Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium. Due to its high antibiotic
resistance and capacity to adapt and survive in hostile conditions, P. aeruginosa is responsible for a
wide range of human infections, such as surgical site infections, bacteremia, urinary tract infections,
and mostly, pneumonia. In COVID-19 patients, P. aeruginosa is a common co-infecting pathogen, associated
with increased disease severity and worse clinical outcomes. 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.
Phage vB_PaeP_PE3 was genetically engineered using the yeast-based phage engineering platform.
The genome of this phage was previously reduced by deleting genes with unknown function, and
here, this phage genome was used as a scaffold for the insertion of the NanoLuc® luciferase. The
gene encoding NanoLuc was swapped with gene gp55, encoding a hypothetical protein with unknown
function. The sensitivity of this phage-based detection system was evaluated through the infection
of serial dilutions of P. aeruginosa suspensions with the synthetic phage, and subsequent
quantification of luminescence (in relative light units, RLU). Our data showed that the reporter
phage was able to reliably detect 10^2 CFU in 1 mL of contaminated sample in less than 8 h.
Overall, the NanoLuc-based reporter phage allows for the rapid and sensitive detection and differentiation
of viable P. aeruginosa cells using a simple protocol, 45 h faster than culture-dependent
approaches. 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
