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

Abstract

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.

Phage Therapy in Germany—Update 2023

“…A test case showed the personalized application of phages against a carbapenem-resistant K. pneumoniae patient isolate, from isolation to in vitro production. Utilizing the advantages of in vitro bacteriophage production, a personalized phage-therapy pipeline can be implemented, and could be applied for on-site production [ 20 , 21 ]. Such a personalized approach is especially important for pathogens with narrow specific phages, such as Klebsiella phages, most of which initiate infection through the highly diverse capsular polysaccharide of K. pneumoniae .…”

“…The ‘Therapeutic Phage Group’ of the Bundeswehr Institute of Microbiology, Munich (head J.B.): In collaboration with the European Commission, Klebsiella phages and their bacterial hosts are characterized, optimized and prepared for therapeutic applications with a one-health perspective (Joint Programming Initiative on Antimicrobial Resistance, JPIAMR, funded KLEOPATRA consortium, , accessed on 12 January 2023”; Design and implementation of effective combination of Phages and Antibiotics for improved TheRApy protocols against KLEbsiella pneumoniae). The aim is to expand the host range of phages, optimize cell-free production, support phage therapy in clinical trials and individual healing-trial protocols with personalized phage preparations, and improve therapeutic approaches of clinical partners [ 20 ].…”

Appelmans Protocol forin vitro Klebsiella pneumoniaephage host range expansion leads to induction of a novel temperate linear plasmid prophagevB_KpnS-KpLi5

“…The above-mentioned diversity of Kp capsule types and further restriction factors of Kp lead to the need for a large amount of the very host specific Kp phages to cover all types required for therapy. However, even a phage bank with more than 350 phages with different capsule types is not sufficient to efficiently lyse the respective Kp target population of patients [5].…”

SpyPhage: A Cell-Free TXTL Platform for Rapid Engineering of Targeted Phage Therapies

“…Such systems would function as heterologous expression platforms, containing phage specific transcription factors, that would enable the production of a variety of different phage within a single minimal CFS derived from a nonpathogenic host (Figure B) . Further still, achieving concurrent whole genome assembly of an engineered genome within a minimal CFS capable of expressing virulent phage particles from the newly assembled genome is a significant milestone yet to be reached. − These strategies would forego the need for cultivation of pathogenic bacterial hosts in a future phage engineering pipeline.…”