Topical liquid formulation of bacteriophages for metered-dose spray delivery.

Bacteriophage (phage) therapy is a promising treatment strategy to combat antibiotic-resistant bacteria. Clinical reports from a century ago, as well as recent reports have revealed safety and efficacy of phage therapy for bacterial wound infections. However, the conventional liquid phage formulation and delivery platforms reported lack of dose control as it easily runs off from the infection site and it is impossible to determine total volume transfer. The aim of this study was to formulate phage liquids for topical delivery using a metered-dose spray. Two types of anti-Pseudomonas phages, PEV1 (myovirus) and PEV31 (podovirus) were formulated in 35% ethanol in water containing non-ionic polymers. The formulations were evaluated for physical properties, ease of spray, dripping upon spraying, drying time, in vitro release profiles, antibacterial activity, and storage stability. The optimized phage-polymer spray formulations were easily sprayable with minimal dripping and fast drying time. Phages were rapidly released from the formulation and inhibited the growth of Pseudomonas aeruginosa. Both PEV1 and PEV31 remained biologically stable in the optimized formulations during storage at 4 °C for eight weeks. This study showed the topical spray formulations containing non-ionic polymers in ethanol/water could be a promising and innovative therapeutic system for delivering phages.

The effects of different doses of inhaled bacteriophage therapy for Pseudomonas aeruginosa pulmonary infections in mice.

OBJECTIVES: Inhaled phage therapy has been revisited as a potential treatment option for respiratory infections caused by multidrug-resistant Pseudomonas aeruginosa; however, there is a distinct gap in understanding the dose-response effect. The aim of this study was to investigate the dose-response effect of Pseudomonas-targeting phage PEV31 delivered by the pulmonary route in a mouse lung infection model. METHODS: Neutropenic BALB/c mice were infected with multidrug-resistant P. aeruginosa (2 × 104 colony-forming units) through the intratracheal route and then treated with PEV31 at three different doses of 7.5 × 104 (Group A), 5 × 106 (Group B), and 5 × 108 (Group C) plaque-forming units, or phosphate-buffered saline at 2 hours postinoculation. Mice (n = 5-7) were euthanized at 2 hours and 24 hours postinfection, and lungs, kidneys, spleen, liver, bronchoalveolar lavage fluid, and blood were collected for bacteria and phage enumeration. RESULTS: At 24 hours postinfection, all phage-treated groups exhibited a significant reduction in pulmonary bacterial load by 1.3-1.9 log10, independent of the delivered phage dose. The extent of phage replication was negatively correlated with the dose administered, with log10 titre increases of 6.2, 2.7, and 9 for Groups A, B, and C, respectively. Phage-resistant bacterial subpopulations in the lung homogenate samples harvested at 24 hours postinfection increased with the treatment dose (i.e. 30%, 74%, and 91% in respective Groups A-C). However, the mutants showed increased susceptibility to ciprofloxacin, impaired twitching motility, and reduced blue-green pigment production. The expression of the inflammatory cytokines (IL-1ß and IL-6, and TNF-α) was suppressed with increasing PEV31 treatment dose. DISCUSSION: This study provides the dose-response effect of inhaled phage therapy that may guide dose selection for treating P. aeruginosa respiratory infections in humans.

The genetic basis of phage susceptibility, cross-resistance and host-range in Salmonella.

Though bacteriophages (phages) are known to play a crucial role in bacterial fitness and virulence, our knowledge about the genetic basis of their interaction, cross-resistance and host-range is sparse. Here, we employed genome-wide screens in Salmonella enterica serovar Typhimurium to discover host determinants involved in resistance to eleven diverse lytic phages including four new phages isolated from a therapeutic phage cocktail. We uncovered 301 diverse host factors essential in phage infection, many of which are shared between multiple phages demonstrating potential cross-resistance mechanisms. We validate many of these novel findings and uncover the intricate interplay between RpoS, the virulence-associated general stress response sigma factor and RpoN, the nitrogen starvation sigma factor in phage cross-resistance. Finally, the infectivity pattern of eleven phages across a panel of 23 genome sequenced Salmonella strains indicates that additional constraints and interactions beyond the host factors uncovered here define the phage host range.

Hydrogel formulations containing non-ionic polymers for topical delivery of bacteriophages.

Hydrogel is an attractive delivery vehicle for phages as it keeps the wound moist, acts as a protective barrier and facilitates wound healing process. The aim of this study was to formulate biologically stable phage hydrogels that enable controlled release of infective phages. Pseudomonas-targeting phages, PEV1 (myovirus) and PEV31 (podovirus) were formulated in hydrogels (109 PFU/g) consisting of non-ionic polymers, including hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), polyethylene oxide (PEO), polyvinyl alcohol (PVA), hydroxypropyl cellulose (HPC) and polyvinylpyrrolidone (PVP). The formulations were evaluated for physical properties, in vitro release profiles, antibacterial activity, and storage stability. Controlled release of phages was observed in 7.5% PEO, 20% PVA and 75% PVP hydrogels with >108 PFU release within 8 h. Poor phage release (7 × 105-4 × 107 PFU) was observed in 5% HPMC, 5% HEC and 30% HPC gels. The biostability of the optimized hydrogels was phage-specific with less titer loss observed for PEV1 (0-0.8 log) than for PEV31 (0.3-1.4 log). Both phages remained stable in PEO, PVA and HPMC hydrogels with <1 log titer reductions when stored at 5 °C. This study showed that 7.5% PEO and 20% PVA hydrogel formulations could be promising therapeutic systems for delivering phages for the treatment of wound infections.

Phage cocktail powder for Pseudomonas aeruginosa respiratory infections.

Phage cocktail broadens the host range compared with a single phage and minimizes the development of phage-resistant bacteria thereby promoting the long-term usefulness of inhaled phage therapy. In this study, we produced a phage cocktail powder by spray drying three Pseudomonas phages PEV2 (podovirus), PEV1 and PEV20 (both myovirus) with lactose (80 wt%) and leucine (20 wt%) as excipients. Our results showed that the phages remained viable in the spray dried powder, with little to mild titer reduction (ranging from 0.11 to 1.3 logs) against each of their specific bacterial strains. The powder contained spherical particles with a small volume median diameter of 1.9 µm (span 1.5), a moisture content of 3.5 ± 0.2 wt%., and was largely amorphous with some crystalline peaks, which were assigned to the excipient leucine, as shown in the X-ray diffraction pattern. When the powder was dispersed using the low- and high-resistance Osmohalers, the fine particle fraction (FPF, wt. % of particles < 5 µm in the aerosols relative to the loaded dose) values were 45.37 ± 0.27% and 62.69 ± 2.1% at the flow rate of 100 and 60 L/min, respectively. In conclusion, the PEV phage cocktail powder produced was stable, inhalable and efficacious in vitro against various MDR P. aeruginosa strains that cause pulmonary infections. This formulation will broaden the bactericidal spectrum and reduce the emergence of resistance in bacteria compared with single-phage formulations reported previously.

Synergistic activity of phage PEV20-ciprofloxacin combination powder formulation-A proof-of-principle study in a P. aeruginosa lung infection model.

Combination treatment using bacteriophage and antibiotics is potentially an advanced approach to combatting antimicrobial-resistant bacterial infections. We have recently developed an inhalable powder by co-spray drying Pseudomonas phage PEV20 with ciprofloxacin. The purpose of this study was to assess the in vivo effect of the powder using a neutropenic mouse model of acute lung infection. The synergistic activity of PEV20 and ciprofloxacin was investigated by infecting mice with P. aeruginosa, then administering freshly spray-dried single PEV20 (106 PFU/mg), single ciprofloxacin (0.33 mg/mg) or combined PEV20-ciprofloxacin treatment using a dry powder insufflator. Lung tissues were then harvested for colony counting and flow cytometry analysis at 24 h post-treatment. PEV20 and ciprofloxacin combination powder significantly reduced the bacterial load of clinical P. aeruginosa strain in mouse lungs by 5.9 log10 (p < 0.005). No obvious reduction in the bacterial load was observed when the animals were treated only with PEV20 or ciprofloxacin. Assessment of immunological responses in the lungs showed reduced inflammation associating with the bactericidal effect of the PEV20-ciprofloxacin powder. In conclusion, this study has demonstrated the synergistic potential of using the combination PEV20-ciprofloxacin powder for P. aeruginosa respiratory infections.

Pharmacokinetics and Time-Kill Study of Inhaled Antipseudomonal Bacteriophage Therapy in Mice.

Inhaled bacteriophage (phage) therapy is a potential alternative to conventional antibiotic therapy to combat multidrug-resistant (MDR) Pseudomonas aeruginosa infections. However, pharmacokinetics (PK) and pharmacodynamics (PD) of phages are fundamentally different from antibiotics and the lack of understanding potentially limits optimal dosing. The aim of this study was to investigate the in vivo PK and PD profiles of antipseudomonal phage PEV31 delivered by pulmonary route in immune-suppressed mice. BALB/c mice were administered phage PEV31 at doses of 107 and 109 PFU by the intratracheal route. Mice (n = 4) were sacrificed at 0, 1, 2, 4, 8, and 24 h posttreatment and various tissues (lungs, kidney, spleen, and liver), bronchoalveolar lavage fluid, and blood were collected for phage quantification. In a separate study combining phage with bacteria, mice (n = 4) were treated with PEV31 (109 PFU) or phosphate-buffered saline (PBS) at 2 h postinoculation with MDR P. aeruginosa Infective PEV31 and bacteria were enumerated from the lungs. In the phage-only study, the PEV31 titer gradually decreased in the lungs over 24 h, with a half-life of approximately 8 h for both doses. In the presence of bacteria, in contrast, the PEV31 titer increased by almost 2-log10 in the lungs at 16 h. Furthermore, bacterial growth was suppressed in the PEV31-treated group, while the PBS-treated group showed exponential growth. Of the 10 colonies tested, four phage-resistant isolates were observed from the lung homogenates sampled at 24 h after phage treatment. These colonies had a different antibiogram to the parent bacteria. This study provides evidence that pulmonary delivery of phage PEV31 in mice can reduce the MDR bacterial burden.

High-throughput mapping of the phage resistance landscape in E. coli.

Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.

Storage stability of phage-ciprofloxacin combination powders against Pseudomonas aeruginosa respiratory infections.

Novel inhalable and synergistic combination powder formulations of phage PEV20 and ciprofloxacin were recently developed to treat Pseudomonas aeruginosa respiratory infections. In the present study, we investigated the storage stability of these powders which comprised ciprofloxacin, lactose and L-leucine in mass ratios of 1:1:1 (Formulation A) or ciprofloxacin and L-leucine in 2:1 without lactose (Formulation B). These powders were produced by spray drying, collected in polypropylene tubes and packed inside aluminium pouches which were heat-sealed at < 20% relative humidity (RH), then stored at 4 °C or 25 °C. The phage viability, aerosol performance and solid-state properties of the powders were examined over 12 months. The biological activity and aerosol performance of both formulations showed no significant change over 12 months of storage at 4 °C. However, after four months of storage at 25 °C, a significant titer loss of 2.2 log10 (p < 0.01) was observed in Formulation B, but the loss in Formulation A was much less (0.5 log10 (p < 0.05)). In contrast, the fine particle fraction (FPF, wt. % particles ≤ 5 µm) of Formulation A was significantly reduced by 11% (p < 0.05) after four months of storage at 25 °C, whereas the aerosol performance of Formulation B remained stable over 12 months. The results showed that ciprofloxacin can sufficiently stabilize phage through vitrification and/or hydrogen bonding at 4 °C. The presence of lactose was beneficial to preserve the phage at 25 °C. In conclusion, spray dried PEV20-ciprofloxacin combination powders were biologically and physico-chemically stable even without lactose as a stabilising excipient, when stored below 20% RH at 4 °C for 12 months.

Development of a broad-spectrum Salmonella phage cocktail containing Viunalike and Jerseylike viruses isolated from Thailand.

Salmonella is one of the most common agents of foodborne disease worldwide. As natural alternatives to traditional antimicrobial agents, bacteriophages (phages) are emerging as highly effective biocontrol agents against Salmonella and other foodborne bacteria. Due to the high diversity within the Salmonella genus and emergence of drug resistant strains, improved efforts are necessary to find broad range and strictly lytic Salmonella phages for use in food biocontrol. Here, we describe the isolation and characterization of two Salmonella phages: ST-W77 isolated on S. Typhimurium and SE-W109 isolated on S. Enteritidis with extraordinary Salmonella specificity. Whole genome sequencing identified ST-W77 as a Myovirus within the Viunalikevirus genus and SE-W109 as a Siphovirus within the Jerseylikevirus genus. Infectivity studies using a panel of S. Typhimurium cell wall mutants revealed both phages require the lipopolysaccharide O-antigen, with SE-W109 also recognizing the flagella, during infection of Salmonella. A combination of both phages was capable of prolonged (one-week) antibacterial activity when added to milk or chicken meat contaminated with Salmonella. Due to their broad host ranges, strictly lytic lifestyles and lack of lysogeny-related genes or virulence genes in their genomes, ST-W77 and SE-W109 are ideal phages for further development as Salmonella biocontrol agents for food production.

Inhalable bacteriophage powders: Glass transition temperature and bioactivity stabilization.

Recent heightened interest in inhaled bacteriophage (phage) therapy for combating antibacterial resistance in pulmonary infections has led to the development of phage powder formulations. Although phages have been successfully bioengineered into inhalable powders with preserved bioactivity, the stabilization mechanism is yet unknown. This paper reports the first study investigating the stabilization mechanism for phages in these powders. Proteins and other biologics are known to be preserved in dry state within a glassy sugar matrix at storage temperatures (T s) at least ~50°C below the glass transition temperature (T g). This is because at (T g - T s) >50°C, molecules are sufficiently immobilized with reduced reactivity. We hypothesized that this glass stabilization mechanism may also be applicable to phages comprising mostly of proteins. In this study, spray dried powders of Pseudomonas phage PEV20 containing lactose and leucine as excipients were stored at 5, 25 or 50°C and 15 or 33% relative humidity (RH), followed by assessment of bioactivity (PEV20 stability) and physical properties. PEV20 was stable with negligible titer loss after storage at 5°C/15% RH for 250 days, while storage at 33% RH caused increased titer losses of 1 log10 and 3 log10 at 5 and 25°C, respectively. The plasticizing effect of water at 33% RH lowered the T g by 30°C, thus narrowing the gap between T s and T g to 19-28°C, which was insufficient for glass stabilization. In contrast, the (T g - T s) values were higher (range, 46-65°C) under the drier condition of 15% RH, resulting in the improved stability which corroborated with the vitrification hypothesis. Furthermore, phage remained stable (≤1 log10) when the (T g - T s) value lay between 26-48°C, but became inactivated as the value fell below 20°C. In conclusion, this study demonstrated that phage can be sufficiently stabilized in spray dried powders by keeping the (T g - T s) value above 46°C, thus supporting the vitrification hypothesis that phages are stabilized by immobilization inside a rigid glassy sugar matrix. These findings provide a guide to better manufacture and storage practices of inhaled phage powder products using for translational medicines.

Inhalable combination powder formulations of phage and ciprofloxacin for P. aeruginosa respiratory infections.

Recently we showed that nebulized ciprofloxacin and phage PEV20 in combination had a synergistic bactericidal effect against antibiotic-resistant Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Compared to nebulization, dry powders for inhalation may improve patient handling characteristics and compliance. In the present study, we co-spray dried ciprofloxacin and phage PEV20 using L-leucine with or without lactose as excipients. Two formulations were identified for testing in this study. The mass ratios were set at 1:1:1 for ciprofloxacin, lactose and L-leucine (Formulation A) or 2:1 for ciprofloxacin and L-leucine without lactose (Formulation B). Concentrations of PEV20 were set at 108 and 109 PFU/mL for two clinical P. aeruginosa strains FADD1-PA001 and JIP865, respectively. Formulations A and B were characterized as partially crystalline and the powders recrystallized at >40% relative humidity (RH). Both formulations exhibited strong synergistic antimicrobial killing effect on the two strains. Formulations A and B maintained bactericidal synergy after dispersion using both low and high resistance Osmohaler™. Powder aerosol performance was examined by next generation impactor (NGI) in low resistance inhaler at 100 L/min and by multi-stage liquid impinger (MSLI) in high resistance inhaler at 60 L/min. Fine particle fractions (FPF) obtained by NGI were 59.7 ±â€¯2.1% and 64.3 ±â€¯2.9% for A and B, respectively. FPF obtained by MSLI were 71.0 ±â€¯3.4% and 73.3 ±â€¯5.0%, respectively. In conclusion, it is feasible to prepare stable and inhalable combination powder formulations of phage PEV20 and ciprofloxacin for potential treatment of respiratory infections caused by multi-drug resistant (MDR) P. aeruginosa.

Structure and Function of the Branched Receptor-Binding Complex of Bacteriophage CBA120.

Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120-TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1-TSP2-TSP3-TSP4 branched complex in CBA120 and its related ViI-like phages.

Bacteriophage PEV20 and Ciprofloxacin Combination Treatment Enhances Removal of Pseudomonas aeruginosa Biofilm Isolated from Cystic Fibrosis and Wound Patients.

Antibiotic resistance in Pseudomonas aeruginosa biofilms necessitates the need for novel antimicrobial therapy with anti-biofilm properties. Bacteriophages (phages) are recognized as an ideal biopharmaceutical for combating antibiotic-resistant bacteria especially when used in combination with antibiotics. However, previous studies primarily focused on using phages against of P. aeruginosa biofilms of laboratory strains. In the present study, biofilms of six P. aeruginosa isolated from cystic fibrosis and wound patients, and one laboratory strain was treated singly and with combinations of anti-Pseudomonas phage PEV20 and ciprofloxacin. Of these strains, three were highly susceptible to the phage, while one was partially resistant and one was completely resistant. Combination treatment with PEV20 and ciprofloxacin enhanced biofilm eradication compared with single treatment. Phage and ciprofloxacin synergy was found to depend on phage-resistance profile of the target bacteria. Furthermore, phage and ciprofloxacin combination formulation protected the lung epithelial and fibroblast cells from P. aeruginosa and promoted cell growth. The results demonstrated that thorough screening of phage-resistance is crucial for designing phage-antibiotic formulation. The addition of highly effective phage could reduce the ciprofloxacin concentration required to combat P. aeruginosa infections associated with biofilm in cystic fibrosis and wound patients.

Storage stability of inhalable phage powders containing lactose at ambient conditions.

The aim of this study was to evaluate the storage stability of inhalable phage powders containing lactose and leucine as excipient. As an FDA-approved excipient for inhalation, lactose is preferred over other sugars. PEV phages active against antibiotic-resistant Pseudomonas aeruginosa was spray dried with lactose (55-90%) and leucine (45-10%). Produced powders were heat-sealed in an aluminium pouch at 15% relative humidity (RH) with subsequent storage at 20 °C/60% RH for 12 months. Lactose concentration in the powder positively influenced the phage stability over time. Formulation containing 90% lactose maintained the viability of PEV61 across the study, while ∼1.2 log10 titer reduction was observed in formulations with less lactose. PEV20 was more prone to inactivation (1.7 log10 titer loss at 12-month) when lactose concentration in the particle was below 80%. The fine particle fraction (% wt. particles <5 µm in aerosol) of phage powders was 52-61% and remained the same after 12-month storage. The results demonstrate that spray dried PEV phage powders containing lactose and leucine are biologically and physically stable over long-term storage at ambient temperature. Furthermore, these spray dried phage powders were shown to be non-toxic to lung alveolar macrophage and epithelial cells in vitro.

Jet nebulization of bacteriophages with different tail morphologies - Structural effects.

It was previously demonstrated that the loss of infectivity of a myovirus PEV44 after jet nebulization was closely related to a change in bacteriophage (phage) structure. In this follow-up study, we further examined the impact of jet nebulization on tailed phages, which constitute 96% of all known phages, from three different families, Podoviridae (PEV2), Myoviridae (PEV40) andSiphoviridae (D29). Transmission electron microscopy (TEM) identified major changes in phage structures after jet nebulization, correlating with their loss of infectivity. For the podovirus PEV2, jet nebulization had a negligible impact on its activity (0.04 log10 pfu/mL loss) and structural change. On the other hand, the proportion of intact phages in the nebulized samples dropped from 50% to ∼27% for PEV40 and from 15% to ∼2% for D29. Phage deactivation of PEV40 measured by the TEM structural damage (0.52 log10 pfu/mL) was lower than that obtained by plaque assay (1.02 log10 pfu/mL), but within the range of variation (±0.5 log10 pfu/mL). However, TEM quantification considerably underestimated the titer reduction of D29 phage, ∼2 log pfu/mL lower than that obtained in plaque assay (3.25 log10 pfu/mL loss). In conclusion, nebulization-induced titre loss was correlated with morphological damage to phages and in particular, the tail length may be an important consideration for selection of phages in inhaled therapy using jet nebulization.

Resolving Digital Staphylococcal Osteomyelitis Using Bacteriophage-A Case Report.

Infections involving diabetic foot ulcers (DFU) are a major public health problem and have a substantial negative impact on patient outcomes. Osteomyelitis in an ulcerated foot substantially increases the difficulty of successful treatment. While literature suggests that osteomyelitis in selected patients can sometimes be treated conservatively, with no, or minimal removal of bone, we do not yet have clear treatment guidelines and the standard treatment failure fallback remains amputation. The authors report on the successful treatment, with a long term follow up, of a 63 YO diabetic female with distal phalangeal osteomyelitis using bacteriophage, a form of treatment offering the potential for improved outcomes in this era of escalating antibiotic resistance and the increasingly recognized harms associated with antibiotic therapy.