Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials

Abstract

We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on succesive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, φKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee.

Bacterial competition and quorum‐sensing signalling shape the eco‐evolutionary outcomes of model in vitro phage therapy

“…Phage therapy—the use of pathogen‐specific parasitic viruses (bacteriophages) as a treatment for bacterial infections—is almost hundred years old and has been used for decades to treat bacterial infections in Eastern European countries such as Georgia and Poland (Abedon, Kuhl, Blasdel, & Kutter, 2011; Alisky, Iczkowski, Rapoport, & Troitsky, 1998; Housby & Mann, 2009). While many studies have demonstrated the safety and benefits of phage therapy (Abedon et al., 2011; Merabishvili et al., 2009; Rose et al., 2014), phages have not yet been incorporated into western medicine partly due to lack of proper clinical trials and historically inconsistent treatment results (Kutateladze & Adamia, 2008). While large‐scale clinical trials are currently under way (e.g.…”

A comparative study of different strategies for removal of endotoxins from bacteriophage preparations

“…The endotoxin quantification by Endozyme was validated by endotoxin quantification by means of Endosafe-PTS, for a selected number of samples. Both detection methods gave similar results within the same order of magnitude (Table S1) The endotoxin removal strategies include either (1) Endotrap HD column purification alone (Merabishvili et al, 2009) (φET), or (2) CsCl density gradient ultracentrifugation alone ) (φC) or (3) followed with Endotrap HD purification (φCET),…”

“…Since the phage titers used for phage therapy are usually around 10 8 pfu/ml (Merabishvili et al, 2009), the phage preparations can be further diluted leading to a further drop of the endotoxin concentration (ranging from 0.0002 to 316 EU/ml for…”

“…Bacteriophage stocks (Table 1) were prepared using the double-agar overlay method as described in Merabishvili et al (2009) (Merabishvili et al, 2009). Briefly, one ml of the phage preparation containing 10 6 plaque forming units (pfu) of bacteriophages The harvested phages were centrifuged for 20 min at 6,000 x g at 4 °C.…”

“…The bacteriophage titer was determined by assaying decinormal serial dilutions (log(0) to log(-12)) of the bacteriophage suspension with the overlay method (Merabishvili et al, 2009). One ml of each dilution was mixed with 3 ml of molten (45 °C) LB 0.6 % top Bacto agar and the host strain (end concentration of 10 7 cfu/ml) in a sterile 14 ml tube.…”