Open Access

Timo Späth

• The growing threat posed by multidrug-resistant (MDR) pathogens, such as Klebsiella pneumoniae (Kp), represents a significant challenge in modern medicine. Traditional antibiotic therapies are often ineffective against these pathogens, leading to high mortality rates. MDR Kp infections pose a novel challenge in military medical contexts, particularly in Medical Biodefense, as they can beThe growing threat posed by multidrug-resistant (MDR) pathogens, such as Klebsiella pneumoniae (Kp), represents a significant challenge in modern medicine. Traditional antibiotic therapies are often ineffective against these pathogens, leading to high mortality rates. MDR Kp infections pose a novel challenge in military medical contexts, particularly in Medical Biodefense, as they can be deliberately spread, leading to resource-intensive care in military centres. Recognizing this issue, the European Defence Agency initiated a prioritised research project in 2023 (EDF Resilience PHAGE- SGA 2023). To address this challenge, the Bundeswehr Institute of Microbiology (IMB) leads BMBF- (Federal Ministry of Education and Research) and EU-funded projects on the use of bacteriophages as adjuvant therapy alongside antibiotics. Since 2017, the IMB has isolated and characterised Kp phages, collecting over 600 isolates and optimizing their production for therapy, in compliance with the EMA (European Medicine Agency) guidelines. This involves in vitro phage genome packaging to minimize endotoxin load, reduce manufacturing costs, and shorten production times. The goal of this work was to establish MinION sequencing (Oxford Nanopore Technology) as a quick and reliable way for initial identification and characterisation of phage genomes. Especially as a quick screening method for isolated on Kp, prior to more precise but also more expensive and time consuming sequencing methods like Illumina. This characterisation is crucial for developing a personalized pipeline aimed at producing magistral or Good Manufacturing Practice (GMP) quality medicinal phage solutions tailored individually for each patient. DNA extraction methods were compared to identify suitable input DNA for sequencing purposes. Additionally, the quality of this DNA was as- sessed to determine its suitability for in vitro phage packaging, which was successfully done achieving a phage titer of 103, confirming that the DNA used for MinION sequencing could indeed be used for acellular packaging. The created genomes were annotated and compared with Illumina sequencing, revealing high similarity in all five individually tested cases. Between the generated sequences only a 4% maximal percentual difference in genome size was observed, while simultaneously showing high similarity in the actual sequence. Throughout the course of this study, a total of 645.15 GB of sequencing data were generated. In total, 38 phages were successfully characterised, with 21 phage genomes assembled and annotated, and saved in the IMB database.…