Recombinant Reporter Phage rTUN1::nLuc Enables Rapid Detection and Real-Time Antibiotic Susceptibility Testing of Klebsiella pneumoniae K64 Strains

Abstract

The emergence of multi-drug-resistant Klebsiella pneumoniae (Kp) strains constitutes an enormous threat to global health as multi-drug resistance-associated treatment failure causes high mortality rates in nosocomial infections. Rapid pathogen detection and antibiotic resistance screening are therefore crucial for successful therapy and thus patient survival. Reporter phagebased diagnostics offer a way to speed up pathogen identification and resistance testing as integration of reporter genes into highly specific phages allows real-time detection of phage replication and thus living host cells. Kp-specific phages use the host's capsule, a major virulence factor of Kp, as a receptor for adsorption. To date, 80 different Kp capsule types (K-serotypes) have been described with predominant capsule types varying between different countries and continents. Therefore, reporter phages need to be customized according to the locally prevailing variants. Recently, we described the autographivirus vB_KpP_TUN1 (TUN1), which specifically infects Kp K64 strains, the most predominant capsule type at the military hospital in Tunis (MHT) that is also associated with high mortality rates. In this work, we developed the highly specific recombinant reporter phage rTUN1::nLuc, which produces nanoluciferase (nLuc) upon host infection and thus enables rapid detection of Kp K64 cells in clinical matrices such as blood and urine. At the same time, rTUN1::nLuc allows for rapid antibiotic susceptibility testing and therefore identification of suitable antibiotic treatment in less than 3 h.

Phage Therapy in Germany—Update 2023

“…The IMB phage group has one of the most extensive collections of 3-4 MRGN K. pneumoniae (>380 isolates with a large variety of capsule types (>80)) and their specific phages (>505 plaque-purified stocks) in Europe. The group is using its phage collection for studies to extend the host range [ 23 ], and engineers reporter phages to monitor K. pneumoniae infections [ 24 ].…”

Enhancing bacteriophage therapeutics through in situ production and release of heterologous antimicrobial effectors

“…In the future, phage-based precision antimicrobials will likely be designed and implemented as personalized treatment options. Therefore, a rapid and reliable companion diagnostic would be helpful to guide phage selection and/or predict therapeutic success 20,34,35 . To assess the performance of self-targeting HEPTs against E. coli in patient urine, we combined our recently developed reporter phage-based diagnostic 20 with ex vivo urine treatment using HEPT E2::colE7 (workflow: Fig.…”

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

“…Another area of research where NLuc has been utilized extensively is in the development of protein fragment complementation assay, typically utilized for monitoring protein-protein interaction in living cells. 24-26 Additional applications of NLuc include nanoparticle conjugates utilized for in vivo imaging 27 , sensitive viral infection detection 28 , to evaluate one of metabolic intermediate in fatty acid metabolism 29 or to test antibiotic susceptibility 30 . Further, NLuc has been successfully applied in the development of various Bioluminescence Resonance Energy Transfer (BRET)-based biomolecular assays 4-7, 31-33 such as to study molecular/organelle interactions 34, 35 , for optogenetics purposes 36 , for drug descovery 37, 38 , as genetic encoded light source for a photodynamic anticancer therapy 39 , biomolecule detection 40 , monitoring molecular tension 41 , and assays to monitor SARS-CoV-2 protease activity 42, 43 .…”