Maia Merabishvili

The Phage Therapy Paradigm: Prêt-à-Porter or Sur-mesure?

The present opinion is the result of discussions on the future of phage therapy (personalized or large-scale uniform therapy?) during the first International Congress on Viruses of Microbes, held at the Institut Pasteur in Paris on June 21–25, 2010. Antibiotics are becoming ineffective as important bacterial pathogens evolve to outsmart them. Yet the antibiotic pipeline is running dry with only a few new antibacterial drugs expected to make it to the market in the foreseeable future. Bacteria that are resistant to all available antibacterial drugs, so-called superbugs, are emerging worldwide. Evolutionary ecology might inform practical attempts to bring these pathogens under stronger human control (1). In this context, various laboratories worldwide and a handful of small pharmaceutical companies are turning to (bacterio)phages (2). Phages are natural viruses that specifically infect bacteria. They are (among) the most abundant and ubiquitous lifelike entities on Earth and coevolve with their hosts, the bacteria. Lytic phages bind to receptors on the bacterial cell surface, inject their genetic material, use the bacterium’s reproductive machinery to replicate and subsequently destroy (lyse) the bacterium, irrespective of its resistance to antibiotics, releasing the newly formed phages to seek out new hosts. In 1919, d’Herelle used phages to treat dysentery in Paris, in what was probably the first attempt to use phages therapeutically. d’Herelle eventually developed a commercial laboratory in Paris that produced phage preparations against

Quality and Safety Requirements for Sustainable Phage Therapy Products

The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge. To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allowsa timely supplying of phage therapy products for ‘personalized therapy’ or for public health or medical emergencies. This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.

Microbiological and Molecular Assessment of Bacteriophage ISP for the Control of Staphylococcus aureus

The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the ‘Twort-like viruses’. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes.

Stability of Staphylococcus aureus Phage ISP after Freeze-Drying (Lyophilization)

Staphylococcus aureus phage ISP was lyophilized, using an Amsco-Finn Aqua GT4 freeze dryer, in the presence of six different stabilizers at different concentrations. Stability of the lyophilized phage at 4°C was monitored up to 37 months and compared to stability in Luria Bertani broth and physiological saline at 4°C. Sucrose and trehalose were shown to be the best stabilizing additives, causing a decrease of only 1 log immediately after the lyophilization procedure and showing high stability during a 27 month storage period.

Characterization of Newly Isolated Lytic Bacteriophages Active against Acinetobacter baumannii

Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively), high burst size (125 and 145, respectively), stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections.

Selection and Characterization of a Candidate Therapeutic Bacteriophage That Lyses the Escherichia coli O104:H4 Strain from the 2011 Outbreak in Germany

In 2011, a novel strain of O104:H4 Escherichia coli caused a serious outbreak of foodborne hemolytic uremic syndrome and bloody diarrhea in Germany. Antibiotics were of questionable use and 54 deaths occurred. Candidate therapeutic bacteriophages that efficiently lyse the E. coli O104:H4 outbreak strain could be selected rather easily from a phage bank or isolated from the environment. It is argued that phage therapy should be more considered as a potential armament against the growing threat of (resistant) bacterial infections.

Use of bacteriophages in the treatment of colistin-only-sensitive Pseudomonas aeruginosa septicaemia in a patient with acute kidney injury—a case report

Sepsis from Pseudomonas aeruginosa bacteraemia may be fatal, especially when no appropriate therapy can be given. In June 2016, a 61-year-old man was hospitalised for Enterobacter cloacae peritonitis and severe abdominal sepsis with disseminated intravascular coagulation, secondary to a diaphragmatic hernia with bowel strangulation. The patient had a prolonged hospital course complicated by gangrene of the peripheral extremities, resulting in the amputation of the lower limbs and the development of large necrotic pressure sores (Fig. 1). Three months after admission, the patient was transferred to the Queen Astrid military hospital for surgical management of the pressure sores. Wound cultures on admission revealed colonisation with, amongst others, multidrug-resistant P. aeruginosa. One month after admission, the patient developed septicaemia with colistin-only-sensitive P. aeruginosa. Intravenous colistin therapy was started. Ten days later, the patient developed acute kidney injury with rising serum creatinine and urea levels (Fig. 2), presumably sepsisand drug-induced. Antibiotic therapy was discontinued to prevent further kidney damage. Unfortunately, on 12 November, P. aeruginosa septicaemia re-emerged with rapid heart rate, low blood pressure, fever and high C-reactive protein (CRP) levels. On 14 November, the patient went into a coma with 9 scores (E2V2M5) on the Glasgow Coma Scale. Because of the risk of colistin nephrotoxicity and the family’s will to avoid intensive therapy interventions such as hemofiltration, bacteriophage therapy was initiated under the umbrella of Article 37 (Unproven Interventions in Clinical Practice) of the Declaration of Helsinki [1]. Fifty microlitres of purified bacteriophage cocktail BFC1 [2], containing two bacteriophages that showed in vitro activity against the patient’s P. aeruginosa

A bacteriophage journey at the European Medicines Agency.

The seriously and globally increasing bacterial multi-drug resistance calls out on concerted counteractive measures: international health authorities give consideration to the therapeutical use of bacteriophage therapy.

Unexploited opportunities for phage therapy

Project “BioHealth - Biotechnology and Bioengineering approaches to improve health quality,” Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. Project “Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB,” Ref. FCOMP-01-0124- FEDER-027462

Molecular Epidemiology and Clinical Impact of Acinetobacter calcoaceticus-baumannii Complex in a Belgian Burn Wound Center.

Multidrug resistant Acinetobacter baumannii and its closely related species A. pittii and A. nosocomialis, all members of the Acinetobacter calcoaceticus-baumannii (Acb) complex, are a major cause of hospital acquired infection. In the burn wound center of the Queen Astrid military hospital in Brussels, 48 patients were colonized or infected with Acb complex over a 52-month period. We report the molecular epidemiology of these organisms, their clinical impact and infection control measures taken. A representative set of 157 Acb complex isolates was analyzed using repetitive sequence-based PCR (rep-PCR) (DiversiLab) and a multiplex PCR targeting OXA-51-like and OXA-23-like genes. We identified 31 rep-PCR genotypes (strains). Representatives of each rep-type were identified to species by rpoB sequence analysis: 13 types to A. baumannii, 10 to A. pittii, and 3 to A. nosocomialis. It was assumed that isolates that belonged to the same rep-type also belonged to the same species. Thus, 83.4% of all isolates were identified to A. baumannii, 9.6% to A. pittii and 4.5% to A. nosocomialis. We observed 12 extensively drug resistant Acb strains (10 A. baumannii and 2 A. nosocomialis), all carbapenem-non-susceptible/colistin-susceptible and imported into the burn wound center through patients injured in North Africa. The two most prevalent rep-types 12 and 13 harbored an OXA-23-like gene. Multilocus sequence typing allocated them to clonal complex 1 corresponding to EU (international) clone I. Both strains caused consecutive outbreaks, interspersed with periods of apparent eradication. Patients infected with carbapenem resistant A. baumannii were successfully treated with colistin/rifampicin. Extensive infection control measures were required to eradicate the organisms. Acinetobacter infection and colonization was not associated with increased attributable mortality.