruginosa in clinical care.
Using the yeast-based phage engineering platform, the phage vB_PaeP_PE3 was genetically
modified by removing genes with unknown function (g1-g12) and then used as a scaffold for the
insertion of the NanoLuc® luciferase gene that was swapped with gene g53. The assembled
reporter phage (vB_PaeP_PE3gp1-gp12,gp53:NLuc) was then used for sensitivity and specificity
assays. The detection limit was evaluated through the infection of serial dilutions of P. aeruginosa
suspensions with the reporter phage, and subsequent quantification of luminescence.
Our data showed that the assembled reporter phage was capable of reliably detect 500 CFU/mL
within 7h or an average 1 CFU/mL after 24h, and no false positives were observed. Similar results
were also obtained when the reporter phage was tested in blood, being capable of detecting an
average of 8 CFU/mL within 24 hours.
Overall, compared to culture-dependent methods, the NanoLuc-based reporter phage allows a fast
and sensitive detection of P. aeruginosa cells using a simple protocol. Therefore, this phage-based
detection system is a promising alternative to the common methods for the accurate detection of
P. aeruginosa in clinical settings.info:eu-repo/semantics/publishedVersio
