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

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 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. 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

Fecundity data

This text file contains both individual and cage level fitness data

Census data

This text file contains the raw juvenile, vegetative adults, and reproductive adults census counts for the entire experiment

Moss scoring guide

This Microsoft Word document provides a guide and examples for non-Arabidopsis growth cover scoring which was used to estimate interspecific competition

Code

This file contains the R code used to visualize and analyze the data

Competition data

This text file contains interspecific competition scores for each growth cage

Predicted functional consequences of SNP polymorphisms on the translational level.

<p>¹silent mutation</p><p>The SNP numbers (#), positions in the reference and the respective variant alleles for the SNP (Mut.) and the reference (Ref.) are shown as well as the potential amino acid (AA.) changes in the affected gene products and the gene designations.</p

SNPs in Pollino isolates detected by whole genome sequencing.

<p>SNPs are numbered according to their position in the reference genome¹ (<i>B</i>. <i>anthracis</i> Ames Ancestor; GenBank accession: AE017334). The bases at the respective positions are shown for Pollino isolates and the reference². SNPs are highlighted in italics.</p

Oligonucleotides used for high resolution melting analysis of Pollino-SNPs.

<p>¹with reference to <i>B</i>. <i>anthracis</i> strain Ames Ancestor</p><p>Oligonucleotides used for high resolution melting analysis of Pollino-SNPs.</p

Hypothesis on an active life cycle of <i>B</i>. <i>anthracis</i> in soil environments.

<p>Soil surrounding the carcass at the lower part of the figure harbors a very high spore burden. The left part of the panel predicts massive soil proliferation of <i>B</i>. <i>anthracis</i> (hypothesis). In this case of an active near-surface life cycle, local accumulation of spores is suggested to be due to repeated rounds of germination, replication and sporulation in the near-surface soil environment. During genome amplification random mutations occur resulting in derived genotypes compared to the genotypes of the initial spore population within the carcass. The right part of the panel depicts events if there was no soil-borne life cycle of the pathogen (competing hypothesis). Inert spores are supposed to accumulate in rainwater depressions. Genotypes differing from the original animal-infecting population cannot be observed in near-surface isolates.</p