Anti-Pseudomonal Bacteriophage Reduces Infective Burden and Inflammatory Response in Murine Lung

Rishi Pabary1,2, Charanjit Singh1, Sandra Morales3, Andrew Bush1,2, Khalid Alshafi4, Diana Bilton4, Eric WFW Alton1, Anthony Smithyman3 and Jane C. Davies1,2#

1National Heart and Lung Institute, Imperial College London

2Department of Paediatric Respiratory Medicine, Royal Brompton Hospital, London

3Special Phage Services, Australia

4Department of Microbiology, Royal Brompton Hospital, London

5Adult Cystic Fibrosis Unit, Royal Brompton Hospital, London

Running Head: Phage reduces murine infection and inflammation

Corresponding Author: Professor Jane C. Davies (j.c.davies@imperial.ac.uk)

Keywords (MESH terms): Bacteriophages, bronchoalveolar lavage, cystic fibrosis, drug resistance (microbial), infection, inflammation

Abstract

Rationale: As antibiotic resistance increases, there is a need for new therapies to treat infection, particularly in cystic fibrosis (CF) where Pseudomonas aeruginosa (Pa) is a ubiquitous pathogen associated with increased morbidity and mortality. Bacteriophages are an attractive alternative treatment as they are specific to the target bacteria and have no documented side-effects.

Methods: Efficacy of phage cocktails was established in vitro. Two Pa strains were taken forward into an acute murine infection model with bacteriophage administered either prophylactically, simultaneously or post-infection. Assessment of infective burden and inflammation in bronchoalveolar lavage fluid (BALF) was undertaken at various times.

Results: With low infective doses, both control mice and those undergoing simultaneous phage treatment cleared Pa infection at 48hrs but there were fewer neutrophils in BALF of phage-treated mice (median [range] 73.2 [35.2-102.1], x104/ml vs. 174 [112.1-266.8] p < 0.01 for clinical strain; median [range] 122.1 [105.4-187.4] x104/ml vs. 206 [160.1-331.6], p < 0.01 for PAO1). With higher infective doses of PAO1, all phage-treated mice cleared infection at 24hrs whereas infection persisted in all control mice; median [range] CFU/ml 1305 [190-4700], p < 0.01. Bacteriophage also reduced CFU/ml in BALF when administered post-infection (24 hours) and both CFU/ml and inflammatory cells in BALF when administered prophylactically. Reduction in soluble inflammatory cytokines in BALF was also demonstrated under different conditions.

Conclusion: Bacteriophages are efficacious in reducing both bacterial load and inflammation in a murine model of Pa lung infection. This study provides proof-of-concept for future clinical trials in patients with CF.

Introduction

Antimicrobial resistance in general has been flagged as a major global health risk by the World Health Organisation (1), with the rising incidence of multi-drug resistant gram negative bacteria, such as Pseudomonas aeruginosa, of particular concern.. Pseudomonas aeruginosa (Pa) is a ubiquitous, gram-negative bacterium that opportunistically infects patients with chronic suppurative lung diseases such as cystic fibrosis (CF), and is clearly associated with increased morbidity and mortality (2). Antimicrobial therapy is usually effective at eradicating initial infection (3) but most patients ultimately become chronically infected as Pa is both inherently resistant to many classes of antibiotics due to its efflux-pump system (4) and rapidly develops mutation-based resistances in the presence of exposure to antimicrobial agents (5). Bacterial infection is closely associated with pulmonary inflammation in CF and, although there is increasing evidence that this paradigm may be simplistic (6), it is clear that neutrophilic inflammation causes lung injury (7) and declines following antibiotic treatment of Pa in CF (8). For CF patients, failure of conventional antibiotics facilitates the development of chronic Pa infection whereby originally free-floating (planktonic) organisms switch to a biofilm mode of growth (9). In addition to increasing antibiotic resistance (10), there are significant side-effects associated with conventional antimicrobials, particularly when they are used repeatedly or over long periods of time. These include renal and oto-toxicity, both of which are commonly encountered in adult clinics. There is thus an urgent need for novel anti-pseudomonal therapies for patients with CF.

Bacteriophages are naturally occurring viruses that specifically target bacterial cells (11). First described by Felix d’Herelle in 1917 (12), they were the focus of several therapeutic studies in the 1920s. However, these were run under conditions not comparable to modern standards and lacked suitable controls and due to the low quality of some products, results were often inconsistent (13). Coupled with the discovery of antibiotics in 1928 (14), this meant that widespread clinical use was mainly limited to Eastern Europe (12, 15).

Bacteriophages offer several advantages over conventional antibiotics: they are highly selective so can be targeted against pathogenic bacteria without disturbing the resident bacterial flora; they multiply exponentially in the presence of host (bacterial) cells rather than decreasing in concentration over time, thereby potentially providing treatment targeted to the sites of need (12); they can adapt and mutate like bacteria, thereby potentially reducing the emergence of resistant bacterial strains (16, 17) and they appear to be relatively free of side-effects (17). Bacteriophages are widely used in food preservation, being applied for example to the surfaces of preserved meats and cheeses (18, 19). Bacteriophage have been shown to be efficacious in vitro against Pa in biofilms (20) and in vivo in murine models of Pa septicaemia: between 50-100% of mice infected with a lethal intraperitoneal dose of Pa survived when administered a single dose of intravenous (21) or intraperitoneal (22) phage up to one hour post-infection. Recent studies of acute lung infection in mice have used bioluminescent strains of Pa to demonstrate phage efficacy; bioluminescence decreased following administration of phage with an associated reduction in bacteria recovered from bronchoalveolar lavage fluid (BALF) and disease severity (as assessed by histological analysis of lung tissue) in phage-treated mice compared with controls (23, 24). However, none of these studies investigated the impact of phage-targeted pseudomonal killing on lung inflammation. This is highly relevant as persistent neutrophilic inflammation has been associated with lung injury (25) and, even during periods of stability, CF patients with chronic Pa infection have higher inflammatory indices than subjects without CF (26). Reduction in bacterial load demonstrated in previous studies does not necessarily equate to attenuation of inflammatory damage. An important unanswered question remains as to whether phage therapy itself induces a host inflammatory response either directly or secondary to phage-induced Pa lysis (leading to release of toxins such as LPS) or reduces the response by hastening bacterial clearance.

Although in vitro models suggest that bacteriophages can be deposited successfully in the human lung by nebulisation (27), no studies of efficacy in lung infection have been undertaken to date under strict regulatory criteria. However, a small randomised controlled trial in the United Kingdom reported that a single topical dose of phage reduced symptoms in patients with persistent Pa ear infections refractory to multiple courses of antibiotics, with no reported adverse events (28). Safety has also previously been reported in children receiving intravenous phage (29).

Based on the previously published data, we consider that bacteriophages could be a useful treatment for Pa in patients with CF. We hypothesised that such treatment would reduce bacterial load as previously described but also thereby reduce inflammation and the detrimental downstream consequences thereof. In this study, we test specifically-designed anti-Pa bacteriophage cocktails in a murine model of Pa lung infection. Pa strains assessed as being susceptible to bacteriophage cocktails in vitro were studied in vivo in order to determine if there were any immunological benefits of phage therapy. We assess the effect on lung bacterial load, systemic spread of infection and pulmonary inflammation and explore the potential both for treatment of infection and for prophylaxis.

Materials and Methods

Ethics Statement

Female BALB/c mice (Harlan, UK) were housed in a specialised animal facility in accordance with European regulations. Food and drink were provided ad libitum. The work was prospectively approved by the United Kingdom Home Office and National Ethics Committee.

Bacteriophage isolation and cocktail selection

Bacteriophages for this study were isolated by Special Phage Services Pty Ltd (Sydney, Australia) from a variety of environmental sources in New South Wales, Australia, using different protocols as previously described. (30) Three different bacteriophage cocktails: cocktail 1 (Pa 24, Pa 25 and Pa 37), cocktail 2 (Pa 39, Pa 67, Pa 77 and Pa 119) and cocktail 3 (Pa 3, Pa 6, Pa 10, Pa 32 and Pa 37) were selected based on their abilities to delay or inhibit appearance of putative phage-resistant cells in liquid or solid media. Each bacteriophage was tested for its morphology and host spectrum of activity against PAO1 and ten P. aeruginosa clinical isolates collected in Australia (Table 1 Supplementary Information). The approximate molecular weight (MW) for each phage was also determined by pulsed-field electrophoresis (31) and each phage shown to be different by restriction digest (data not shown).

In vitro Phage Susceptibility Testing

Before use in vivo, susceptibility of our chosen bacterial isolates to the bacteriophage cocktails was initially confirmed using conventional plaque assays (32)). PAO1, a well-described laboratory reference strain (33, 34), and five Pa strains isolated from the sputa of adult in-patients with CF at the Royal Brompton Hospital, London, were tested against the three novel bacteriophage cocktails. Pure isolates were inoculated into 10mls tryptone soy broth (TSB: Oxoid, UK) and cultured overnight at 37oC with agitation. Optical density (OD) of the broths was measured spectrophotometrically (Spectronic, UK) and adjusted to 0.1 (equivalent to approximately 1x108 colony forming units (CFU)/ml) by dilution with sterile TSB. 100µl of the diluted broth was added to 3mls semi-solid agar (prepared by dissolving 3g of TSB powder (Sigma, UK) and 0.4g agar (Sigma, UK) in 100mls deionised water and autoclaving) that had been maintained at 55oC in a water bath before pouring onto Pseudomonas-specific agar (PSA: Oxoid, UK). After cooling, 10µl aliquots of each bacteriophage cocktail (6.2 x 1010 plaque-forming units (PFU)/ml at neat and serially log10 diluted down to 10-6) were pipetted onto the prepared bacterial lawns and incubated overnight at 37oC. The cocktail that was most broadly efficacious with lab strain PAO1 and the most susceptible strain isolated from CF patients (henceforth termed “clinical strain”) were taken forward for these proof-of-principle in vivo studies.

In vivo Methodology

Following overnight culture of the two selected bacterial strains in TSB, broth was centrifuged (Meadowrose Scientific, UK) at 2000g at 4oC for ten minutes and the resultant cell pellet resuspended in 10mls of phosphate buffered saline (PBS: Gibco, UK). OD was adjusted by dilution with PBS; the relationship between CFU/ ml and OD was previously determined by serial dilution and colony counting as per Miles and Misra (35).

Adult BALB/C mice were anaesthetised by isoflurane inhalational. In a pilot, dose-finding study, n=3/ group received 50µl by nasal gavage (sniffing) of 1x109, 5x108,1x108 or 5x107 CFU/ml. Mice in the first 3 groups were either deceased or unwell 24hrs post-infection. A maximum inoculum of 5x107 CFU/ml was therefore selected for initial experimental use

Mice were infected by intranasal sniffing initially with 50l of 5x107 CFU/ml (2.5 x 106 CFU; ‘low dose’); in later experiments where bronchoalveolar lavage (BAL) was carried out 24hrs post-infection, we were able to apply 50l of 5x108 CFU/ml (2.5 x 107 CFU; ‘high dose’). 20l (1.2x109 PFU) intranasal phage therapy or buffer (controls) was administered either simultaneously, 24hrs post-infection or 48hrs pre-infection. BAL was carried out either 24 or 48hrs post-infection using the following technique: terminal general anaesthesia was achieved by intraperitoneal administration of Hypnorm (Vetapharma, UK) and Hypnovel (Roche, UK). After cessation of circulation, the trachea was surgically exposed and cannulated with a 22g AbbocathTM (Hospira, UK). Bronchoalveolar lavage (BAL) was performed with 500l PBS instillation and aspirated three times. Spleens were dissected and harvested into 500l PBS.

Processing of Samples

100l BAL was serially log10 diluted and 5 x 10l drops cultured overnight at 37oC on PSA plates as per Miles and Misra (35). Non-quantitative culture on PSA agar was also performed on homogenised explanted spleens to determine systemic spread.

Remaining BAL was centrifuged at 4oc, 2000g for ten minutes. 100l aliquots of supernatant were stored at -80oC for subsequent batched analysis of inflammatory cytokines. Cytokines were selected based on their inclusion in a commercially available multiplex ELISA platform (MesoScale Discovery (MSD) mouse pro-inflammatory 7-plex ultra-sensitive assay). The remaining cell pellet was resuspended in 200l PBS. 20l of this solution was added to 40l tryphan blue (Sigma, UK) and 20l PBS (1 in 4 dilution) and total inflammatory cells counted with Neubauer haemocytometer. A further 100ul was used for differential cell count following cytospin (Shandon, UK) for five minutes at 400rpm. Slides were fixed with methanol and stained using May-Grunwald-Giesma Quickstain kit prior to mounting with DPX (Sigma, UK). 300 cells per slide were counted by one investigator following blinding of the slides by a second investigator; unblinding took place at the end of each part of the study.

Statistical Analyses

Based on modest group sizes and assuming non-Gaussian data distribution, Mann-Whitney t-test was performed on all datasets using Prism 6.0 (GraphPad, United States). Eight mice was the arbitrary number decided upon for each arm of each condition being tested; if clear differences became apparent with fewer (minimum of six mice in each arm), the study was stopped in accordance with ethical standards of animal research. Median data and range are presented. The null hypothesis was rejected if p<0.05.

Results

Lytic activity of bacteriophage cocktail in vitro

All three bacteriophage cocktails were effective against PAO1 at phage dilutions from neat to 10-5. This result matched expectations given the reported activity of the individual phages against this strain (Table 1 Online Supplement). When tested against the clinical isolates, bacteriophage cocktail 1 was active against the 5 clinical isolates/strains tested whilst bacteriophage cocktail 2 and 3 infected only 3 out of the 5 isolates/strains. Sensitivities of each clinical strain tested to each phage cocktail are shown in Table 1:

The broad-spectrum of activity of a bacteriophage cocktail has been suggested as an important characteristic to overcome the limitations of specificity associated with bacteriophages. Based on the susceptibility results obtained, bacteriophage cocktail 1 was selected for in vivo use. Similarly, as there are reports suggesting good correlation between in vitro activity and in vivo phage efficacy (36), the isolate/strain PA12B-4973 was selected for in vivo experimentation as the phage cocktail 1 was very efficient against this isolate/strain even at a very low concentration (10-6).

Simultaneous Administration of Bacteriophage and Pa

Two experimental conditions were tested. Initially, mice were infected with 2.5 x 106 bacteria (50 l of 5x107 CFU/ml) PAO1 (n=16) or the clinical strain (n=12) and immediately afterwards, whilst under the same inhalational anaesthetic, 20l phage (n=14) or buffer (n=14) was administered. Samples were harvested at 48hrs. BALF culture demonstrated that all phage-treated mice and most control mice cleared Pseudomonas; 2/6 control mice infected with the clinical strain had persistent infection but with low bacterial load (20 and 40 CFU/ml) on quantitative culture. Systemic spread, as indicated by positive splenic cultures, was not seen in either group. However, inflammation was significantly reduced in the phage-treated animals. Total inflammatory cells (predominantly neutrophils) were lower with both bacterial strains (Table 2 in Supplemental Information Section and Figure 1) as were several cytokines although this was only observed with the clinical strain (Tables 3a and 3b in Supplemental Information Section and Figure 2).

These data provided evidence for a phage effect, but the ability of control animals to clear this dose of Pa meant that no signal on bacterial killing could be demonstrated. Therefore, we next infected mice with a higher dose of PAO1 (2.5x107 CFU/ml) and chose an earlier, 24hr, time point for sampling. Mice infected with higher inoculums of the clinical strain became terminally unwell in less than 24hrs and thus only PAO1 was used for ongoing work. Under these conditions, all control mice had detectable Pa infection (median [range] 1305 [190-4700] CFU/ml). In contrast, no bacteria were cultured from BAL from any phage treated mice (Figure 3a; p <0.01). There was no growth from splenic cultures in either group. IL-10 (p < 0.01) and IL-1 (p < 0.05) were significantly reduced in phage-treated mice compared with controls (Figure 3b) but there was no difference in the five other cytokines measured or in inflammatory cell counts (Tables 4 and 5 in Supplemental Information Section). Having demonstrated efficacy with simultaneous administration, and recognising how poorly this mirrored any clinical context, we went on to assess delayed and prophylactic phage administration.

Delayed Administration of Bacteriophage

High dose (2.5x107 CFU/ml) PAO1 was inoculated intranasally and bacteriophage or buffer administered 24hrs hours later. Samples were obtained a further 24hrs after this. In contrast to control mice, who all had positive BAL cultures (5950 [40 – 194000] CFU/ml), complete clearance was seen in 6/7 (86%) of phage treated mice (and median CFU/ml was significantly lower (0 [0-160] CFU/ml, p < 0.01, Figure 4a). Two control mice had growth of Pa from splenic culture, indicating systemic spread of infection. This was not seen in any of the phage-treated animals. There was a reduction in IL-10 (p < 0.05) and KC (keratinocyte chemoattractant) (p < 0.01) in phage-treated mice (Figure 4b) but no reduction in other inflammatory cytokines or in cell counts (Tables 6 and 7 in Supplemental Information Section).

‘Prophylactic’ Administration of Bacteriophage

Bacteriophage or buffer was instilled 48hrs prior to intranasal infection with high dose (2.5x107 CFU/ml) PAO1. Samples were obtained 24 hours after bacterial infection. Two control mice died in this 24 hour period. Of those surviving, all had persistent and high levels of bacteria in BAL (1.8 x 106 [1140 – 1.64x1010] CFU/ml). In contrast, 5/7 (71%) of phage pre-treated mice had successfully cleared the infection and those which had not, had only low levels of bacteria detected (0 [0-20] CFU/ml, p < 0.01, Figure 5a). Four of five (80%) surviving control mice had positive splenic cultures indicating systemic spread. This was not seen in any of the phage-treated mice (n=7).

KC (Figure 5b) (p <0.01) and total and differential cell counts (Figure 6) in BALF of mice pre-treated with phage were significantly reduced compared with controls (Table 8 in Supplementary Information Section and Figure 6) although there was no difference in other cytokines (Table 8 in Supplementary Information Section).

Discussion

We have shown that delivery of selected bacteriophage cocktails during, before or after lung infection with Pa has a significant impact on local bacterial burden, systemic spread of infection and lung inflammatory responses.

We first confirmed the expected activity of three bacteriophage cocktails in vitro against the laboratory strain, PAO1, and demonstrated the activity of the three cocktails against some but not all of clinical isolates of Pa taken from patients with CF. The ability of a phage to form plaques on a lawn of the target bacteria is seen as the basic requirement for phage therapy. Furthermore, correlation between bacteriophage activity in vitro and subsequent success in vivo has been reported before (36). This study supports the importance of this correlation, although care should be taken not to assume this is the only property required for efficacy (37). Subsequently, bacteriophage reduced infective burden and inflammatory response in a murine infection model when using an initial theoretical multiplicity of infection (MOI) of ~100. At lower bacterial doses, no difference in infective burden was demonstrated, as mice were capable of spontaneous clearance, but there was a significant reduction in neutrophils. At higher infective doses, the objective of achieving persistent infection was achieved, but only in control mice; all phage-treated mice retained the ability to clear their lungs of infection. Similarly, in experiments where phage or buffer was administered post-infection, there were significantly lower CFU/ml in BALF of phage-treated mice compared with controls, although no difference was seen in inflammatory cells. Finally, the efficacy of prophylactic phage was also demonstrated; all treated mice survived a high dose of inoculum and had significantly lower CFU/ml and neutrophils in BALF compared to controls.

In keeping with the observation that BALB/c mice are inherently resistance to Pa infection (38), most mice in this study were able to clear a low dose of intranasally administered Pa with no evidence of systemic spread even in the absence of phage treatment. However, such mice demonstrated neutrophilic inflammation at 48 hours in response to both strains of Pa administered. This inflammatory response was significantly reduced when bacteriophage were administered simultaneously. This is significant because, although inflammation and infection may be dissociated in CF (39, 40), the role of neutrophils in mediating tissue injury is clear and therefore treatments that reduce their number may be of benefit (41). However, as trials of leukotriene B4 receptor antagonists demonstrate, this paradigm may be over-simplistic (42)

In addition, reduced levels of BALF IL-10, IL-6, TNF- and IL-12p70 were demonstrated in phage-treated mice infected with the clinical strain of Pa, with a trend towards reduced KC.TNF- plays a key role in the acute phase response, promoting recruitment of neutrophils to sites of infection (43, 44) and is also one of the physiological stimuli for IL-6 production, along with bacterial endotoxin (45) . IL-12p70 is the biologically active form of IL-12 which is important in Th1 immune responses to bacteria and viruses (46) whilst KC is a major neutrophil chemoattractant (47). The reduction in neutrophil count and cytokine levels in BALF of phage-treated mice 48hrs following infection with a clinical Pa strain suggests that bacteriophage complements the inherent resistance of these mice to Pa, hastening clearance and thereby diminishing the inflammatory response. That there was no significant reduction in cytokine levels in phage-treated mice infected with PAO1 most likely reflects a difference in virulence between the two strains of bacteria as differences did become apparent when the inoculum of PAO1 was increased.

When numbers of nasally instilled PAO1 were increased ten-fold and BAL was performed earlier at 24hrs, control mice had significant numbers of Pa present in the BALF, whereas all phage-treated mice had completely cleared the infection. Lower levels of inflammation (IL-1 and IL-10 and a trend in IL-6) were also observed.

In addition to the co-administration experiments, we demonstrated efficacy when phage were administered either after bacterial infection, mimicking a clinical ‘treatment’ scenario or beforehand, as ‘prophylaxis’. Both resulted in a significant impact on bacterial load and inflammatory response and suggest potential clinical utility. The prophylaxis experiments also indicate that phage is relatively stable in the murine lung (for at least 24hrs). This raises a concern that carryover phage might be present when plating BAL from infected animals, which has the potential to reduce CFU counts ex vivo. The way in which samples were processed aimed to minimise the risk of phage-bacteria interactions in vitro but it was not possible to demonstrate that no carryover phage was present in cultured BALF. This question has been addressed previously; studies using bioluminescent strains to monitor phage efficacy in real time (23, 48) demonstrate that phage activity clearly occurs in the lungs and is not the result of ex vivo culturing only. This issue is analogous to culturing BALF or sputum from patients already on antibiotics. The fact that bacteria do not grow in vitro leads to the conclusion that infection is not present; it is not possible to be sure if this is because of efficacy in vivo or an in vitro effect after samples are collected. Molecular assay testing to address this issue may be applied to future experimental models.

What we have not done in this set of experiments is model chronic infection with mucoid or biofilm modes of growth. Transgenic CF mice in general do not recapitulate the lung disease characteristic of human CF, and most investigators have resorted to the use of artificial means of establishing chronic infection such as agar beads. Whilst potentially useful for studying host responses, we decided against this model for the testing of a topically applied therapeutic, penetration of which may have been adversely affected by the presence of the agar. We may, in the future be able to study such mechanisms in alternative animal models such as the β-ENaC over-expressing mouse or the CF pig or ferret. Data from other fields suggesting that bacteriophage are effective against biofilm-growing organisms (20, 49-51) provide encouraging support for this approach.

Whilst all mice infected with Pa and simultaneously treated with bacteriophage cleared infection (Figure 3a), colonies remained present in BALF of some mice who received delayed or prophylactic dosing of phage (Figures 4a and 5a) albeit in far lower quantities than untreated mice. This is most likely indicative of incomplete clearance due to higher bacterial load in mice where phage treatment was delayed and/or because BAL was performed at an earlier time point (24hrs rather than 48hrs) but the possibility that the recovered Pa had evolved phage-resistance cannot be discounted. The recovered colonies were not retested in vitro for phage susceptibility but this will be done in future experiments as the question of whether sensitive bacterial strains become resistant to bacteriophage over time is key to clinical application.

Although the majority of the data supports a reduction and benefit in the general inflammatory response when bacteriophages are used, different conditions led to variable changed in specific soluble inflammatory markers. Five cytokines were lower in phage-treated mice infected with clinical Pa whereas no phage-related differences were seen with PAO1 at the same inoculum; given the severity of illness noted in mice infected with higher doses of the clinical strain, this could be attributed to differences in virulence of the Pa strains. At higher inoculums of PAO1, IL-10 and IL-1b were lower in phage-treated animals following simultaneous administration, IL-10 and KC were lower when phage was given 24hrs post-infection and only KC was lower with prophylactic phage administration. Difficulties in standardisation of animals, exacerbating inherent biological variability under each condition, may have contributed to this; although all mice were adult female BALB/C, exact age and weight could not be matched which may have affected response. There may also have been underpowering for some of these effects due to our attempts to limit animal numbers used in the experiments.

Reduction in IL-10 in phage-treated animals was seen across several conditions tested. This initially seemed counter-intuitive as IL-10 inhibits production of pro-inflammatory cytokines (including IL-1, IL-6, IL-12 and TNF-) by T-cells, thereby down-regulating the acute immune response (52); there was close correlation of IL-10 with IL-1, IL-6 and TNF- (r2 0.734 – 0.787) but not with IL-12p70 (r2 = 0.368) in this study. However, recent evidence suggests that IL-10 response is related to the severity of a preceding pro-inflammatory response (52), which is subsequently down-regulated by IL-10 to prevent ongoing inflammation; hence high levels are associated with protracted infection and blockade of IL-10 may in fact promote clearance of bacteria (53). If this is the case, and there remains no consensus in the literature due to the complexity of the IL-10 signalling (52), then reduced IL-1, IL-6 and TNF- in experiments with the clinical strain, reduced IL-1 and a trend towards reduced IL-6 (p = 0.06) when the inoculum of PAO1 was increased with simultaneous dosing of phage and a trend towards reduced IL-1 and IL-6 with later dosing of phage, could account for reduced “anti-inflammatory” IL-10 in this study; as there was less initial inflammation in phage-treated mice, less IL-10 was detected. Further support for this theory is the fact that IL-10, of all the measured cytokines in this study, correlated most strongly with absolute neutrophil count across each of the tested conditions (r2 = 0.503).

From a translational perspective, there were three key findings from this study. Firstly, no evidence of murine toxicity following rapid lysis of Pa by bacteriophage was seen, suggesting that this approach may be safe in a human clinical trial. Secondly, a beneficial effect of phage treatment once infection was established provides support of bacteriophage as a therapy. Thirdly, and perhaps most encouragingly, administration prior to infection is efficacious (both aiding clearance once infection is encountered and reducing neutrophilic inflammation), raising the possibility of prophylaxis, perhaps only at times of increased infection risk, for example during viral infection, which has been linked to acquisition of Pa. UK Registry data (54) currently demonstrates a window of opportunity in childhood and early adolescence, before the majority of patients have become chronically infected with Pa, for such a prophylactic approach. Clearly, further work is needed to establish the longevity of phage in the non-bacterial infected host, the frequency with which this would have to be administered and potential host responses (either inflammatory or immune) associated with acute administration or long-term use. It will also be crucial to assess the development of phage-resistance in any persisting bacteria. Recent studies have demonstrated proof-of-concept for prophylactic phage therapy in humans, particularly for gastrointestinal infections (55); regular dosing from a young age of anti-Pa bacteriophage cocktails, selected with knowledge of local strains and sensitivities, is therefore an attractive strategy by which to attempt to reduce the incidence of infection and burden of long-term morbidity and mortality associated with chronic infection.

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Table

Clinical Isolate

Cocktail 1

Cocktail 2

Cocktail 3

PA 12B-4854

10-2

No effect

No effect

PA 12B-4973

10-6

10-4

10-6

PA 12B-5001

10-5

10-5

10-6

PA 12B-5025

10-2

10-2

10-4

PA 12B-5099

10-2

No effect

No effect

Table 1: Susceptibility of five clinical strains of Pa to three bacteriophage cocktails. Cocktail 1 was more broadly efficacious and PA12B-4973 (from here on known as clinical strain) was most broadly sensitive, and therefore these were used for ongoing work.

Figure Legends

Figure 1: Differential cell counts (median/range) from BAL performed at 48hrs in mice inoculated with 2.5 x 106 of a clinical strain of Pa and simultaneously treated with 20l bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer.

Figure 2: Pro-inflammatory cytokines (median/range) from BAL performed at 48hrs in mice inoculated with 2.5 x 106 of a clinical strain of Pa and simultaneously treated with 20l bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer.

Figure 3a: Colony counts/ml from BAL performed at 24hrs in mice inoculated with 2.5 x 107 of PAO1 and simultaneously treated with 20ul bacteriophage cocktail (containing 1.24 x 109 PFU) or 20l SM buffer. If no colonies were visible to the naked eye, this is reported as 0 CFU/ml; the theoretical limit of detection was 100 CFU/ml as 10l drops of BALF were cultured.

Figure 3b: Pro-inflammatory cytokines (median/range) from BAL performed at 24hrs in mice inoculated with 2.5 x 107 of PAO1 and simultaneously treated with 20l bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer.

Figure 4a: Colony counts/ml from BAL performed at 48hrs in mice inoculated with 2.5 x 107 of PAO1 and treated with 20l bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer 24hrs after the initial infection. If no colonies were visible to the naked eye, this is reported as 0 CFU/ml; the theoretical limit of detection was 100 CFU/ml as 10l drops of BALF were cultured.

Figure 4b: Pro-inflammatory cytokines (median/range) from BAL performed at 48hrs in mice inoculated with 2.5 x 107 of PAO1 and treated with 20l bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer 24hrs after the initial infection.

Figure 5a: Colony counts/ml from BAL performed at 24hrs in mice inoculated with 2.5 x 107 of PAO1 and treated with 20l bacteriophage cocktail (containing 1.24 x 109 PFU) or 20l SM buffer prophylactically, 48hrs prior to infection. If no colonies were visible to the naked eye, this is reported as 0 CFU/ml; the theoretical limit of detection was 100 CFU/ml as 10l drops of BALF were cultured.

Figure 5b: KC (median/range) from BAL performed at 24hrs in mice inoculated with 2.5 x 107 of PAO1 and treated with 20l bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer prophylactically, 48hrs prior to infection.

Figure 6: Differential cell counts (median/range) from BAL performed at 24hrs in mice inoculated with 2.5 x 107 of PAO1 and treated with 20l bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer prophylactically, 48hrs prior to infection.

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