Administration of Bacteriophages via Nebulization during Mechanical Ventilation: In Vitro Study and Lung Deposition in Macaques.

Bacteriophages have been identified as a potential treatment option to treat lung infection in the context of antibiotic resistance. We performed a preclinical study to predict the efficacy of delivery of bacteriophages against Pseudomonas aeruginosa (PA) when administered via nebulization during mechanical ventilation (MV). We selected a mix of four anti-PA phages containing two Podoviridae and two Myoviridae, with a coverage of 87.8% (36/41) on an international PA reference panel. When administered via nebulization, a loss of 0.30-0.65 log of infective phage titers was measured. No difference between jet, ultrasonic and mesh nebulizers was observed in terms of loss of phage viability, but a higher output was measured with the mesh nebulizer. Interestingly, Myoviridae are significantly more sensitive to nebulization than Podoviridae since their long tail is much more prone to damage. Phage nebulization has been measured as compatible with humidified ventilation. Based on in vitro measurement, the lung deposition prediction of viable phage particles ranges from 6% to 26% of the phages loaded in the nebulizer. Further, 8% to 15% of lung deposition was measured by scintigraphy in three macaques. A phage dose of 1 × 109 PFU/mL nebulized by the mesh nebulizer during MV predicts an efficient dose in the lung against PA, comparable with the dose chosen to define the susceptibility of the strain.

Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial.

BACKGROUND: Wound infections are the main cause of sepsis in patients with burns and increase burn-related morbidity and mortality. Bacteriophages, natural bacterial viruses, are being considered as an alternative therapy to treat infections caused by multidrug-resistant bacteria. We aimed to compare the efficacy and tolerability of a cocktail of lytic anti-Pseudomonas aeruginosa bacteriophages with standard of care for patients with burns. METHODS: In this randomised phase 1/2 trial, patients with a confirmed burn wound infection were recruited from nine burn centres in hospitals in France and Belgium. Patients were eligible if they were aged 18 years or older and had a burn wound clinically infected with P aeruginosa. Eligible participants were randomly assigned (1:1) by use of an interactive web response system to a cocktail of 12 natural lytic anti-P aeruginosa bacteriophages (PP1131; 1 × 106 plaque-forming units [PFU] per mL) or standard of care (1% sulfadiazine silver emulsion cream), both given as a daily topical treatment for 7 days, with 14 days of follow-up. Masking of treatment from clinicians was not possible because of the appearance of the two treatments (standard of care a thick cream, PP1131 a clear liquid applied via a dressing), but assignments were masked from microbiologists who analysed the samples and patients (treatment applied while patients were under general anaesthetic). The primary endpoint was median time to sustained reduction in bacterial burden by at least two quadrants via a four-quadrant method, assessed by use of daily swabs in all participants with a microbiologically documented infection at day 0 who were given at least one sulfadiazine silver or phage dressing (modified intention-to-treat population). Safety was assessed in all participants who received at least one dressing according to protocol. Ancillary studies were done in the per-protocol population (all PP1131 participants who completed 7 days of treatment) to assess the reasons for success or failure of phage therapy. This trial is registered with the European Clinical Trials database, number 2014-000714-65, and ClinicalTrials.gov, number NCT02116010, and is now closed. FINDINGS: Between July 22, 2015, and Jan 2, 2017, across two recruitment periods spanning 13 months, 27 patients were recruited and randomly assigned to receive phage therapy (n=13) or standard of care (n=14). One patient in the standard of care group was not exposed to treatment, giving a safety population of 26 patients (PP1131 n=13, standard of care n=13), and one patient in the PP1131 group did not have an infection at day 0, giving an efficacy population of 25 patients (PP1131 n=12, standard of care n=13). The trial was stopped on Jan 2, 2017, because of the insufficient efficacy of PP1131. The primary endpoint was reached in a median of 144 h (95% CI 48-not reached) in the PP1131 group versus a median of 47 h (23-122) in the standard of care group (hazard ratio 0·29, 95% CI 0·10-0·79; p=0·018). In the PP1131 group, six (50%) of 12 analysable participants had a maximal bacterial burden versus two (15%) of 13 in the standard of care group. PP1131 titre decreased after manufacturing and participants were given a lower concentration of phages than expected (1 × 102 PFU/mL per daily dose). In the PP1131 group, three (23%) of 13 analysable participants had adverse events versus seven (54%) of 13 in the standard of care group. One participant in each group died after follow-up and the deaths were determined to not be related to treatment. The ancillary study showed that the bacteria isolated from patients with failed PP1131 treatment were resistant to low phage doses. INTERPRETATION: At very low concentrations, PP1131 decreased bacterial burden in burn wounds at a slower pace than standard of care. Further studies using increased phage concentrations and phagograms in a larger sample of participants are warranted. FUNDING: European Commission: Framework Programme 7.

Inhaled phage therapy: a promising and challenging approach to treat bacterial respiratory infections.

INTRODUCTION: Bacterial respiratory tract infections (RTIs) are increasingly difficult to treat due to evolving antibiotic resistance. In this context, bacteriophages (or phages) are part of the foreseen alternatives or combination therapies. Delivering phages through the airways seems more relevant to accumulate these natural antibacterial viruses in proximity to their bacterial host, within the infectious site. Areas covered: This review addresses the potential of phage therapy to treat RTIs and discusses preclinical and clinical results of phages administration in this context. Recent phage formulation and aerosolization attempts are also reviewed, raising technical challenges to achieve efficient pulmonary deposition via inhalation. Expert opinion: Overall, the inhalation of phages as antibacterial treatment seems both clinically relevant and technically feasible. Several crucial points still need to be investigated, such as phage product pharmacokinetics and immunogenicity. Furthermore, given phage-specific features, appropriate regulatory and manufacturing guidelines will need to be defined. Finally, randomized controlled clinical trials should be carried out to establish phage therapy's clinical positioning in the antimicrobial arsenal against RTIs.

Synergistic Interaction Between Phage Therapy and Antibiotics Clears Pseudomonas Aeruginosa Infection in Endocarditis and Reduces Virulence.

Background: Increasing antibiotic resistance warrants therapeutic alternatives. Here we investigated the efficacy of bacteriophage-therapy (phage) alone or combined with antibiotics against experimental endocarditis (EE) due to Pseudomonas aeruginosa, an archetype of difficult-to-treat infection. Methods: In vitro fibrin clots and rats with aortic EE were treated with an antipseudomonas phage cocktail alone or combined with ciprofloxacin. Phage pharmacology, therapeutic efficacy, and resistance were determined. Results: In vitro, single-dose phage therapy killed 7 log colony-forming units (CFUs)/g of fibrin clots in 6 hours. Phage-resistant mutants regrew after 24 hours but were prevented by combination with ciprofloxacin (2.5 × minimum inhibitory concentration). In vivo, single-dose phage therapy killed 2.5 log CFUs/g of vegetations in 6 hours (P < .001 vs untreated controls) and was comparable with ciprofloxacin monotherapy. Moreover, phage/ciprofloxacin combinations were highly synergistic, killing >6 log CFUs/g of vegetations in 6 hours and successfully treating 64% (n = 7/11) of rats. Phage-resistant mutants emerged in vitro but not in vivo, most likely because resistant mutations affected bacterial surface determinants important for infectivity (eg, the pilT and galU genes involved in pilus motility and LPS formation). Conclusions: Single-dose phage therapy was active against P. aeruginosa EE and highly synergistic with ciprofloxacin. Phage-resistant mutants had impaired infectivity. Phage-therapy alone or combined with antibiotics merits further clinical consideration.

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 allows a 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.

Rapid identification of international multidrug-resistant Pseudomonas aeruginosa clones by multiple-locus variable number of tandem repeats analysis and investigation of their susceptibility to lytic bacteriophages.

The objective of this study was to determine the genetic diversity of multidrug-resistant (MDR) Pseudomonas aeruginosa strains isolated over a period of 12 months in two French hospitals and to test their susceptibility to bacteriophages. A total of 47 MDR isolates recovered from hospitalized patients were genotyped using multiple-locus variable number of tandem repeats analysis. The genotypes were distributed into five clones (including 19, 5, 5, 3, and 3 isolates, respectively) and 12 singletons. Comparison to 77 MDR strains from three other countries, and MLST analysis of selected isolates showed the predominance of international MDR clones. The larger clone, CC235, contained 59 isolates displaying different antibiotic resistance mechanisms, including the presence of the GES1, VIM-2, VIM-4, and IMP-1 ß-lactamases. Three newly isolated P. aeruginosa bacteriophages were found to lyse 42 of the 44 analyzed strains, distributed into the different clonal complexes. This pilot study suggests that systematic genotyping of P. aeruginosa MDR strains could improve our epidemiological understanding of transmission at both the local (hospital) and the national level and that phage therapy could be an alternative or a complementary treatment to antibiotics for treating MDR-infected patients.