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Background Multidrug-resistant Klebsiella pneumoniae spp. (kp) are emerging agents of severe infections of the respiratory, urinary tract and wounds that can progress to fatal septicemia. The use of bacteriophages is currently being considered as an effective alternative or adjuvant to antibiotic therapy. Results In this study, we report capsule (K)-typing of 163 carbapenem-resistant Kp (CRKP) isolated 2014–2018 at the Military Hospital of Instruction of Tunis (MHT), Tunisia, by partial amplification and sequencing of the Kp wzi gene. The most prevalent K-type overall was K64 with 50.3% followed by K17 and K27 (22.7 and 11.0%, respectively). K64 Kp strains were most common and associated with increased case/fatality rates, especially at the intensive care unit (ICU). Using a K64 Kp strain we isolated and characterized a lytic Kp phage, vB_KpP_TUN1 (phage TUN1), from wastewater samples of the ICU at the MHT. TUN1 belongs to the Autographiviridae family and specifically digests K64 Kp capsules most probably via a depolymerase encoded by gp47. Furthermore, we successfully assembled phage TUN1 in a non-replicative host (E. coli) raising the possibility of in vitro assembly in the absence of live bacterial hosts. We propose that phage TUN1 is a promising candidate to be used as an adjuvant or an alternative to antibiotic therapy in CRKP infections, facilitating regulatory approval of phage therapy. Conclusions K64, K17 and K27 are the most common wzi capsule types in this geographical location in Northern Africa. The lytic phage TUN1 efficiently lyses K64 Kp strains associated with increased case/fatality rates at body temperature. Together with its ability to be rescued in a non-replicative host these features enhance the utility of this phage as an antibacterial agent.

Despite numerous advances in personalized phage therapy, smooth logistics are challenging, particularly for multidrug-resistant Gram-negative bacterial infections requiring high numbers of specific lytic phages. We conducted this study to pave the way for efficient logistics for critically ill patients by (1) closely examining and improving a current pipeline under realistic conditions, (2) offering guidelines for each step, leading to safe and high-quality phage supplies, and (3) providing a tool to evaluate the pipeline’s efficiency. Due to varying stipulations for quality and safety in different countries, we focused the pipeline on all steps up to a required phage product by a cell-free extract system. The first of three study runs included patients with respiratory bacterial infections from four intensive care units, and it revealed a cumulative time of up to 23 days. Ultimately, adjustment of specific set points of the vulnerable components of the pipeline, phage isolation, and titration increased the pipeline’s efficiency by 15% and decreased the maximum required time to 13 days. We present a site-independent practical approach to establish and optimize pipelines for personalized phage delivery, the co-organization of pipeline components between different institutions, non-binding guidelines for every step, and an efficiency check for phage laboratories.

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Background Multidrug-resistant Klebsiella pneumoniaespp (Kp) are emerging agents of severe infections of the respiratory and urinary tract or wounds, and progression to fatal septicemia. The use of bacteriophages is currently being considered as an effective alternative or adjuvant to antibiotic treatment. Results In this study, we report K-typing of 163 carbapenem-resistant Kp (CRKP) isolated 2014–2018 at the Military Hospital of Instruction of Tunis (MHT), Tunisia, using wzi gene sequencing. Thereby, we found that K64 Kp strains are associated with increased case/fatality rates, especially at the intensive care unit (ICU). Furthermore, we characterized a lytic Kp phage, vB_KpP_TUN1 (phage TUN1), isolated from waste water samples of the ICU at the MHT. Whole genome sequencing showed that this phage belongs to the Podoviridae family and encodes one putative depolymerase specifically digesting K64 Kp capsules. Furthermore, we could successfully assemble phage TUN1 in a non-replicative host (E. coli) raising the possibility of in vitro assembly in the absence of live bacterial hosts. Therefore, phage TUN1 is a promising candidate to be used as an adjuvant or an alternative to antibiotic therapy in CRKP infections, facilitating regulatory approval of phage therapy. Conclusions The lytic phage TUN1, efficiently lyses K64 Kp strains associated with increased case/fatality rates at body temperature. Together with its ability to be rescued in a non-replicative host these features enhance the utility of this phage as antibacterial agent.