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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
10
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
15
16
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
39
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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
122
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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
183
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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
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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
521
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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
Figure 4a: Colony counts/ml from BAL performed at 48hrs in mice inoculated with 2.5 x 107 of 542
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
Figure 5a: Colony counts/ml from BAL performed at 24hrs in mice inoculated with 2.5 x 107 of 549
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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