Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria.

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.

Practical Assessment of an Interdisciplinary Bacteriophage Delivery Pipeline for Personalized Therapy of Gram-Negative Bacterial Infections.

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.

Isolation and characterization of lytic phage TUN1 specific for Klebsiella pneumoniae K64 clinical isolates from Tunisia.

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.