Article
Personalized bacteriophage therapy to treat pandrug-resistant spinal P. aeruginosa infection
https://doi.org/10.21203/rs.3.rs-1231457/v1
This work is licensed under a CC BY 4.0 License
posted 08 Feb, 2022
You are reading this latest preprint version
A pandrug-resistant P. aeruginosa spinal abscess was treated with surgery and personalized phage therapy that was added to cefiderocol and colistin. After a 2-stage approach, despite bacterial persistence with expression of small colony variants, the patient cured with local and intravenous injections of purified bacteriophages as part of the treatment.
Bacteriophage
phage
phage therapy
antimicrobial resistance
orthopedic infections
The phage library of a fundamental microbiology laboratory located in Switzerland was screened to find active phages on the patient’s strain. Among >100 different phages active against P. aeruginosa, five were able to efficiently lyse the patient’s strain. The genome of three of them (named vB_Pae_4029, vB_Pae_4032 and vB_Pae_4034) were fully sequenced and annotated, which allowed checking for their truly lytic nature (absence of integrase and recombinase genes) and the absence of undesired genes, such as virulence determinants or toxins (data not shown). The phages looked as podovirus on the Electron Microscopy (EM) micrographs and belonged to the family Schitoviridae, genus Litunavirus based on the genome sequences (Figure 1, panel C-E). The phagogram and the Efficiency of Plating (EOP) tests performed as previously described [5] on the patient strain for the three unpurified phages revealed lytic activity (Figure 1, panel F-H and supplementary table 1). Production of the APIs, in compliance with a Belgian monograph describing the production process and QC system for incorporation in magistral preparations was done in the laboratory of a military hospital, under the supervision of the French national health care authority (Agence Nationale de Sécurité du Médicament et des produits de santé; ANSM), in collaboration with French hospital pharmacists [4, 5]. Of note, this monograph received on 10 January 2018 a formal positive advice from the Belgian Minister of Public Health asked the Federal Agency for Medicines and Health Products (FAMHP). It was conceived by representatives of the Queen Astrid Military Hospital located in Brussels, the FAMHP and Sciensano, formerly known as the Belgian Scientific Institute of Public Health. Here the three P. aeruginosa bacteriophages were produced using bacterial P. aeruginosa host strains PAO1 and ATCC® 15442™, according to the monograph for bacteriophage APIs. Bacteriohages were propagated using the double-agar overlay method in in animal-free growth medium (APS-LB broth, BD). The subsequent purification and concentration process consisted of two centrifugation steps at 6,000 and 40,000 x g for 20 and 90 min, respectively, separated by filtration through 0.45 and 0.22 µm polyethersulfone (PES) filters. The resulting bacteriophage pellet was resuspended in Dulbecco's Phosphate Buffered Saline (DPBS) buffer to obtain high titer (1012 pfu/ml) bacteriophage stocks. Next, bacteriophage stocks were transferred to the clean room facility of the military hospital and conditioned as APIs by diluting to a final concentration of 109 pfu/ml, followed by filtration through 0.22 µm PES filters, endotoxin purification using Endotrap columns (Lionex, Germany) and final filtration through 0.22 µm medical filters. Samples of each of the three bacteriophage APIs were sent to Sciensano for QC testing, including determination of pH, endotoxin level (EU/ml), and microbial burden. The pH values for the bacteriophage APIs ranged from 7.29 to 7.35, the endotoxin content endotoxin concentration ranged from 134 to 3400 UI/mL and no bacterial growth was observed in any of the samples. Based on the above results the three bacteriophage APIs were approved by Sciensano and deemed safe for application in humans, including intravenous use [4, 5]. In addition, we obtained from the ANSM a Temporary Authorization for Use (ATU) for cefiderocol, a new cephalosporin evaluated in clinical trials [6], that showed activity against the patient’s P. aeruginosa strain (MIC = 1 mg/L, broth microdilution).
Potential conflict of interest
All authors report no conflict of interest.
There is NO Competing Interest.
Supplementary tables
Supplementary video file
Reporting Summary
posted 08 Feb, 2022
You are reading this latest preprint version