anti-pseudomonal bacteriophage reduces infecti ve...

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Anti-Pseudomonal Bacteriophage Reduces Infective Burden and Inflammatory Response in 1

Murine Lung 2

Rishi Pabary1,2, Charanjit Singh1, Sandra Morales3, Andrew Bush1,2, Khalid Alshafi4, Diana Bilton4, 3

Eric WFW Alton1, Anthony Smithyman3 and Jane C. Davies1,2# 4

1National Heart and Lung Institute, Imperial College London 5

2Department of Paediatric Respiratory Medicine, Royal Brompton Hospital, London 6

3Special Phage Services, Australia 7

4Department of Microbiology, Royal Brompton Hospital, London 8

5Adult Cystic Fibrosis Unit, Royal Brompton Hospital, London 9

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Running Head: Phage reduces murine infection and inflammation 11

Corresponding Author: Professor Jane C. Davies ([email protected]) 12

Keywords (MESH terms): Bacteriophages, bronchoalveolar lavage, cystic fibrosis, drug 13 resistance (microbial), infection, inflammation 14

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AAC Accepted Manuscript Posted Online 16 November 2015Antimicrob. Agents Chemother. doi:10.1128/AAC.01426-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract 17

Rationale: As antibiotic resistance increases, there is a need for new therapies to treat 18

infection, particularly in cystic fibrosis (CF) where Pseudomonas aeruginosa (Pa) is a ubiquitous 19

pathogen associated with increased morbidity and mortality. Bacteriophages are an attractive 20

alternative treatment as they are specific to the target bacteria and have no documented side-21

effects. 22

Methods: Efficacy of phage cocktails was established in vitro. Two Pa strains were taken 23

forward into an acute murine infection model with bacteriophage administered either 24

prophylactically, simultaneously or post-infection. Assessment of infective burden and 25

inflammation in bronchoalveolar lavage fluid (BALF) was undertaken at various times. 26

Results: With low infective doses, both control mice and those undergoing simultaneous phage 27

treatment cleared Pa infection at 48hrs but there were fewer neutrophils in BALF of phage-28

treated mice (median [range] 73.2 [35.2-102.1], x104/ml vs. 174 [112.1-266.8] p < 0.01 for 29

clinical strain; median [range] 122.1 [105.4-187.4] x104/ml vs. 206 [160.1-331.6], p < 0.01 for 30

PAO1). With higher infective doses of PAO1, all phage-treated mice cleared infection at 24hrs 31

whereas infection persisted in all control mice; median [range] CFU/ml 1305 [190-4700], p < 32

0.01. Bacteriophage also reduced CFU/ml in BALF when administered post-infection (24 hours) 33

and both CFU/ml and inflammatory cells in BALF when administered prophylactically. Reduction 34

in soluble inflammatory cytokines in BALF was also demonstrated under different conditions. 35


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Conclusion: Bacteriophages are efficacious in reducing both bacterial load and inflammation in 36

a murine model of Pa lung infection. This study provides proof-of-concept for future clinical 37

trials in patients with CF. 38

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Introduction 40

Antimicrobial resistance in general has been flagged as a major global health risk by the World 41

Health Organisation (1), with the rising incidence of multi-drug resistant gram negative 42

bacteria, such as Pseudomonas aeruginosa, of particular concern.. Pseudomonas aeruginosa 43

(Pa) is a ubiquitous, gram-negative bacterium that opportunistically infects patients with 44

chronic suppurative lung diseases such as cystic fibrosis (CF), and is clearly associated with 45

increased morbidity and mortality (2). Antimicrobial therapy is usually effective at eradicating 46

initial infection (3) but most patients ultimately become chronically infected as Pa is both 47

inherently resistant to many classes of antibiotics due to its efflux-pump system (4) and rapidly 48

develops mutation-based resistances in the presence of exposure to antimicrobial agents (5). 49

Bacterial infection is closely associated with pulmonary inflammation in CF and, although there 50

is increasing evidence that this paradigm may be simplistic (6), it is clear that neutrophilic 51

inflammation causes lung injury (7) and declines following antibiotic treatment of Pa in CF (8). 52

For CF patients, failure of conventional antibiotics facilitates the development of chronic Pa 53

infection whereby originally free-floating (planktonic) organisms switch to a biofilm mode of 54

growth (9). In addition to increasing antibiotic resistance (10), there are significant side-effects 55

associated with conventional antimicrobials, particularly when they are used repeatedly or over 56

long periods of time. These include renal and oto-toxicity, both of which are commonly 57

encountered in adult clinics. There is thus an urgent need for novel anti-pseudomonal therapies 58

for patients with CF. 59


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Bacteriophages are naturally occurring viruses that specifically target bacterial cells (11). First 60

described by Felix d’Herelle in 1917 (12), they were the focus of several therapeutic studies in 61

the 1920s. However, these were run under conditions not comparable to modern standards 62

and lacked suitable controls and due to the low quality of some products, results were often 63

inconsistent (13). Coupled with the discovery of antibiotics in 1928 (14), this meant that 64

widespread clinical use was mainly limited to Eastern Europe (12, 15). 65

Bacteriophages offer several advantages over conventional antibiotics: they are highly selective 66

so can be targeted against pathogenic bacteria without disturbing the resident bacterial flora; 67

they multiply exponentially in the presence of host (bacterial) cells rather than decreasing in 68

concentration over time, thereby potentially providing treatment targeted to the sites of need 69

(12); they can adapt and mutate like bacteria, thereby potentially reducing the emergence of 70

resistant bacterial strains (16, 17) and they appear to be relatively free of side-effects (17). 71

Bacteriophages are widely used in food preservation, being applied for example to the surfaces 72

of preserved meats and cheeses (18, 19). Bacteriophage have been shown to be efficacious in 73

vitro against Pa in biofilms (20) and in vivo in murine models of Pa septicaemia: between 50-74

100% of mice infected with a lethal intraperitoneal dose of Pa survived when administered a 75

single dose of intravenous (21) or intraperitoneal (22) phage up to one hour post-infection. 76

Recent studies of acute lung infection in mice have used bioluminescent strains of Pa to 77

demonstrate phage efficacy; bioluminescence decreased following administration of phage 78

with an associated reduction in bacteria recovered from bronchoalveolar lavage fluid (BALF) 79

and disease severity (as assessed by histological analysis of lung tissue) in phage-treated mice 80

compared with controls (23, 24). However, none of these studies investigated the impact of 81


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phage-targeted pseudomonal killing on lung inflammation. This is highly relevant as persistent 82

neutrophilic inflammation has been associated with lung injury (25) and, even during periods of 83

stability, CF patients with chronic Pa infection have higher inflammatory indices than subjects 84

without CF (26). Reduction in bacterial load demonstrated in previous studies does not 85

necessarily equate to attenuation of inflammatory damage. An important unanswered question 86

remains as to whether phage therapy itself induces a host inflammatory response either 87

directly or secondary to phage-induced Pa lysis (leading to release of toxins such as LPS) or 88

reduces the response by hastening bacterial clearance. 89

Although in vitro models suggest that bacteriophages can be deposited successfully in the 90

human lung by nebulisation (27), no studies of efficacy in lung infection have been undertaken 91

to date under strict regulatory criteria. However, a small randomised controlled trial in the 92

United Kingdom reported that a single topical dose of phage reduced symptoms in patients 93

with persistent Pa ear infections refractory to multiple courses of antibiotics, with no reported 94

adverse events (28). Safety has also previously been reported in children receiving intravenous 95

phage (29). 96

Based on the previously published data, we consider that bacteriophages could be a useful 97

treatment for Pa in patients with CF. We hypothesised that such treatment would reduce 98

bacterial load as previously described but also thereby reduce inflammation and the 99

detrimental downstream consequences thereof. In this study, we test specifically-designed 100

anti-Pa bacteriophage cocktails in a murine model of Pa lung infection. Pa strains assessed as 101

being susceptible to bacteriophage cocktails in vitro were studied in vivo in order to determine 102


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if there were any immunological benefits of phage therapy. We assess the effect on lung 103

bacterial load, systemic spread of infection and pulmonary inflammation and explore the 104

potential both for treatment of infection and for prophylaxis. 105

Materials and Methods 106

Ethics Statement 107

Female BALB/c mice (Harlan, UK) were housed in a specialised animal facility in accordance 108

with European regulations. Food and drink were provided ad libitum. The work was 109

prospectively approved by the United Kingdom Home Office and National Ethics Committee. 110

Bacteriophage isolation and cocktail selection 111

Bacteriophages for this study were isolated by Special Phage Services Pty Ltd (Sydney, Australia) 112

from a variety of environmental sources in New South Wales, Australia, using different 113

protocols as previously described. (30) Three different bacteriophage cocktails: cocktail 1 (Pa 114

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 115

10, Pa 32 and Pa 37) were selected based on their abilities to delay or inhibit appearance of 116

putative phage-resistant cells in liquid or solid media. Each bacteriophage was tested for its 117

morphology and host spectrum of activity against PAO1 and ten P. aeruginosa clinical isolates 118

collected in Australia (Table 1 Supplementary Information). The approximate molecular weight 119

(MW) for each phage was also determined by pulsed-field electrophoresis (31) and each phage 120

shown to be different by restriction digest (data not shown). 121

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In vitro Phage Susceptibility Testing 123

Before use in vivo, susceptibility of our chosen bacterial isolates to the bacteriophage cocktails 124

was initially confirmed using conventional plaque assays (32)). PAO1, a well-described 125

laboratory reference strain (33, 34), and five Pa strains isolated from the sputa of adult in-126

patients with CF at the Royal Brompton Hospital, London, were tested against the three novel 127

bacteriophage cocktails. Pure isolates were inoculated into 10mls tryptone soy broth (TSB: 128

Oxoid, UK) and cultured overnight at 37oC with agitation. Optical density (OD) of the broths was 129

measured spectrophotometrically (Spectronic, UK) and adjusted to 0.1 (equivalent to 130

approximately 1x108 colony forming units (CFU)/ml) by dilution with sterile TSB. 100µl of the 131

diluted broth was added to 3mls semi-solid agar (prepared by dissolving 3g of TSB powder 132

(Sigma, UK) and 0.4g agar (Sigma, UK) in 100mls deionised water and autoclaving) that had 133

been maintained at 55oC in a water bath before pouring onto Pseudomonas-specific agar (PSA: 134

Oxoid, UK). After cooling, 10µl aliquots of each bacteriophage cocktail (6.2 x 1010 plaque-135

forming units (PFU)/ml at neat and serially log10 diluted down to 10-6) were pipetted onto the 136

prepared bacterial lawns and incubated overnight at 37oC. The cocktail that was most broadly 137

efficacious with lab strain PAO1 and the most susceptible strain isolated from CF patients 138

(henceforth termed “clinical strain”) were taken forward for these proof-of-principle in vivo 139

studies. 140

In vivo Methodology 141

Following overnight culture of the two selected bacterial strains in TSB, broth was centrifuged 142

(Meadowrose Scientific, UK) at 2000g at 4oC for ten minutes and the resultant cell pellet 143


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resuspended in 10mls of phosphate buffered saline (PBS: Gibco, UK). OD was adjusted by 144

dilution with PBS; the relationship between CFU/ ml and OD was previously determined by 145

serial dilution and colony counting as per Miles and Misra (35). 146

Adult BALB/C mice were anaesthetised by isoflurane inhalational. In a pilot, dose-finding study, 147

n=3/ group received 50µl by nasal gavage (sniffing) of 1x109, 5x108,1x108 or 5x107 CFU/ml. Mice 148

in the first 3 groups were either deceased or unwell 24hrs post-infection. A maximum inoculum 149

of 5x107 CFU/ml was therefore selected for initial experimental use 150

Mice were infected by intranasal sniffing initially with 50μl of 5x107 CFU/ml (2.5 x 106 CFU; ‘low 151

dose’); in later experiments where bronchoalveolar lavage (BAL) was carried out 24hrs post-152

infection, we were able to apply 50μl of 5x108 CFU/ml (2.5 x 107 CFU; ‘high dose’). 20μl (1.2x109 153

PFU) intranasal phage therapy or buffer (controls) was administered either simultaneously, 154

24hrs post-infection or 48hrs pre-infection. BAL was carried out either 24 or 48hrs post-155

infection using the following technique: terminal general anaesthesia was achieved by 156

intraperitoneal administration of Hypnorm (Vetapharma, UK) and Hypnovel (Roche, UK). After 157

cessation of circulation, the trachea was surgically exposed and cannulated with a 22g 158

AbbocathTM (Hospira, UK). Bronchoalveolar lavage (BAL) was performed with 500μl PBS 159

instillation and aspirated three times. Spleens were dissected and harvested into 500μl PBS. 160

Processing of Samples 161

100μl BAL was serially log10 diluted and 5 x 10μl drops cultured overnight at 37oC on PSA plates 162

as per Miles and Misra (35). Non-quantitative culture on PSA agar was also performed on 163

homogenised explanted spleens to determine systemic spread. 164


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Remaining BAL was centrifuged at 4oc, 2000g for ten minutes. 100μl aliquots of supernatant 165

were stored at -80oC for subsequent batched analysis of inflammatory cytokines. Cytokines 166

were selected based on their inclusion in a commercially available multiplex ELISA platform 167

(MesoScale Discovery (MSD) mouse pro-inflammatory 7-plex ultra-sensitive assay). The 168

remaining cell pellet was resuspended in 200μl PBS. 20μl of this solution was added to 40μl 169

tryphan blue (Sigma, UK) and 20μl PBS (1 in 4 dilution) and total inflammatory cells counted 170

with Neubauer haemocytometer. A further 100ul was used for differential cell count following 171

cytospin (Shandon, UK) for five minutes at 400rpm. Slides were fixed with methanol and 172

stained using May-Grunwald-Giesma Quickstain kit prior to mounting with DPX (Sigma, UK). 300 173

cells per slide were counted by one investigator following blinding of the slides by a second 174

investigator; unblinding took place at the end of each part of the study. 175

Statistical Analyses 176

Based on modest group sizes and assuming non-Gaussian data distribution, Mann-Whitney t-177

test was performed on all datasets using Prism 6.0 (GraphPad, United States). Eight mice was 178

the arbitrary number decided upon for each arm of each condition being tested; if clear 179

differences became apparent with fewer (minimum of six mice in each arm), the study was 180

stopped in accordance with ethical standards of animal research. Median data and range are 181

presented. The null hypothesis was rejected if p<0.05. 182

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Results 184

Lytic activity of bacteriophage cocktail in vitro 185

All three bacteriophage cocktails were effective against PAO1 at phage dilutions from neat to 186

10-5. This result matched expectations given the reported activity of the individual phages 187

against this strain (Table 1 Online Supplement). When tested against the clinical isolates, 188

bacteriophage cocktail 1 was active against the 5 clinical isolates/strains tested whilst 189

bacteriophage cocktail 2 and 3 infected only 3 out of the 5 isolates/strains. Sensitivities of each 190

clinical strain tested to each phage cocktail are shown in Table 1: 191

The broad-spectrum of activity of a bacteriophage cocktail has been suggested as an important 192

characteristic to overcome the limitations of specificity associated with bacteriophages. Based 193

on the susceptibility results obtained, bacteriophage cocktail 1 was selected for in vivo use. 194

Similarly, as there are reports suggesting good correlation between in vitro activity and in vivo 195

phage efficacy (36), the isolate/strain PA12B-4973 was selected for in vivo experimentation as 196

the phage cocktail 1 was very efficient against this isolate/strain even at a very low 197

concentration (10-6). 198

Simultaneous Administration of Bacteriophage and Pa 199

Two experimental conditions were tested. Initially, mice were infected with 2.5 x 106 bacteria 200

(50 μl of 5x107 CFU/ml) PAO1 (n=16) or the clinical strain (n=12) and immediately afterwards, 201

whilst under the same inhalational anaesthetic, 20μl phage (n=14) or buffer (n=14) was 202

administered. Samples were harvested at 48hrs. BALF culture demonstrated that all phage-203


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treated mice and most control mice cleared Pseudomonas; 2/6 control mice infected with the 204

clinical strain had persistent infection but with low bacterial load (20 and 40 CFU/ml) on 205

quantitative culture. Systemic spread, as indicated by positive splenic cultures, was not seen in 206

either group. However, inflammation was significantly reduced in the phage-treated animals. 207

Total inflammatory cells (predominantly neutrophils) were lower with both bacterial strains 208

(Table 2 in Supplemental Information Section and Figure 1) as were several cytokines although 209

this was only observed with the clinical strain (Tables 3a and 3b in Supplemental Information 210

Section and Figure 2). 211

These data provided evidence for a phage effect, but the ability of control animals to clear this 212

dose of Pa meant that no signal on bacterial killing could be demonstrated. Therefore, we next 213

infected mice with a higher dose of PAO1 (2.5x107 CFU/ml) and chose an earlier, 24hr, time 214

point for sampling. Mice infected with higher inoculums of the clinical strain became terminally 215

unwell in less than 24hrs and thus only PAO1 was used for ongoing work. Under these 216

conditions, all control mice had detectable Pa infection (median [range] 1305 [190-4700] 217

CFU/ml). In contrast, no bacteria were cultured from BAL from any phage treated mice (Figure 218

3a; p <0.01). There was no growth from splenic cultures in either group. IL-10 (p < 0.01) and IL-219

1β (p < 0.05) were significantly reduced in phage-treated mice compared with controls (Figure 220

3b) but there was no difference in the five other cytokines measured or in inflammatory cell 221

counts (Tables 4 and 5 in Supplemental Information Section). Having demonstrated efficacy 222

with simultaneous administration, and recognising how poorly this mirrored any clinical 223

context, we went on to assess delayed and prophylactic phage administration. 224


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Delayed Administration of Bacteriophage 225

High dose (2.5x107 CFU/ml) PAO1 was inoculated intranasally and bacteriophage or buffer 226

administered 24hrs hours later. Samples were obtained a further 24hrs after this. In contrast to 227

control mice, who all had positive BAL cultures (5950 [40 – 194000] CFU/ml), complete 228

clearance was seen in 6/7 (86%) of phage treated mice (and median CFU/ml was significantly 229

lower (0 [0-160] CFU/ml, p < 0.01, Figure 4a). Two control mice had growth of Pa from splenic 230

culture, indicating systemic spread of infection. This was not seen in any of the phage-treated 231

animals. There was a reduction in IL-10 (p < 0.05) and KC (keratinocyte chemoattractant) (p < 232

0.01) in phage-treated mice (Figure 4b) but no reduction in other inflammatory cytokines or in 233

cell counts (Tables 6 and 7 in Supplemental Information Section). 234

‘Prophylactic’ Administration of Bacteriophage 235

Bacteriophage or buffer was instilled 48hrs prior to intranasal infection with high dose (2.5x107 236

CFU/ml) PAO1. Samples were obtained 24 hours after bacterial infection. Two control mice died 237

in this 24 hour period. Of those surviving, all had persistent and high levels of bacteria in BAL 238

(1.8 x 106 [1140 – 1.64x1010] CFU/ml). In contrast, 5/7 (71%) of phage pre-treated mice had 239

successfully cleared the infection and those which had not, had only low levels of bacteria 240

detected (0 [0-20] CFU/ml, p < 0.01, Figure 5a). Four of five (80%) surviving control mice had 241

positive splenic cultures indicating systemic spread. This was not seen in any of the phage-242

treated mice (n=7). 243

KC (Figure 5b) (p <0.01) and total and differential cell counts (Figure 6) in BALF of mice pre-244

treated with phage were significantly reduced compared with controls (Table 8 in 245


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Supplementary Information Section and Figure 6) although there was no difference in other 246

cytokines (Table 8 in Supplementary Information Section). 247

Discussion 248

We have shown that delivery of selected bacteriophage cocktails during, before or after lung 249

infection with Pa has a significant impact on local bacterial burden, systemic spread of infection 250

and lung inflammatory responses. 251

We first confirmed the expected activity of three bacteriophage cocktails in vitro against the 252

laboratory strain, PAO1, and demonstrated the activity of the three cocktails against some but 253

not all of clinical isolates of Pa taken from patients with CF. The ability of a phage to form 254

plaques on a lawn of the target bacteria is seen as the basic requirement for phage therapy. 255

Furthermore, correlation between bacteriophage activity in vitro and subsequent success in 256

vivo has been reported before (36). This study supports the importance of this correlation, 257

although care should be taken not to assume this is the only property required for efficacy (37). 258

Subsequently, bacteriophage reduced infective burden and inflammatory response in a murine 259

infection model when using an initial theoretical multiplicity of infection (MOI) of ~100. At 260

lower bacterial doses, no difference in infective burden was demonstrated, as mice were 261

capable of spontaneous clearance, but there was a significant reduction in neutrophils. At 262

higher infective doses, the objective of achieving persistent infection was achieved, but only in 263

control mice; all phage-treated mice retained the ability to clear their lungs of infection. 264

Similarly, in experiments where phage or buffer was administered post-infection, there were 265

significantly lower CFU/ml in BALF of phage-treated mice compared with controls, although no 266


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difference was seen in inflammatory cells. Finally, the efficacy of prophylactic phage was also 267

demonstrated; all treated mice survived a high dose of inoculum and had significantly lower 268

CFU/ml and neutrophils in BALF compared to controls. 269

In keeping with the observation that BALB/c mice are inherently resistance to Pa infection (38), 270

most mice in this study were able to clear a low dose of intranasally administered Pa with no 271

evidence of systemic spread even in the absence of phage treatment. However, such mice 272

demonstrated neutrophilic inflammation at 48 hours in response to both strains of Pa 273

administered. This inflammatory response was significantly reduced when bacteriophage were 274

administered simultaneously. This is significant because, although inflammation and infection 275

may be dissociated in CF (39, 40), the role of neutrophils in mediating tissue injury is clear and 276

therefore treatments that reduce their number may be of benefit (41). However, as trials of 277

leukotriene B4 receptor antagonists demonstrate, this paradigm may be over-simplistic (42) 278

In addition, reduced levels of BALF IL-10, IL-6, TNF-α and IL-12p70 were demonstrated in 279

phage-treated mice infected with the clinical strain of Pa, with a trend towards reduced KC.TNF-280

α plays a key role in the acute phase response, promoting recruitment of neutrophils to sites of 281

infection (43, 44) and is also one of the physiological stimuli for IL-6 production, along with 282

bacterial endotoxin (45) . IL-12p70 is the biologically active form of IL-12 which is important in 283

Th1 immune responses to bacteria and viruses (46) whilst KC is a major neutrophil 284

chemoattractant (47). The reduction in neutrophil count and cytokine levels in BALF of phage-285

treated mice 48hrs following infection with a clinical Pa strain suggests that bacteriophage 286

complements the inherent resistance of these mice to Pa, hastening clearance and thereby 287


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diminishing the inflammatory response. That there was no significant reduction in cytokine 288

levels in phage-treated mice infected with PAO1 most likely reflects a difference in virulence 289

between the two strains of bacteria as differences did become apparent when the inoculum of 290

PAO1 was increased. 291

When numbers of nasally instilled PAO1 were increased ten-fold and BAL was performed 292

earlier at 24hrs, control mice had significant numbers of Pa present in the BALF, whereas all 293

phage-treated mice had completely cleared the infection. Lower levels of inflammation (IL-1β 294

and IL-10 and a trend in IL-6) were also observed. 295

In addition to the co-administration experiments, we demonstrated efficacy when phage were 296

administered either after bacterial infection, mimicking a clinical ‘treatment’ scenario or 297

beforehand, as ‘prophylaxis’. Both resulted in a significant impact on bacterial load and 298

inflammatory response and suggest potential clinical utility. The prophylaxis experiments also 299

indicate that phage is relatively stable in the murine lung (for at least 24hrs). This raises a 300

concern that carryover phage might be present when plating BAL from infected animals, which 301

has the potential to reduce CFU counts ex vivo. The way in which samples were processed 302

aimed to minimise the risk of phage-bacteria interactions in vitro but it was not possible to 303

demonstrate that no carryover phage was present in cultured BALF. This question has been 304

addressed previously; studies using bioluminescent strains to monitor phage efficacy in real 305

time (23, 48) demonstrate that phage activity clearly occurs in the lungs and is not the result of 306

ex vivo culturing only. This issue is analogous to culturing BALF or sputum from patients already 307

on antibiotics. The fact that bacteria do not grow in vitro leads to the conclusion that infection 308


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is not present; it is not possible to be sure if this is because of efficacy in vivo or an in vitro 309

effect after samples are collected. Molecular assay testing to address this issue may be applied 310

to future experimental models. 311

What we have not done in this set of experiments is model chronic infection with mucoid or 312

biofilm modes of growth. Transgenic CF mice in general do not recapitulate the lung disease 313

characteristic of human CF, and most investigators have resorted to the use of artificial means 314

of establishing chronic infection such as agar beads. Whilst potentially useful for studying host 315

responses, we decided against this model for the testing of a topically applied therapeutic, 316

penetration of which may have been adversely affected by the presence of the agar. We may, 317

in the future be able to study such mechanisms in alternative animal models such as the β-ENaC 318

over-expressing mouse or the CF pig or ferret. Data from other fields suggesting that 319

bacteriophage are effective against biofilm-growing organisms (20, 49-51) provide encouraging 320

support for this approach. 321

Whilst all mice infected with Pa and simultaneously treated with bacteriophage cleared 322

infection (Figure 3a), colonies remained present in BALF of some mice who received delayed or 323

prophylactic dosing of phage (Figures 4a and 5a) albeit in far lower quantities than untreated 324

mice. This is most likely indicative of incomplete clearance due to higher bacterial load in mice 325

where phage treatment was delayed and/or because BAL was performed at an earlier time 326

point (24hrs rather than 48hrs) but the possibility that the recovered Pa had evolved phage-327

resistance cannot be discounted. The recovered colonies were not retested in vitro for phage 328


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susceptibility but this will be done in future experiments as the question of whether sensitive 329

bacterial strains become resistant to bacteriophage over time is key to clinical application. 330

Although the majority of the data supports a reduction and benefit in the general 331

inflammatory response when bacteriophages are used, different conditions led to variable 332

changed in specific soluble inflammatory markers. Five cytokines were lower in phage-treated 333

mice infected with clinical Pa whereas no phage-related differences were seen with PAO1 at 334

the same inoculum; given the severity of illness noted in mice infected with higher doses of the 335

clinical strain, this could be attributed to differences in virulence of the Pa strains. At higher 336

inoculums of PAO1, IL-10 and IL-1b were lower in phage-treated animals following 337

simultaneous administration, IL-10 and KC were lower when phage was given 24hrs post-338

infection and only KC was lower with prophylactic phage administration. Difficulties in 339

standardisation of animals, exacerbating inherent biological variability under each condition, 340

may have contributed to this; although all mice were adult female BALB/C, exact age and 341

weight could not be matched which may have affected response. There may also have been 342

underpowering for some of these effects due to our attempts to limit animal numbers used in 343

the experiments. 344

Reduction in IL-10 in phage-treated animals was seen across several conditions tested. This 345

initially seemed counter-intuitive as IL-10 inhibits production of pro-inflammatory cytokines 346

(including IL-1β, IL-6, IL-12 and TNF-α) by T-cells, thereby down-regulating the acute immune 347

response (52); there was close correlation of IL-10 with IL-1β, IL-6 and TNF-α (r2 0.734 – 0.787) 348

but not with IL-12p70 (r2 = 0.368) in this study. However, recent evidence suggests that IL-10 349


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response is related to the severity of a preceding pro-inflammatory response (52), which is 350

subsequently down-regulated by IL-10 to prevent ongoing inflammation; hence high levels are 351

associated with protracted infection and blockade of IL-10 may in fact promote clearance of 352

bacteria (53). If this is the case, and there remains no consensus in the literature due to the 353

complexity of the IL-10 signalling (52), then reduced IL-1β, IL-6 and TNF-α in experiments with 354

the clinical strain, reduced IL-1β and a trend towards reduced IL-6 (p = 0.06) when the inoculum 355

of PAO1 was increased with simultaneous dosing of phage and a trend towards reduced IL-1β 356

and IL-6 with later dosing of phage, could account for reduced “anti-inflammatory” IL-10 in this 357

study; as there was less initial inflammation in phage-treated mice, less IL-10 was detected. 358

Further support for this theory is the fact that IL-10, of all the measured cytokines in this study, 359

correlated most strongly with absolute neutrophil count across each of the tested conditions (r2 360

= 0.503). 361

From a translational perspective, there were three key findings from this study. Firstly, no 362

evidence of murine toxicity following rapid lysis of Pa by bacteriophage was seen, suggesting 363

that this approach may be safe in a human clinical trial. Secondly, a beneficial effect of phage 364

treatment once infection was established provides support of bacteriophage as a therapy. 365

Thirdly, and perhaps most encouragingly, administration prior to infection is efficacious (both 366

aiding clearance once infection is encountered and reducing neutrophilic inflammation), raising 367

the possibility of prophylaxis, perhaps only at times of increased infection risk, for example 368

during viral infection, which has been linked to acquisition of Pa. UK Registry data (54) currently 369

demonstrates a window of opportunity in childhood and early adolescence, before the majority 370

of patients have become chronically infected with Pa, for such a prophylactic approach. Clearly, 371


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further work is needed to establish the longevity of phage in the non-bacterial infected host, 372

the frequency with which this would have to be administered and potential host responses 373

(either inflammatory or immune) associated with acute administration or long-term use. It will 374

also be crucial to assess the development of phage-resistance in any persisting bacteria. Recent 375

studies have demonstrated proof-of-concept for prophylactic phage therapy in humans, 376

particularly for gastrointestinal infections (55); regular dosing from a young age of anti-Pa 377

bacteriophage cocktails, selected with knowledge of local strains and sensitivities, is therefore 378

an attractive strategy by which to attempt to reduce the incidence of infection and burden of 379

long-term morbidity and mortality associated with chronic infection. 380

381

382


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References 383

1. Organisation WH. Antimicrobial Resistance2013. Available from: 384 http://www.who.int/mediacentre/factsheets/fs194/en/. 385 2. Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson RL. Pseudomonas aeruginosa and 386 other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol. 387 2002;34(2):91-100. 388 3. Langton Hewer SC, Smyth AR. Antibiotic strategies for eradicating Pseudomonas aeruginosa in 389 people with cystic fibrosis. The Cochrane database of systematic reviews. 2009(4):CD004197. 390 4. Poole K. Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and 391 related organisms. J Mol Microbiol Biotechnol. 2001;3(2):255-64. 392 5. Livermore DM. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: 393 our worst nightmare? Clinical infectious diseases : an official publication of the Infectious Diseases 394 Society of America. 2002;34(5):634-40. 395 6. Rao S, Grigg J. New insights into pulmonary inflammation in cystic fibrosis. Arch Dis Child. 396 2006;91(9):786-8. 397 7. Downey DG, Bell SC, Elborn JS. Neutrophils in cystic fibrosis. Thorax. 2009;64(1):81-8. 398 8. Ordoñez CL, Henig NR, Mayer-Hamblett N, Accurso FJ, Burns JL, Chmiel JF, 399 Daines CL, Gibson RL, McNamara S, Retsch-Bogart GZ, Zeitlin PL, Aitken ML. Inflammatory and 400 microbiologic markers in induced sputum after intravenous antibiotics in cystic fibrosis. American 401 journal of respiratory and critical care medicine. 2003;168(12):1471-5. 402 9. O'Toole GA, Kolter R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa 403 biofilm development. Mol Microbiol. 1998;30(2):295-304. 404 10. Nordmann P, Naas T, Fortineau N, Poirel L. Superbugs in the coming new decade; multidrug 405 resistance and prospects for treatment of Staphylococcus aureus, Enterococcus spp. and Pseudomonas 406 aeruginosa in 2010. Current opinion in microbiology. 2007;10(5):436-40. 407 11. Bradbury J. "My enemy's enemy is my friend." Using phages to fight bacteria. Lancet. 408 2004;363(9409):624-5. 409 12. Fruciano DE, Bourne S. Phage as an antimicrobial agent: d'Herelle's heretical theories and their 410 role in the decline of phage prophylaxis in the West. The Canadian journal of infectious diseases & 411 medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale / 412 AMMI Canada. 2007;18(1):19-26. 413 13. Lobocka M, Szybalski WT. Advances in Virus Research. Bacteriophages, part A. Preface. 414 Advances in virus research. 2012;82:xiii-xv. 415 14. Fleming A. On the antibacterial action of cultures of a penicillium, with special reference to their 416 use in the isolation of B. influenzae. British Journal of Experimental Pathology. 1929;10(3):226-36. 417 15. Kutateladze M, Adamia R. Phage therapy experience at the Eliava Institute. Medecine et 418 maladies infectieuses. 2008;38(8):426-30. 419 16. Parisien A, Allain B, Zhang J, Mandeville R, Lan CQ. Novel alternatives to antibiotics: 420 bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides. Journal of applied 421 microbiology. 2008;104(1):1-13. 422 17. Thiel K. Old dogma, new tricks--21st Century phage therapy. Nature biotechnology. 423 2004;22(1):31-6. 424 18. Sillankorva SM, Oliveira H, Azeredo J. Bacteriophages and their role in food safety. Int J 425 Microbiol. 2012;2012:863945. 426 19. Greer GG. Bacteriophage control of foodborne bacteriat. J Food Prot. 2005;68(5):1102-11. 427


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

22

20. Fu W, Forster T, Mayer O, Curtin JJ, Lehman SM, Donlan RM. Bacteriophage cocktail for the 428 prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system. 429 Antimicrobial agents and chemotherapy. 2010;54(1):397-404. 430 21. Meitert E, Petrovici M, Sima F, Costache G, Savulian C. Investigation on the therapeutical 431 efficiency of some adapted bacteriophages in experimental infection with Pseudomonas aeruginosa. 432 Archives roumaines de pathologie experimentales et de microbiologie. 1987;46(1):17-26. 433 22. Wang J, Hu B, Xu M, Yan Q, Liu S, Zhu X, Sun Z, Reed E, Ding L, Gong J, Li QQ, 434 Hu J. Use of bacteriophage in the treatment of experimental animal bacteremia from imipenem-435 resistant Pseudomonas aeruginosa. International journal of molecular medicine. 2006;17(2):309-17. 436 23. 437 Debarbieux L, Leduc D, Maura D, Morello E, Criscuolo A, Grossi O, Balloy V, Touqui L. Bacteriophages can 438 treat and prevent Pseudomonas aeruginosa lung infections. The Journal of infectious diseases. 439 2010;201(7):1096-104. 440 24. Morello E, Saussereau E, Maura D, Huerre M, Touqui L, Debarbieux L. Pulmonary bacteriophage 441 therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards treatment and 442 prevention. PloS one. 2011;6(2):e16963. 443 25. Craig A, Mai J, Cai S, Jeyaseelan S. Neutrophil recruitment to the lungs during bacterial 444 pneumonia. Infection and immunity. 2009;77(2):568-75. 445 26. Jones AM, Martin L, Bright-Thomas RJ, Dodd ME, McDowell A, Moffitt KL, Elborn 446 JS, Webb AK. Inflammatory markers in cystic fibrosis patients with transmissible Pseudomonas 447 aeruginosa. Eur Respir J. 2003;22(3):503-6. 448 27. Golshahi L, Seed KD, Dennis JJ, Finlay WH. Toward modern inhalational bacteriophage therapy: 449 nebulization of bacteriophages of Burkholderia cepacia complex. Journal of aerosol medicine and 450 pulmonary drug delivery. 2008;21(4):351-60. 451 28. Wright A, Hawkins CH, Anggard EE, Harper DR. A controlled clinical trial of a therapeutic 452 bacteriophage preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a 453 preliminary report of efficacy. Clinical otolaryngology : official journal of ENT-UK ; official journal of 454 Netherlands Society for Oto-Rhino-Laryngology & Cervico-Facial Surgery. 2009;34(4):349-57. 455 29. Fortuna W, Miedzybrodzki R, Weber-Dabrowska B, Gorski A. Bacteriophage therapy in children: 456 facts and prospects. Medical science monitor : international medical journal of experimental and clinical 457 research. 2008;14(8):RA126-32. 458 30. Kutter ESA. Bacteriophages: Biology and Applications. 1 ed: CRC Press; December 28 2004. 528 459 p. 460 31. Finney M. Pulsed-field gel electrophoresis. Current protocols in molecular biology / edited by 461 Frederick M Ausubel Roger Brent, Robert E. Kingston, David D. Moore, J.G. Seidman, John A. Smith, 462 Kevin Struhl. 2001;Chapter 2:Unit2 5B. 463 32. H AM. Bacteriophages. 1st ed. New York: Interscience Publishers Inc; 1959. 592 p. 464 33. Holloway BW. Genetic recombination in Pseudomonas aeruginosa. Journal of general 465 microbiology. 1955;13(3):572-81. 466 34. Klockgether J, Munder A, Neugebauer J, Davenport CF, Stanke F, Larbig KD, Heeb 467 S, Schöck U, Pohl TM, Wiehlmann L, Tümmler B. Genome diversity of Pseudomonas aeruginosa PAO1 468 laboratory strains. J Bacteriol. 2010;192(4):1113-21. 469 35. Miles AA, Misra SS, Irwin JO. The estimation of the bactericidal power of the blood. The Journal 470 of hygiene. 1938;38(6):732-49. 471 36. Henry M, Lavigne R, Debarbieux L. Predicting In Vivo Efficacy of Therapeutic Bacteriophages 472 Used To Treat Pulmonary Infections. Antimicrobial agents and chemotherapy. 2013;57(12):5961-8. 473 37. Bull JJ, Gill JJ. The habits of highly effective phages: population dynamics as a framework for 474 identifying therapeutic phages. Frontiers in microbiology. 2014;5. 475


http://aac.asm.org/

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38. Morissette C, Skamene E, Gervais F. Endobronchial inflammation following Pseudomonas 476 aeruginosa infection in resistant and susceptible strains of mice. Infection and immunity. 477 1995;63(5):1718-24. 478 39. Khan TZ, Wagener JS, Bost T, Martinez J, Accurso FJ, Riches DW. Early pulmonary inflammation 479 in infants with cystic fibrosis. Am J Respir Crit Care Med. 1995;151(4):1075-82. 480 40. Rosenfeld M, Gibson RL, McNamara S, Emerson J, Burns JL, Castile R, et al. Early pulmonary 481 infection, inflammation, and clinical outcomes in infants with cystic fibrosis. Pediatr Pulmonol. 482 2001;32(5):356-66. 483 41. Segel GB, Halterman MW, Lichtman MA. The paradox of the neutrophil's role in tissue injury. 484 Journal of leukocyte biology. 2011;89(3):359-72. 485 42. Konstan MW, Doring G, Heltshe SL, Lands LC, Hilliard KA, Koker P, Bhattacharya S, Staab A, 486 Hamilton A. A randomized double blind, placebo controlled phase 2 trial of BIIL 284 BS (an LTB4 receptor 487 antagonist) for the treatment of lung disease in children and adults with cystic fibrosis. Journal of cystic 488 fibrosis : official journal of the European Cystic Fibrosis Society. 2014;13(2):148-55. 489 43. van Furth R, van Zwet TL, Buisman AM, van Dissel JT. Anti-tumor necrosis factor antibodies 490 inhibit the influx of granulocytes and monocytes into an inflammatory exudate and enhance the growth 491 of Listeria monocytogenes in various organs. The Journal of infectious diseases. 1994;170(1):234-7. 492 44. Staugas RE, Harvey DP, Ferrante A, Nandoskar M, Allison AC. Induction of tumor necrosis factor 493 (TNF) and interleukin-1 (IL-1) by Pseudomonas aeruginosa and exotoxin A-induced suppression of 494 lymphoproliferation and TNF, lymphotoxin, gamma interferon, and IL-1 production in human leukocytes. 495 Infection and immunity. 1992;60(8):3162-8. 496 45. Hedges S, Svensson M, Svanborg C. Interleukin-6 response of epithelial cell lines to bacterial 497 stimulation in vitro. Infection and immunity. 1992;60(4):1295-301. 498 46. Watford WT, Moriguchi M, Morinobu A, O'Shea JJ. The biology of IL-12: coordinating innate and 499 adaptive immune responses. Cytokine & growth factor reviews. 2003;14(5):361-8. 500 47. Rovai LE, Herschman HR, Smith JB. The murine neutrophil-chemoattractant chemokines LIX, KC, 501 and MIP-2 have distinct induction kinetics, tissue distributions, and tissue-specific sensitivities to 502 glucocorticoid regulation in endotoxemia. Journal of leukocyte biology. 1998;64(4):494-502. 503 48. Alemayehu D, Casey PG, McAuliffe O, Guinane CM, Martin JG, Shanahan F, Coffey 504 A, Ross RP, Hill C. Bacteriophages φMR299-2 and φNH-4 can eliminate Pseudomonas aeruginosa in the 505 murine lung and on cystic fibrosis lung airway cells. MBio. 2012;3(2):e00029-12. 506 49. Lu TK, Collins JJ. Dispersing biofilms with engineered enzymatic bacteriophage. Proceedings of 507 the National Academy of Sciences of the United States of America. 2007;104(27):11197-202. 508 50. Hughes KA, Sutherland IW, Jones MV. Biofilm susceptibility to bacteriophage attack: the role of 509 phage-borne polysaccharide depolymerase. Microbiology. 1998;144 ( Pt 11):3039-47. 510 51. Zhang Y, Hu Z. Combined treatment of Pseudomonas aeruginosa biofilms with bacteriophages 511 and chlorine. Biotechnology and bioengineering. 2013;110(1):286-95. 512 52. Couper KN, Blount DG, Riley EM. IL-10: the master regulator of immunity to infection. Journal of 513 immunology. 2008;180(9):5771-7. 514 53. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA. Anti-inflammatory and pro-inflammatory roles 515 of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Current opinion in pharmacology. 516 2009;9(4):447-53. 517 54. Registry UC. Annual Data Report 2013. 2014:56. 518 55. Sulakvelidze A, Alavidze Z, Morris JG, Jr. Bacteriophage therapy. Antimicrobial agents and 519 chemotherapy. 2001;45(3):649-59. 520

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522


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Table 523

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

524

Table 1: Susceptibility of five clinical strains of Pa to three bacteriophage cocktails. Cocktail 1 525

was more broadly efficacious and PA12B-4973 (from here on known as clinical strain) was most 526

broadly sensitive, and therefore these were used for ongoing work. 527

Figure Legends 528

Figure 1: Differential cell counts (median/range) from BAL performed at 48hrs in mice 529

inoculated with 2.5 x 106 of a clinical strain of Pa and simultaneously treated with 20μl 530

bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer. 531

Figure 2: Pro-inflammatory cytokines (median/range) from BAL performed at 48hrs in mice 532

inoculated with 2.5 x 106 of a clinical strain of Pa and simultaneously treated with 20μl 533

bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer. 534

Figure 3a: Colony counts/ml from BAL performed at 24hrs in mice inoculated with 2.5 x 107 of 535

PAO1 and simultaneously treated with 20ul bacteriophage cocktail (containing 1.24 x 109 PFU) 536

or 20μl SM buffer. If no colonies were visible to the naked eye, this is reported as 0 CFU/ml; the 537

theoretical limit of detection was 100 CFU/ml as 10μl drops of BALF were cultured. 538


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Figure 3b: Pro-inflammatory cytokines (median/range) from BAL performed at 24hrs in mice 539

inoculated with 2.5 x 107 of PAO1 and simultaneously treated with 20μl bacteriophage cocktail 540

(containing 1.24 x 109 PFU) or SM buffer. 541


PAO1 and treated with 20μl bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer 543

24hrs after the initial infection. If no colonies were visible to the naked eye, this is reported as 0 544

CFU/ml; the theoretical limit of detection was 100 CFU/ml as 10μl drops of BALF were cultured. 545

Figure 4b: Pro-inflammatory cytokines (median/range) from BAL performed at 48hrs in mice 546

inoculated with 2.5 x 107 of PAO1 and treated with 20μl bacteriophage cocktail (containing 1.24 547

x 109 PFU) or SM buffer 24hrs after the initial infection. 548


PAO1 and treated with 20μl bacteriophage cocktail (containing 1.24 x 109 PFU) or 20μl SM 550

buffer prophylactically, 48hrs prior to infection. If no colonies were visible to the naked eye, this 551

is reported as 0 CFU/ml; the theoretical limit of detection was 100 CFU/ml as 10μl drops of 552

BALF were cultured. 553

Figure 5b: KC (median/range) from BAL performed at 24hrs in mice inoculated with 2.5 x 107 of 554

PAO1 and treated with 20μl bacteriophage cocktail (containing 1.24 x 109 PFU) or SM buffer 555

prophylactically, 48hrs prior to infection. 556


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Figure 6: Differential cell counts (median/range) from BAL performed at 24hrs in mice 557

inoculated with 2.5 x 107 of PAO1 and treated with 20μl bacteriophage cocktail (containing 1.24 558

x 109 PFU) or SM buffer prophylactically, 48hrs prior to infection. 559

560


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