phage therapy is effective in a mouse model of bacterial equine

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Title 1

Phage therapy is effective in a mouse model of bacterial equine keratitis. 2

Running Head 3

Phage therapy for mouse model of bacterial keratitis. 4

5

Takaaki Furusawa1, Hidetomo Iwano1*, Yutaro Hiyashimizu1, Kazuki Matsubara1, 6

Hidetoshi Higuchi2, Hajime Nagahata2, Hidekazu Niwa3, Yoshinari Katayama3, Yuta 7

Kinoshita3, Katsuro Hagiwara4, Tomohito Iwasaki5, Yasunori Tanji6, Hiroshi Yokota1, 8

Yutaka Tamura7 9

10

Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Rakuno Gakuen 11

University, Ebetsu, Japan1; Laboratory of Veterinary Hygiene, School of Veterinary 12

medicine, Rakuno Gakuen University, Ebetsu, Japan2; Microbiology Division, Tochigi 13

Branch, Epizootic Research Center, Equine Research Institute, Japan Racing 14

Association, Tochigi, Japan3; Laboratory of Veterinary Virology, School of Veterinary 15

medicine, Rakuno Gakuen University, Ebetsu, Japan4; Laboratory of Applied 16

Biochemistry, School of Food Science and Human Wellness, Rakuno Gakuen University, 17

Ebetsu, Japan5; Department of Bioengineering, Tokyo Institute of Technology, 18

AEM Accepted Manuscript Posted Online 24 June 2016Appl. Environ. Microbiol. doi:10.1128/AEM.01166-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Yokohama, Japan6; Laboratory of Food Microbiology and Food Safety, School of 19

Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Japan7 20

21

# Address correspondence to Hidetomo Iwano, [email protected] 22

23

Abstract 24

Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, 25

and pseudomonas. Of these bacteria, Pseudomonas aeruginosa sometimes causes rapid 26

corneal corruption and, in some cases, blindness. Antimicrobial resistance can make 27

treatment very difficult. Therefore, new strategies to control bacterial infection are 28

required. A bacteriophage (phage) is a virus that specifically infects and kills bacteria. 29

Since phage often can lyse antibiotic-resistant bacteria because the killing mechanism is 30

different, we examined the use of phage to treat horse bacterial keratitis. We isolated 31

Myoviridae or Podoviridae phages, which together have a broad host range. They 32

adsorb efficiently to host bacteria; over 80% of ΦR18 phage were adsorbed to host cells 33

after 30 seconds. In our keratitis mouse model, administration of phage within 3 hours 34

also could kill bacteria and suppress keratitis. A phage MOI of 100 times the host 35

bacteria number could kill host bacteria effectively. A cocktail of two phages suppressed 36


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bacteria in the keratitis model mouse. These data demonstrated that phages in this study 37

could completely prevent the keratitis caused by P. aeruginosa in a keratitis mouse 38

model. Furthermore, these results suggest phage may be more effective prophylaxis for 39

horse keratitis than is the current preventive use of antibiotics. Such treatment may 40

reduce use of antibiotics and, therefore, antibiotic resistance. Further studies are 41

required to assess phage therapy as a candidate for treatment of horse keratitis. 42

43

Importance 44

Now, antibiotic-resistant bacteria is emerging all over the world. Bacteriophages have 45

great potential for resolution of this problem. Bacteriophage, or phage, is a virus that 46

infects bacteria specifically. As one of the novel therapeutic strategy against race horse 47

keratitis caused by Pseudomonas aeruginosa, we will propose application of phages for 48

treatment. Phages isolated in this work had in vitro effectiveness for broad P. 49

aeruginosa strains. Indeed, great reduction of bacterial proliferation was shown in 50

phage therapy for mouse models of the P. aeruginosa keratitis. Therefore, to reduce 51

antibiotic usage, phage therapy should be investigated and developed further. 52

53

Introduction 54


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Ulcerative keratitis frequently occurs in race horses. Horses have large eyes and a 55

laterally prominent position of the globe. In the race horse, active movement of the head 56

may increase exposure of the corneas to microbes (1). After a race, horses receive a 57

water eye wash and antibiotic eye drops. Despite this, some horses may develop 58

bacterial keratitis. Although there are regional differences, bacterial keratitis is mainly 59

caused by staphylococci, streptococci and the pseudomonas (2). Pseudomonas 60

aeruginosa is a gram-negative aerobic bacillus having flagella for motility (3). 61

Moreover, since this pathogen secretes proteolytic enzymes, the keratitis caused by 62

P. aeruginosa develops rapidly (4). Antibiotic resistance can occur and, in such cases, 63

keratitis could cause corneal perforation or loss of sight. Horses of the Japan Racing 64

Association (JRA) cannot run in races if both eyes become blind (1). Even if horses 65

recover, their eyes could have granulation tissue and their sight would be limited. 66

Antimicrobial resistance in bacteria is a concern not only in veterinary medicine, 67

but also in general, worldwide (5, 6). In 1928, penicillin was developed and soon after 68

that was used globally. But recently, methicillin-resistant Staphylococcus aureus 69

(MRSA), vancomycin-resistant enterococci (VRE), multidrug-resistant P. aeruginosa 70

(MDRP) and others have emerged (7). These bacteria can put the lives of humans and 71

animals at great risk; MRSA is especially widespread in human hospitals. Furthermore, 72


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MRSA carriage is potentially an occupational risk for veterinary personnel (8). When 73

antibiotic-resistance emerged, novel antibiotics to treat these bacteria were developed. 74

But it is difficult to develop effective new antibiotics every time a new resistance 75

mechanism emerges. Now, many pharmaceutical companies are withdrawing from the 76

antimicrobial development business; therefore, to treat bacterial infections, there is a 77

demand for new strategies such as predatory bacteria, antimicrobial peptides, phage 78

therapy, phage endolysins, gene-editing enzymes and metals (9, 10). 79

Phage therapy is one of these strategies. A bacteriophage (phage) is a virus that 80

specifically infects and kills bacteria. In 1896, Ernest Hankin reported a bactericidal 81

property associated within Indian river water (11, 12). Thereafter, phages were 82

co-discovered by Felix d’Herelle and Frederick Twort (13) and phage therapy was 83

proposed by d’Herelle in 1931 (14). Many phages isolated previously are classified into 84

order Caudovirales (15). Their size is about less than 200 nm and many are composed 85

of a head, tail and a long tail fiber. At the bacterial surface, the long tail fiber recognizes 86

receptor molecules and is adsorbed. The host range is partly determined by the 87

interaction of the long tail fiber and specific receptor molecules such as outer membrane 88

protein (Omp) or lipopolysaccharide (LPS). After adsorption, in lytic cycle, the phage 89

injects its nucleic acid and, through replication, many new phage are released with 90


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bacterial lysis (16). After their discovery, the use of phages for therapy had been studied 91

mainly in Eastern European countries, but waned because of the triumph of penicillin. 92

Today, we are globally threatened by antibiotic-resistant bacteria and phage therapy is 93

being reassessed as a new strategy for treatment of infectious disease (13, 17). Indeed, 94

researchers have reported the efficacy of phage therapy for experimental bacterial 95

disease (18-22). Examples include applications for use of the phages ListShieldTM 96

(Intralytix, Inc., Baltimore, MD) for Listeria monocytogenes and EcoShieldTM 97

(Intralytix, Inc., Baltimore, MD) for Escherichia coli O157:H7, to protect against 98

foodborne disease (23, 24). In addition, not only phage, but also phage endolysin is 99

assessed for therapeutic use, which is used at the end of phage lytic cycle (25). The 100

quick approval of more applications of phage therapy for bacterial infections that are 101

hard to be treated is strongly expected. 102

In the current study, P. aeruginosa phages that together have a broad host range 103

were isolated from inflow sewage water and one of them had the ability to adsorb onto 104

the bacterial surface quickly. In in vivo experiments using a mouse model of bacterial 105

horse keratitis, we demonstrated great effectiveness of phage therapy. 106

107

Materials and Methods 108


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Ethical treatment of animals. This study was carried out in strict accordance with the 109

recommendations in the Guidelines for Proper Conduct of Animal Experiments, Science 110

Council of Japan (http://www.scj.go.jp/ja/info/kohyo/pdf/kohyo-20-k16-2e.pdf). The 111

protocol was approved by the Committee on the Ethics of Animal Experiments of 112

Rakuno Gakuen University (approval number VH25A3, approved August 21, 2013). 113

Welfare-related assessments and interventions that were carried out prior to, during, or 114

after the experiment. 115

Animals. Specific pathogen free (SPF) 8-week-old C57BL/6 male mice which had 116

same weight and were healthy were purchased from Sankyo Labo Service Corporation, 117

INC (Tokyo, Japan), and housed under pathogen-free conditions and 3 mice per cage at 118

the animal facility of Rakuno Gakuen University (Hokkaido, Japan). All animals were 119

housed in a temperature-controlled room under a 12:12 light-dark cycle for at least 1 120

week to acclimate to the surroundings and with free access to food and water. 121

Bacterial strains and culture media. Bacterial strains were isolated from dogs (“Pa” 122

strains, received from the Laboratory of Food Microbiology and Food Safety at Rakuno 123

Gakuen University) and from horses (“NE-“ strains, received from the Equine Research 124

Institute, Japan Racing Association). The P. aeruginosa strains Pa12, Pa18, Pa26 and 125

Pa50 were used for phage preparation and the P. aeruginosa strain NE-126 was used for 126


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measurement of adsorption rate and infection experiments. Luria-Bertani (LB) medium 127

was used for bacteria or phage culture and used for counting colony forming units 128

(CFU) of P. aeruginosa harvested from eye tissue. SM buffer (10 mM MgSO4, 100 mM 129

NaCl, 0.01% gelatin, and 50 mM Tris-HCl [pH 7.5]) was used for phage dilution. To 130

assess phage plaque formation, an LB medium containing 1.5% agar or 0.5% agarose 131

ME (Iwai Chemicals Company, Tokyo, Japan) was used to form the lower and upper 132

layer, respectively. 133

Isolation of phages. Applying previously reported method (26), phages were isolated 134

from sewage samples, obtained from sewage-treatment plants at Sapporo and Ebetsu, 135

Japan, using P. aeruginosa strains (strain Pa12, Pa18, Pa26 and Pa50) as hosts. This 136

procedure was approved by these plants (27). Of these phages, ΦR18 and ΦS12-1 were 137

sequenced previously (accession number; LC102729 and LC102730) (28). 138

Host specificity of phages. Procedure was carried out according to preciously reported 139

method (27, 29). 5 µL of a phage suspension (1010 plaque forming units (PFU)/mL) was 140

dropped onto a double-layer agar plate containing a P. aeruginosa strain. This was then 141

incubated at 37°C overnight. After incubation, the infected area was characterized as 142

one of four categories; clear plaques, turbid plaques, faint plaques, or no plaques. 143

Large-scale culture and purification of P. aeruginosa phages. A fresh LB broth 144


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culture of a P. aeruginosa strain was mixed with phage and added to 3 mL of soft LB 145

agar (0.5%). Then, this was overlaid onto LB agar (1.5%) to be uniform. The plate was 146

incubated at 37°C overnight. After incubation, 3 ml of SM buffer was added onto the 147

plate with plaques and plates were statically kept for 1 – 2 h at room temperature. Then, 148

the soft agar layer was scraped off and homogenized with SM buffer by a glass spreader. 149

The homogenized soft agar was collected with SM buffer using a truncated 1 mL 150

pipette tip. This was centrifuged at 10,000 × g, 4°C, for 5 min. The supernatant was 151

transferred to a new tube and a drop of chloroform (CHCl3) was added; this was stored 152

at 4°C. This was then purified by density gradient ultracentrifugation using CsCl. 153

The phage suspension was placed on top of a discontinuous CsCl gradient (ρ = 154

1.46, 1.55, and 1.63) and purified by CsCl density gradient ultracentrifugation (RCF 155

(relative centrifuge force); Rmax 111,000 × g, Rav 81,900 × g and 25 °C, 1 h). After 156

phage band collection, the phage was dialyzed against SM buffer overnight. The phage 157

concentration was measured with plaque assay. 158

Electron microscopy. The purified phage sample in SM buffer was loaded onto a 159

collodion membrane and stained with 2% uranyl acetate. Electron micrograph images 160

were obtained with a Hitachi H-800 transmission electron microscope (Hitachi Ltd., 161

Tokyo, Japan) at 75 kV. Phages were classified according to the report by Ackermann 162


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(15). 163

Adsorption rate of a phage on P. aeruginosa. Phage ΦR18 in suspension in SM buffer 164

(1.1 × 107 PFU/mL) were mixed 1:1 with NE-126 cells (1.2 × 109 colony forming units 165

(CFU)/mL), and incubated at room temperature. Aliquots (200 µL) were taken at 166

specific time points; 15 sec, 30 sec, and 1, 3 and 5 minutes. Aliquots were diluted 1:100 167

with SM buffer. The number of free infectious phage virions in the supernatant of the 168

diluted phage-cell mixture was determined with double layer method after the mixture 169

was centrifuged (9,800 × g, 10 min.). 170

Mouse model of P. aeruginosa keratitis. P. aeruginosa strain NE-126 cells were grown 171

in 6 mL LB medium at 37°C, and the cell number was determined by measurement of 172

the optical density of the medium at 600 nm (OD600). When the number was about 2 × 173

108 CFU/mL, the culture was centrifuged at 10,000 × g for 5 min. The cell pellet was 174

washed with 1 ml phosphate buffered saline (PBS) and then re-centrifuged under the 175

same conditions. The pellet was resuspended and diluted by PBS to 2 × 106 CFU/mL. 176

C57BL/6 mice were anesthetized 30 minutes before infection with a mixture of 177

three types of anesthetic agents; 6 mg/kg midazolam (Dormicum, astellas, Tokyo, 178

Japan), 0.45 mg/kg medetomidin (Dorbene, Kyoritsu Seiyaku Corporation, Tokyo, 179

Japan) and 7.5 mg/kg butorphanol (Vetorpale, Meiji Seika Pharma Co., Ltd., Tokyo, 180


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Japan). Mouse eyes were infected using a protocol modified from Minhao et al (30). 181

Using a stereoscopic microscope, corneas were wounded with three 1 mm incisions 182

using a sterile 25-gauge needle. A 5 µL aliquot containing 104 CFU of P. aeruginosa 183

NE-126 was applied to the corneal surface (control; n = 11). Next, 109 plaque forming 184

units (PFU) of phages in 5 µL was applied to the corneal surface 30 min (n = 16), 1 h (n 185

= 10), 3 h (n = 9), 6 h (n = 10) or 12 h (n = 5) after the initial infection. After treatments, 186

mice were awoken with 0.9 mg/kg atipamezole (Atipame, Kyoritsu Seiyaku 187

Corporation, Tokyo, Japan). The eyes were observed for corneal opacity 24 h after the 188

second infection, and were harvested 48 h after the second infection. Multiplicity of 189

Infection (MOI) study was similarly carried out except that phage (102 PFU; n = 6, 104 190

PFU; n = 6, or 106 PFU; n = 6) were applied 30 min after bacteria was applied (control; 191

n = 4). In phage cocktail study, the number of mice were 6 for control, 5 for ΦR18 192

group, 6 for ΦR26 group, and 8 for phage cocktail group. 193

Determination of the number of viable bacteria in the infected cornea. Individual 194

corneas were homogenized with a tissue homogenizer in PBS. Serial 10-fold dilutions 195

of the samples were plated on LB agar and cultured at 37°C for 24 h. Results are 196

reported as CFU per eye. 197

Coculture of phages and bacteria. The examination was carried out according to 198


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method of Synnott et al (29) with slightly modification. P. aeruginosa strain NE-149 199

(107 CFU/mL) and phages ΦR18, ΦR26 (109 PFU/mL)or cocktailed phages (5 × 108 200

PFU/mL each) in 4 mL LB medium were cultured at 37°C with measuring optical 201

density at 660 nm (OD660) with a TVS062CA biophotorecorder (Advantec, Tokyo, 202

Japan), shaking at 40 rpm. The OD660 was monitored at 30-min intervals for a minimum 203

of 24 h. The examination was repeated three times each. 204

Analysis of Data. All statistical analysis was performed using the software R version 205

3.2.3 (https://www.R-project.org/) for windows. Data was analyzed using ANOVA with 206

Dunnett multiple comparisons test or Tukey multiple comparisons. 207

208

Results 209

Host specificity of phages. First, to collect phages from various P. aeruginosa strains, 210

we obtained P. aeruginosa that caused disease in dogs and horses. The phages ΦR12, 211

ΦS12-1, ΦS12-3, ΦR18, ΦR26 and ΦS50 were isolated from P. aeruginosa strains Pa12, 212

Pa18, Pa26 and Pa50 (Fig. S1). The host range of the phages was examined using 213

various P. aeruginosa strains isolated from a horse’s lesions on various parts of the body 214

(Fig. 1a). The result of the experiments to determine host range of each phage was 215

82.8 % (24/29) “Clear” plaque formation. Regarding serotypes, we guessed phage lytic 216


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activity not to correlate. Representative pictures of formed spots were shown in Fig. 1 217

panels b-d. 218

Of these phages, ΦR18 had a relatively broad specificity. Therefore, we used 219

ΦR18 in the experiments to measure adsorption rate and to determine in vivo effects of 220

phage therapy. 221

Electron microscopy. Of the 6 phages, ΦR12, ΦS12-1, ΦS12-3 and ΦR18 were 222

observed with electron microscopy and classified morphologically (Fig. 2). ΦR12, 223

ΦS12-1 and ΦS12-3 had a polyhedral head, 78.8 ± 2.0, 91.7 ± 0.8 and 84.0 ± 1.3 nm in 224

diameter, and a contractile tail of 147.5 ± 2.3, 154.2 ± 0.8 and 148.3 ± 1.3 nm in length, 225

respectively. Therefore, they belonged to the family Myodoviridae. In contrast, ΦR18 226

had a polyhedral head 78.5 ± 1.5 nm in diameter and a short tail of 29.8 ± 1.6 nm in 227

length. Therefore, this phage was assigned to the family Podoviridae. 228

Adsorption rate of a phage on P. aeruginosa. When phage suspensions are dropped 229

into the eye, they must be adsorbed to bacteria quickly before running out of the eye in 230

tears. Therefore, we measured the adsorption rate of ΦR18 on a P. aeruginosa strain 231

NE-126, which was isolated from keratitis of a race horse. At 30 seconds, over 80% of 232

ΦR18 was adsorbed to the host cells (Fig. 3). This suggested that the phage had 233

adsorbed to the bacteria very effectively, which was promising for its potential use to 234


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prevent keratitis. 235

Effects of bacteriophage in a mouse model of P. aeruginosa keratitis. To estimate the 236

efficacy of phage therapy, we carried out an in vivo experiment using a mouse model of 237

P. aeruginosa keratitis. Mice were treated according to the protocol schedule described 238

above (Fig. 4a). 239

Observations of eyes showed that phage administration 30 min after infection 240

prevented keratitis (Fig. 4b). Bacterial counts showed the phage was effective until 3 241

hours after infection (Fig. 4c and Table 1). These results showed that phage treatment 242

was effective using the current schedule for eye wash and administration of antibiotics 243

following races. 244

Effective multiplicity of infection (MOI) range. Phage efficacy was also investigated 245

with regard to the bacteria-to-phage ratio. Bacterial number decreased depending on the 246

phage number or PFU (Fig. 5). Results suggest that MOI=100 was sufficiently effective 247

and, compared with the control group, bacterial proliferation was suppressed 99.78%. 248

Efficacy of cocktailed phages. Using phage cocktail has several advantages. Firstly, 249

because many phages have diverse host ranges, a cocktail of phages could be effective 250

in diverse strains of bacteria. Secondly, there could be synergism between cocktailed 251

phages (23). Thirdly, use of mixed phages could prevent emergence of phage-resistant 252


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bacteria (24). Therefore, we investigated whether our cocktailed phages were effective 253

or not. After considering the practicalities, two phages (ΦR18 and ΦR26) and one 254

bacterial strain, P. aeruginosa NE-149, were used. ΦR18 alone formed only “Faint” 255

plaques, but ΦR26 formed “Clear” plaques, against strain NE-149. Results are shown in 256

Fig. 6: ΦR18 suppressed bacterial multiplication to 20.1%, and ΦR26 to 0.22%, 257

compared with control group. The two phages cocktailed suppressed bacterial 258

multiplication to 0.004%. 259

Coculture of phages and bacteria. We carried out the experiment that phages and 260

bacteria were cultured together to confirm the emergence of resistance against phages. 261

The abilities of ΦR18, ΦR26 and cocktailed phages to lyse P. aeruginosa causing race 262

horse keratitis were tested using liquid LB medium cultures (Fig. 7). The OD660 of 263

bacteria-only control rose immediately followed by that of ΦR18 (○). The OD660 of 264

ΦR18 (×) was increased slight slowly than control and the peak was at 10 h, after that, 265

decreased. ΦR26 (▲) and cocktailed phages (■) repressed proliferation of bacteria until 266

8 h. 267

268

Discussion 269

One of the main symptoms of the keratitis caused by P. aeruginosa is ring 270


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abscesses on the corneal surfaces. When the condition worsens, ulcerative keratitis can 271

develop (1, 4). In addition, antibiotic-resistant bacteria have been detected during 272

treatment (1, 31, 32). Novel treatments for infectious diseases are now required. To 273

address this situation, we assessed the potency of phage for prevention of race horse 274

keratitis. In the present study, phages showed remarkable efficacy against bacterial 275

keratitis caused by P. aeruginosa. Phage ΦR18, isolated from sewage samples, 276

prevented bacteria replicating on the corneal surface if administered in eyes drops 277

within 3 hours after bacterial inoculation. Furthermore, cocktailed phages showed no 278

interference and covered the other phage. Based on these results in a mouse keratitis 279

model, we demonstrated the possibility that phage therapy for race horse keratitis may 280

be useful. 281

First, we isolated 6 phages that have different host ranges from sewage samples. 282

The phage recognizes bacterial cell wall structures; therefore these phages could 283

recognize distinct parts of these structures (16). That is to say, a phage which has a 284

broad host range would recognize conserved bacterial structures. In some P. aeruginosa 285

strains, our phages produced no clear plaque. Therefore, those strains may not have 286

surface structures in common with most P. aeruginosa; or, possibly, after the phages 287

infected, those strains prevented phage replication. Serotype surface antigens may play 288


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a role; group H P. aeruginosa might have uncommon structures and, on the other hand, 289

group G P. aeruginosa may have relatively conserved structures. In a clinical situation, 290

these bacteria will be a problem for use of phage therapy. To lyse these bacteria, phages 291

effective for all P. aeruginosa strains that we have, at least, should be identified and 292

collected. These phages may recognize important features of the P. aeruginosa surface. 293

Using a mixture of phages would lyse more P. aeruginosa strains and prevent 294

development of phage resistant bacteria (29). The phages used in the current study lysed 295

P. aeruginosa from keratitis and also from other diseases; therefore, phage therapy may 296

potentially be applied widely. 297

According to the classification by Ackermann (15), phages having contractile tails 298

are Myoviridae, those having long and noncontractile tails are Siphoviridae and those 299

having short tails are Podoviridae. These tailed phages, Caudovirales, comprise 96% of 300

all phages isolated (15). On the other hand, filamentous phages belonging to the genus 301

Inovirus infect gram-negative bacteria through the bacterial pilus (33). In the current 302

study, electron microscopy showed that ΦR12, ΦS12-1 and ΦS12-3 are Myoviridae, and 303

ΦR18 are Podoviridae. To use phage therapeutically, production of phage neutralizing 304

antibody would not be avoided (34). It is reported that production of antibody is 305

associated with host immunity and phage type (35). If phage therapy is applied, whether 306


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the genome sequence contains virulence factors should first be determined. Phages in 307

this work have none of already-known virulence factors (28). 308

For JRA horses, eye ointment containing a mixture of erythromycin and colistin is 309

prophylactically used for an eye wash after a race. Generally, eye-drops or eye-ointment 310

do not retain an effective concentration for a long time. For example, eye-drops are 311

effective for only 30 min because the antibiotics wash out with tears (36). In this study, 312

the phage used had high adsorption efficiency. Indeed, phage can proliferate if host 313

bacteria exist. Phage therapy probably could replace the present antibiotic treatment as 314

prophylaxis. 315

Administration of phage within 3 hours after infection mostly eradicated the 316

pathogen. Therefore, if a horse’s eyes were washed immediately after a race, phages 317

could replace the antibiotics currently used. On the other hand, 6 and 12 hours after 318

infection, phage did not prevent bacterial growth. It would be reasonable to suppose that 319

since P. aeruginosa invaded the deep corneal stromal, phage could not infiltrate there. P. 320

aeruginosa are motile and produce proteolytic enzymes such as elastase B, alkaline 321

metalloprotease (37), protease IV (38), and P. aeruginosa small protein (PASP) (39), 322

and have a type III secretion system (40). These enable P. aeruinosa to rapidly cause 323

keratitis and to invade deep layers. In addition, the model mouse of P. aeruginosa 324


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keratitis was established and similar pathology would occur in race horse keratitis. 325

The in vivo experiment described above was carried out with 109 PFU/5µL phage 326

and 104 CFU/5µL P. aeruginosa suspensions. But we found that phages are required at 327

100 times the bacteria concentration, at least, to be sufficiently effective. In a clinical 328

situation, there would be little chance that 104 CFU bacteria infect an eye at a given 329

time. Therefore, when phages are used for prevention of keratitis, fewer PFU would be 330

enough for effective therapy. 331

Cocktailed phages also had a great effect on prevention of bacterial proliferation. 332

Phages forming “clear” plaques and those forming “faint” plaques did not interfere with 333

each other. Cocktailing phages with combined broad host ranges would lyse more P. 334

aeruginosa strains. 335

Culturing bacteria with ΦR18 showed resistance to ΦR18 immediately, but the 336

bacteria proliferation was slightly inhibited compared with control (Fig. 7). ΦR26 and 337

cocktailed phages, on the other hand, prevented resistant bacteria for longer time than 338

ΦR18. These results were relatively correlated with host specificity of spot test data (Fig. 339

1) and in vivo keratitis model experiment (Fig. 6). However, the differences of lysis 340

activity between R26 and cocktailed phages in vitro experiments were not clear. The 341

bacterial proliferation speed in vivo experiments is very faster than that in the body, 342


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because of rich nutrition and nothing inhibition. Therefore, the clear effect of cocktailed 343

phage in vitro experiments might not be seen. These mechanisms are further issues that 344

should be investigated in future. 345

While antibiotics have been successful for various bacterial infections, 346

investigators in Eastern European countries, especially Georgia, diligently have 347

continued to study phage therapy (17). Phage therapy probably will have wonderful 348

advantages for modern veterinary medicine, and this is worthy of continuing study. 349

Firstly, since the mechanisms of phage lysis are different from antibiotic mechanisms, 350

phage therapy can be effective for infections caused by antimicrobial resistant bacteria. 351

Secondly, if phages are used instead of antibiotics, the amount of antibiotics used 352

decreases. Thirdly, the numbers of veterinarians carrying antibiotic resistant bacteria 353

will also decrease. As a result, application of phage therapy may actually restore the 354

effectiveness of antibiotics. 355

Now, many researchers have studiously examined the phages and their peptide, 356

endolysin. Endolysin is produced the end of phage-lytic cycle. This enzyme cleaves 357

bacterial peptidoglycan, lysing bacteria osmotically. Furthermore, endolysin-resistant 358

bacteria have never reported. Therefore, therapeutic application of endolysin is very 359

attractive (41). Previously, we identified endolysins encoded by ΦR18 and ΦS12-1 360


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phages. Both endolysins were composed of lysozyme-like domains (27, 28). Since some 361

investigators study recombinant or designed endolysin (41, 42), we will also exam 362

recombinant endolysins. 363

In summary, we demonstrated that the phages used in this study rapidly adsorbed 364

to P. aeruginosa and widely killed P. aeruginosa isolated from horse lesions. 365

Furthermore, one of the phages was shown to completely prevent keratitis in a keratitis 366

mouse model. These results lead us to expect a higher efficiency for phage prophylaxis, 367

compared to the current preventive use of antibiotics, for horse keratitis. Use of phage 368

may lead to reduction of use of antibiotics, and may reduce the number of antibiotic 369

resistant bacteria. Further studies are needed to fully develop methods of phage 370

purification and applications of phage therapy. 371

372

Acknowledgements 373

We thank the wastewater treatment centers at Sapporo and Ebetsu for giving us 374

the sewage samples. This study was supported in part by the Grant-in-Aid for Scientific 375

Research (B), numbers 22380174 and 25292182. 376

377

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542

543

Figure legends 544

FIG 1 Phage specificity against P. aeruginosa isolated from horse lesions. (a) C; Clear 545

plaques (white boxes) indicates combinations resulting in highest lysis activity, 546

followed by T; turbid plaques (light gray boxes), and F; faint plaques (dark gray boxes). 547

Black boxes indicate combinations yielding no plaque formation (N). (b-d) 548

Representative pictures of formed spots are shown. 549

550

FIG 2 Morphology of phages: electron microscopy. Bar: 100 nm. ΦR12, ΦS12-1 and 551

ΦS12-3: Myoviridae. ΦR18: Podoviridae. 552

553

FIG 3 Adsorption rate of ΦR18. Adsorption rate of bacteriophage ΦR18 on 554

Pseudomonas aeruginosa strain NE-126, measured as described in Materials and 555

Methods. 556

557

FIG 4 Effects of bacteriophages on mouse model of P. aeruginosa keratitis. (a) The 558

experiment was carried out according to this schedule. (b) Typical cornea images 24h 559


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and 48h after infection treated with only bacteria or bacteria and phages are shown. (c) 560

The efficacy of a single dose of phage is shown. Differences between control values and 561

phage treated values are denoted by * (statistically significant, p < 0.05), # (p = 0.054) 562

or † (p = 0.068) using Dunnett test. Circles represent the data obtained from each 563

mouse; horizontal bars represent mean values calculated from all mice in each group. 564

565

FIG 5 Effective range of multiplicity of infection (MOI). The cornea was exposed to 104 566

colony forming units (CFU) of P. aeruginosa strain NE-126 and 102 plaque forming 567

units (PFU) (MOI = 0.01), 104 PFU (MOI = 1) or 106 PFU (MOI = 100) of phages. 568

Efficacy of phages was shown by bacterial counts 48 h after infection. Differences 569

between control values and phage treated values are denoted by * (statistically 570

significant, p < 0.05) using Tukey multiple comparison. Bars indicate standard error of 571

the mean. 572

573

FIG 6 Efficacy of cocktailed phages. The cornea was exposed to 104 colony forming 574

units (CFU) of P. aeruginosa strain NE-149 and 109 plaque forming units (PFU) of 575

phage R18 and/or phage R26. Bacterial count of control group and phage groups. CFU 576

rate was shown in percentage toward control CFU. Differences between control values 577

and phage treated values are denoted by * (statistically significant, p < 0.05) using 578


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Tukey multiple comparison. Bars indicate standard error of the mean. 579

580

FIG 7 Coculturing bacteriophages and P. aeruginosa. The bacterial strain is NE-149. ○, 581

bacterium-only controls; ×, ΦR18 added to bacteria; ▲, ΦR26 added to bacteria; ■, 582

both ΦR18 and ΦR26 added to bacteria. 583

584


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TABLE 1 Actual bacterial counts. 585

control 30 min 1 h 3 h 6 h 12 h 190,000 2,400 0 0 1,890,000 3,430,000850,000 0 0 100 3,890,000 750,000

1,640,000 0 8,400 100 13,000 730,000490,000 0 0 63,000 1,000 210,000710,000 0 0 4,500 0 2,850,000100,000 0 10 2,390 3,000148,000 0 140 4,900 0

2,180,000 0 360 620 24,000430,000 0 10 12,400 1,930,000380,000 0 180 12,000

2,120,000 0 0

3,000 0 0

5,400

Actual bacterial counts per an eye were shown. 30 min, 1 h and 3 h group were found 586

bacterial count reduced, compared with other groups. 587


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Figure 1

derivation serotype ΦR12 ΦS12-1 ΦS12-3 ΦR18 ΦR26 ΦS50

NE-12 cornea A C C C C C C

NE-15 cornea G N N F C C F

NE-16 cornea G C C C C C C

NE-69 cornea G C C C C C C

NE-94 cornea D N T T T N N

NE-95 cornea G N F F C N N

NE-126 cornea - C C C C C C

NE-149 cornea A C C C F C T

NE-150 cornea D F T C F C T

NE-153 cornea H N N N N N N

NE-167 cornea G T F F T C C

NE-193 cornea - F F N C N N

NE-85 pneumonia G C C C C C C

NE-98 BALF I N F F T F F

NE-100 BALF G C C C C C C

NE-102 BALF G C C C T C C

NE-112 BALF D N T C C C C

NE-117 sinusitis C C F F N F C



NE-124guttural pouch

empyemaH N N N N N N

NE-125 BALF B N C C C C C

NE-129 empyema C C C T C T T

NE-132lung

parenchymaG T C C C C C

NE-137 lung abscess - C T T C T T

NE-138 BALF B N N N N N N

NE-158 dermatitis - N T F T C T

NE-169 BALF - N F F C C T

NE-188 sinusitis - T C C C C C

clear plaque turbid plaque faint plaque

a

b dc


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phage therapy is effective in a mouse model of bacterial equine

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