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

The emergence of multi-drug-resistant () 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 phage-based 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. -specific phages use the host's capsule, a major virulence factor of , as a receptor for adsorption. To date, 80 different 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 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::, which produces nanoluciferase (nLuc) upon host infection and thus enables rapid detection of K64 cells in clinical matrices such as blood and urine. At the same time, rTUN1:: allows for rapid antibiotic susceptibility testing and therefore identification of suitable antibiotic treatment in less than 3 h.