Layperson summary:
Bacteriophages (or phages) are viruses, which infect specifically bacteria. They can be used for bacteriophage therapy of infections caused by pathogenic bacteria independent of their resistance to antibiotics. Bacteriophages are so specific, therapeutic phage collections have to be screened for lytic phages for every clinical isolate received. If a phage which kills the bacterium of interest cannot be identified in the phage collections, local sources will be screened, because phages can be found where their hosts live.
Before a phage is used for treatment, it has to be well characterized. We look at phage particles using electronmicoscropy, we test the host range, to see if it lyses the target bacterium reliably, and we determine the phage genome sequence.
Academic summary:
The spread of multiantibiotic resistant bacteria is a global phenomenon and requires new therapeutic options. An alternative to antibiotics are bacteriophages, which specifically lyse bacteria. In Eastern Europe and increasingly in the US, the UK and in the European Union (EU), bacteriophage based therapeutics are used by physicians. Regulatory issues and unsolved questions regarding safety and good manufaturing practice (GMP) currently prevent the regulatory approval of bacteriophage therapy in the EU. Here, bacteriophage based therapeutics are only used by clinical academics in specific clinical situations.
Questions of safety and clinical situations that warrant use of bacteriophage therapy are being adressed in publicly funded projects like 'Phage4Cure' and 'PhagoBurn' and 'Phagoflow' in Germany, France and Belgium. The Leibniz Institute DSMZ collects and characterizes phages for these studies as a basis for phage cocktails of several phages with different lytic mechanisms. ITEM at Fraunhofer Institute - Helmholtz Centre Braunschweig has just established a GMP phage production facility in Germany.
Independent academic groups at the Bundeswehr Institute of Microbiology, the BfR in Berlin, the University of Helsinki, the Walter Reed Army Institute of Research and at Invitris- Munich isolate bacteriophage from environmental samples and characterize them using the techniques described below.
These public and independent groups are members of the Phage41Health - The Phage Therapy Consortium.
Phage isolation and characterization
Phages for therapy are ideally mechanically sturdy with DNA genomes ensuring stable storage at 4°C or lyophilized and able to withstand some measure of shearing and breakdown during filtration and freezing. Phage isolation and characterization described below is designed for unenveloped, short tail, DNA bacteriophages mostly represented in the family Caudovirales.
Isolation Environmental surface and waste water are centrifuged at 7.000 x g for 10 min at 4°C to remove crude sediment, followed by filtration through 0.2 μm filters. The sterile filtrate contains bacteriophages and other viruses (1,2)
Enrichment and host range studies Phages are enriched on the host of choice and plaque assay is used to isolate and quantitate specific phage from sterile filtrates. 100 μl Filtrat and 300 μl of a target bacterial strain at OD600 0.4-0.6 are added to 2.5 ml of (40°C) LB-Agar (0.6%) with 1mM CaCl₂ and poured onto a 2% LB Agar plate. Plates are incubated inverted at 37°C for 24h (1,2). Phage host range is determined by testing on a panel of bacterial target strains and establishing the extent of phage replication using growth curves.
Genome extraction and sequencing DNA is extracted from phage particles using commercially available kits. Phage stocks of > 1 x 108 pfu/ml are required. Phage genomes are sequenced using minION ® - and Illumina®/MiSeq® - based systems.
Electron microscopy Purified phage solution is added to carbon-coated Cu400 TEM grids, followed by a negative stain. This is followed by vacuum drying and imaging with a transmission electron microscope at 100 kV and a magnification between × 15,500 and × 21,000.
Production Phage products for therapy are preferably produced in bacterial strains isolated from the patient. This is to ensure that no toxin or antimicrobial resistance genes are transferred to the bacteria colonizing the patient. If the phage cannot be produced in the patient strain, for example if propagation is poor or the bacteria grows poorly, the isolation host for phage can be used.
There are three different methods for phage production: i. in liquid media, ii. in solid media with petri dishes (plating method) or iii. with cell culture bottles.
Liquid phage propagation is the most widely used method and is the easiest to scale up. Here, the host bacteria and the phages are cultured together in liquid media, under optimal temperature and agitation for the growth of the bacteria. It is also usually the first production method tested for new phages, but it does not usually give the best yields.
Another widely used method is the plating method, where the bacteria and phages are mixed in growth agar-media and plated on top of agar-media covered petri dishes. These petri dishes are grown overnight in optimal conditions for the bacteria.
The third method for producing phages can be done in cell culture bottles, which have a growth agar-media lawn and the bacteria is added in to the bottle in liquid form. The bottle is then incubated in optimal conditions for the bacteria, before adding the phages and culturing them together.
Clinical application Suitable characterized bacteriophages are made available to clinical cooperation partners in collaborating hospitals (eg. Bundeswehrkrankenhaus Berlin) and the Leibniz Institute DMSZ collection centre for use in compassionate use protocols or clinical phage therapy studies.
Treatment requirements If a patient is eligible for phage therapy and the treating physician approves an compassionate experimental therapeutic approach, and suitable and high-quality phages can be found for the bacteria of interest in the required time frame, a phage-based personalized approach might be considered upon request of the patient. The Phage Consortium advises patients and physicians on potential approaches and necessary measures for all kinds of infections on a case-by-case basis.
We strongly advise physicians to contact The Phage Consortium for information prior to phage therapy ([email protected]).
The from of delivery The form of delivery depends on the infection site and expert opinions from phage, pharmaceutical and clinical professionals. In published studies, phages have been applied orally, i.v., rectally, topically, and by inhalation. The schedule of treatment, monitoring, and follow-up is delevoped in an interdisciplinary way.
References
- Kęsik-Szeloch A, Drulis-Kawa Z, Weber-Dąbrowska B, Kassner J, Majkowska-Skrobek G, Augustyniak D, Lusiak-Szelachowska M, Zaczek M, Górski A, Kropinski AM. Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae. Virol J. 2013 Mar 28;10:100. doi: 10.1186/1743-422X-10-100.
- Eckstein S, Stender J, Mzoughi S, et al. Isolation and characterization of lytic phage TUN1 specific for Klebsiella pneumoniae K64 clinical isolates from Tunisia. BMC Microbiol. 2021;21(1):186. Published 2021 Jun 21. doi:10.1186/s12866-021-02251-w