Implications of Bacteriophage- and Bacteriophage Component-Based Therapies 1

For The Clinical Microbiology Laboratory 2

Katherine M. Caflisch,1 #Robin Patel

2, 3 3

1Department of Molecular Pharmacology and Experimental Therapeutics, 4

Mayo Clinic, Rochester, MN 5

2Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, 6

Mayo Clinic, Rochester, MN 7

3Division of Infectious Diseases, Department of Medicine, 8

Mayo Clinic, Rochester, MN 9

Running head: Phage therapy and the clinical laboratory 10

#Address correspondence to: 11

Robin Patel, M.D. 12

Mayo Clinic 13

200 1st St SW 14

Rochester, MN 55905 15

patel.robin@mayo.edu 16

Telephone: 507-284-4272 17

Fax: 507-538-0579 18

JCM Accepted Manuscript Posted Online 15 May 2019J. Clin. Microbiol. doi:10.1128/JCM.00229-19Copyright © 2019 American Society for Microbiology. All Rights Reserved.

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ABSTRACT 19

Treatment of bacterial infections is increasingly challenged by resistance to currently available 20

antibacterial agents. Not only are such agents less likely to be active today than they were in the 21

past, but their very use has selected for and continues to select for further resistance. Additional 22

strategies for management of bacterial illness must be identified. In this review, bacteriophage-23

based therapies are presented as one promising approach. In anticipation of their potential 24

expansion into clinical medicine, clinical microbiologists may wish to acquaint themselves with 25

bacteriophages and their antibacterial components, and specifically with methods for testing 26

them. Here, we reviewed the literature spanning 01/2007 to 03/2019 on bacteriophage and 27

phage-encoded protein therapies of relevance to clinical microbiology. 28

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Following early therapeutic application of bacteriophage (phage) in France in the 1930s, 29

industrial investment in phage production took place across Europe, Russia and the United States 30

(1). The popularity of human phage therapy waned in Western countries with the advent of 31

antibiotics; as chemicals, antibiotics were easier to test in the clinical laboratory and administer 32

than were bacteriophages. At the time, there was also an underdeveloped understanding of phage 33

biology, and lack of standardized in vitro methods to assess bacteriophage activity, alongside 34

clinical trials methodologies to evaluate them; what clinical data was available yielded 35

inconsistent results (1). Today, phages continue to be used clinically in parts of Eastern Europe 36

as well as historic (e.g., Poland, the Republic of Georgia) and present-day Russia (1-4), and are 37

also used as preservatives in agronomy and food processing (5), as well as in veterinary medicine 38

(6). With the current challenge of antibiotic resistance, the case for human phage therapy in 39

Western medicine has been re-opened. In addition, the potential use of phage components as 40

antibacterial agents is being evaluated. While there are no FDA-approved bacteriophages or 41

phage component products available for human clinical application in the US, some are being 42

administered on an expanded access basis, with others in clinical trials. 43

Clinical microbiologists have historically used phage for phage typing to differentiate 44

between bacterial strains as part of outbreak investigations, though with the advent of molecular 45

techniques, including most recently whole genome sequencing, younger clinical microbiologists 46

may be unfamiliar with phage-based testing. In anticipation of potential expansion of phage 47

therapy into clinical medicine, clinical microbiologists may wish to acquaint themselves with 48

bacteriophages and their antibacterial components, and specifically with methods for testing 49

them. Here, we reviewed the literature spanning 01/2007 to 03/2019 on bacteriophage and 50

phage-encoded protein therapy of relevance to clinical microbiology. 51

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Bacteriophages are a clade of bacteriotropic viruses of individual sizes ranging from 20 52

to 200 nm that infect their hosts with strain- or species-, and, rarely, genus-level or family-level 53

specificity (7, 8). More than 6,000 phages have been characterized to date, with genome sizes 54

ranging from a few thousand to 480,000 nucleotides or more (7). Residing in close association 55

with bacteria, phages are found throughout the biosphere, including in bodies of water and 56

sewage, and in and on humans and animals, totaling an estimated 1x1031

virions and 57

outnumbering bacterial cells by ten-fold (9). Given their abundance and diversity, it is not 58

surprising that most are as yet undescribed. Phage bioprospecting efforts are ongoing; this 59

typically involves adding target bacteria in a concentrated nutrient broth to a solid (e.g., soil) or 60

liquid (i.e., water) sample followed by incubation for several hours (10), and then assessment for 61

phage using the double overlay plaque assay described below (Fig. 1), with isolated phages 62

purified by serial plating. Thirteen (as many as nineteen, by some estimates) phage families are 63

distinguished by morphology, presence or absence of an envelope, as well as size and 64

composition of their genome (DNA or RNA, single-stranded [ss] or double-stranded [ds]) (11). 65

That “phages are nature’s version of precision medicine” manifests advantages and 66

disadvantages to their therapeutic application (12). While their specificity may minimize off-67

target effects on commensal bacteria compared to antibiotics, this same property may limit their 68

empiric administration by requiring testing of the bacterium with which a particular patient is 69

infected before selecting the specific phage to treat that patient, requiring availability of 70

laboratory testing and potentially engendering treatment delays. The narrow spectrum of 71

individual phages may, however, be overcome by concurrent use of combinations of phages in 72

phage cocktails (12). 73

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Although bacteriophages have been traditionally classified as lytic or lysogenic, a more 74

precise terminology is strictly lytic and temperate (13) (Fig. 2). Temperate phages enter bacteria 75

and become incorporated into the host genome assuming a quiescent prophage form for a period 76

of time, occasionally extruding progeny phages in the absence of lysis (7). Prophages may be 77

excised from the genome stochastically in a process known as spontaneous induction or 78

following cellular stress (7). Phage excision may occur precisely, or may result in splicing and 79

packaging of adjacent bacterial genes within newly synthesized virions; ultimately, the lytic 80

cycle is engaged, bringing about lysis of the host. Many phages are capable of lytic and 81

temperate cycles, the switch between them governed by such factors as intracellular 82

concentrations of second messenger molecules, host co-infection by other viruses, extracellular 83

phage density, and environmental resources (14). Temperate phages may encode virulence 84

(including toxin) genes, protect their host from infection by other phages (including lytic 85

phages), and promote bacterial fitness; furthermore, Sweere et al. recently demonstrated that Pf4 86

phage infecting Pseudomonas aeruginosa sourced from human wounds contributed to chronic 87

infection by binding Toll-like receptor 3, reducing phagocytosis and tumor necrosis factor 88

secretion (15). Strictly lytic phages only engage the lytic cycle; that is, they enter their hosts, 89

seize control of bacterial replication machinery and express proteins that destabilize the cell 90

envelope, resulting in immediate bacteriolysis and dissemination of new virions for subsequent 91

infection. Because they effect temporally-constrained cell death, are not frequent vectors for 92

horizontal gene transfer, and are not known to interact with eukaryotic cells, strictly lytic phages 93

are typically used for phage therapy. Still, generalized transduction of host genetic material may 94

occur with strictly lytic phages as a consequence of inadvertent encapsulation of host genes 95

within newly produced viral capsids (1, 7). 96

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The clinical use of phage protein components, including endolysins (described below) 97

and depolymerases [further categorized as hydrolases (or polysaccharases), endosialidases, 98

endorhamnosidases, and polysaccharide lyases (16)], and holins (enzymes which insert holes 99

into bacterial cell membranes), has been recently considered (17). The use of phage components 100

eliminates the risk of genomic integration and gene transduction, while featuring ease of dosing, 101

quality control and, potentially, storage. In addition, phage protein components may purport 102

more predictable activity against strains of targeted species compared to phage. Drawbacks of 103

lysin therapy include abbreviated circulation due to human host proteases and the absence of 104

endogenous viral amplification associated with conventional phage therapy, in addition to 105

immunogenicity, abrogating future efficacy of the same phage protein component (18). 106

Endolysins, alternatively known as lysins or peptidoglycan hydrolases, hydrolyze 107

bacterial cell walls, with those targeting Gram-positive bacteria generally composed of an 108

enzymatically active domain and a cell wall binding domain, and those targeting Gram-negative 109

bacteria lacking the latter (19). Endolysins are classified on the basis of which murein bond they 110

cleave (20). There are two phage proteins in clinical trials at present, Tonabacase (N-Rephasin® 111

SAL-200, iNtRON Biotechnology) (21), and Exebacase (CF-301, ContraFect Corporation) (22), 112

both of which are endolysin formulations specific to Staphylococcus aureus; Tonabacase cleaves 113

amide and peptide bonds with Exebacase cleaving amide bonds (23-25). 114

Adoption of phage therapy into human medicine would likely have implications for the 115

clinical microbiology laboratory if performance of phage susceptibility testing (PST) were to be 116

required to select phage or phage combinations for individual patients’ isolates and to assess 117

whether resistance to particular phages or phage combinations has been selected for in cases of 118

individual patient treatment failure. Susceptibility testing of patient isolates to fixed composition 119

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phage cocktails could be considered, with screening against individual phages from a phage 120

library performed only in cases that are not susceptible to generic cocktails (26). 121

If an individualized phage therapy approach were to be adopted, healthcare facilities 122

would not only need to have access to PST, but possibly also to engage in phage preparation. 123

What role clinical laboratories versus pharmacies or other entities might play in preparation of 124

individualized phage preparations for patient administration as is customary in some other 125

countries, a paradigm that is discussed further below, remains to be determined (27). Once 126

matched with target bacteria, phages would need to be amplified to obtain viral titers sufficiently 127

high for human administration. This entails incubation of phage with its bacterial target followed 128

by purification for pyrogen removal. Care needs to be taken to avoid the use of lysogenized 129

bacteria for phage amplification, lest genes carried by integrated temperate phages encoding 130

bacterial resistance or toxins become inadvertently intermingled with lytic virions. Since 131

physical phage isolation using centrifugation and filtration does not remove all bacterial 132

components (e.g., endotoxin), methods for removal of these need to be applied, using elution 133

filters and phase separation with 1-octanol, dialysis against ethanol and NaCl and speed-134

vacuuming, or monovalent cations, for example (28, 29). 135

No standard method for PST exists. A common approach is the double overlay plaque 136

assay or a modification thereof (including the spot plate method, used for screening high 137

numbers of phage-bacterium combinations), in which known concentrations of phage and 138

bacteria are combined with semisoft agar and incubated, with the presence or absence of plaques 139

denoting bacterial susceptibility or lack thereof, respectively (Fig. 1). Another method is the 140

cross-streak method, in which phage lysate is plated across an agar plate and bacteria are plated 141

in perpendicular fashion, with susceptibility inferred based on zone of inhibition size (30). 142

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Merabishvili et al. describe the use of large square Petri dishes with 2% Luria broth agar 143

inoculated with target bacteria in horizontal strips, air-dried and then spotted with 5 ml of 107 144

pfu/ml suspensions of individual bacteriophages under consideration. Following incubating for 145

16 to 18 hours at 37C, the obtained lysis zones are evaluated and scored as confluent lysis, 146

opaque lysis, semi-confluent lysis, several plaques or negative. These approaches can be time- 147

and labor-intensive and may not be user-friendly in clinical laboratories. The OmniLog™ system 148

(Biolog, Haywood, CA) has been adapted for PST by assaying varying phage concentrations and 149

bacteria, in the presence of a tetrazolium dye, in microtiter plates. If bacterial growth occurs, 150

cells respire and reduce the tetrazolium dye, forming a strong color; conversely, with phage lysis, 151

respiration is slowed or stopped, and color formation diminished. This method also serves as the 152

basis of the Host Range Quick Test, which is capable of screening a single bacterial sample 153

against as many as 5,000 bacteriophages in less than 18 hours (Adaptive Phage Therapeutics, 154

Gaithersburg, MD). While the technology is not commercially available, it may portend the 155

efficiency and phage screening capacity of clinical laboratories in the future. 156

An unanswered question pertaining to PST regards the need for establishment of positive, 157

intermediate and negative phage infectivity thresholds, as is standard for interpretation of the 158

minimum inhibitory concentrations of traditional antibiotics. The convention in phage research is 159

to measure the multiplicity of infection (MOI), that is, ratios of phages to bacteria. This metric 160

may be challenging to apply in phage therapy because one wouldn’t necessarily know how many 161

bacteria are being treated, that number could change over time, and some bacteria may be more 162

or less accessible to phage than others (31). Additionally, unlike traditional antibiotics, phage can 163

amplify in the host, such that the dose increases in vivo - the very attractiveness of phage 164

therapy. In the end, phage will likely be delivered as a specific number of phage; how that 165

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delivery will take place is the subject of ongoing studies, and may vary depending on the site and 166

type of infection. This too could influence interpretation of results of laboratory studies on phage 167

activity. Further, it is likely that phage will be applied in challenging-to-treat infections, which, 168

in some cases, will involve biofilms. As such, methods for determining phage anti-biofilm 169

activity may be needed, which is not even standardized for traditional antibiotics. 170

In contrast to PST, testing for susceptibility to phage-encoded proteins is more similar to 171

conventional antibacterial susceptibility testing. For example, a standard or modified broth 172

microdilution method is used for susceptibility testing of Tonabacase (21) and Exebacase (32), 173

respectively, the modification comprised of the addition of 0.5 mM DL-dithiothreitol and 25% 174

horse serum to the cation-adjusted Mueller Hinton broth (33). Quality control ranges for 175

Exebacase are being developed by the Clinical and Laboratory Standards Institute (34). As a 176

result of the use of broth microdilution, lysin susceptibility testing should be amenable to 177

automation on commercial susceptibility testing instruments (35). 178

Exercising proper storage and maintenance is as crucial as selecting phage(s) active 179

against target bacteria toward successful phage therapy. Given the proteinaceous composition of 180

viruses, shear forces or variation in temperature and pH to which phages are subject during 181

handling may impact titers as a result of protein misfolding or denaturation. Their shelf life may 182

similarly vary by bacteriophage species which may be anticipated by phage size and morphology 183

(29, 36-39). Storage methods utilized in the Republic of Georgia where phage therapy is used 184

clinically have included freeze-drying, spray-drying, deep freezing, refrigeration, and short-term 185

ambient storage (personal communication, Marina Tediashvili), though some studies suggest that 186

phase changes, including freezing or drying, may result in drops in titer, at least among certain 187

phages (37). These effects may be abrogated by the addition of excipients, such as skim milk 188

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(37, 40). Phage viability over long-term storage appears to be maximized at 4°C, -80°C, or -189

196°C (41). It is likely that conditions for ideal stability will need to be evaluated for each phage 190

considered for clinical use. Lysins, in contrast to whole phages, may exhibit stability at a wider 191

range of temperatures and pH (22). 192

Whether or not phages will ultimately be adopted into the therapeutic armamentarium in 193

Western medicine is unknown. Case studies of phage therapy continue to be added to the 194

published literature (Table 1a). Patey et al. note that the generally positive results reported from 195

isolated applications stand in contrast to the negative results of some clinical trials (Table 1b) 196

(42). The routes of phage administration and dosing intervals, as well as the diversity of target 197

bacteria and infection-types portend that investigational use of phage therapy will likely continue 198

for some time before its approval for clinical use in the United States. PST methods ought to be 199

standardized in order to guide studies and enable results between studies to be compared and 200

observations generalized and extrapolated. 201

Phages may be used as a complementary approach to currently-authorized antimicrobial 202

strategies rather than a replacement (42). As viruses, they and their components may be 203

recognized by the immune system and eliminated via innate and/or adaptive defenses before 204

reaching sites of infection, and their use may also compromise future use of similar species in the 205

same patient. Phages are also six-fold larger than small-molecule antibiotics, limiting their 206

distribution and ability to diffuse across some physiological membranes such as the blood-brain 207

barrier, and subsequently, their utility to treat certain types of infection (3, 43). Finally, their host 208

specificity requires identification of the pathogen prior to treatment such that empiric therapy 209

may be challenging (43, 44). 210

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How phage therapy would be regulated in the United States is presently unclear, with 211

some questioning whether this can be done within the existing drug and device infrastructure of 212

the United States Food and Drug Administration, founded on the concept of drugs as small 213

molecules of fixed composition (42, 44, 45). Subjecting the production and oversight of 214

therapeutic phage to Current Good Manufacturing Practice and evaluation within conventional 215

randomized clinical trials will be associated with significant cost and incite process development 216

inefficiencies (e.g., in a phage cocktail, each phage as well as the final cocktail may require FDA 217

approval; the composition of off-the-shelf phage products might require updating at regular 218

intervals to avoid resistance and to maintain clinical relevance to current pathogens), culminating 219

in approval delays (42, 44, 46, 47). Countries in which phages are used therapeutically operate 220

under different regulatory guidelines, such as Belgium’s magistral preparations, the adoption of 221

which some favor on a broader scale (46, 48). By this course, a phage prescription is composed 222

by the clinician, and the corresponding formulation produced by an associated pharmacy using 223

master phage stocks that are quality controlled by an accredited laboratory (48). Regulation will 224

also depend upon the level at which oversight of phage treatment is controlled: individual 225

patients, hospitals, local or national governments, or some combination thereof (49, 50). While 226

offering critique or suggestions for the regulation of phage therapy is beyond the scope of this 227

review, clinical microbiologists should be aware of the possibilities receiving consideration as 228

well as their predicted impact on laboratory practice. 229

Although more translational research must be completed before clinical implementation 230

of bacteriophage therapy is feasible, it is not too early to consider how this antibacterial strategy 231

may shift current treatment paradigms. Such a shift will likely impact the clinical microbiology 232

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laboratory, upon which the burden of PST, reporting parameters, and phage storage (at least for 233

in vitro testing) will rest. 234

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ACKNOWLEDGEMENTS 235

The authors gratefully acknowledge Larry J. Prokop, MLS, for his medical library 236

expertise and assistance in curating the collection of articles which inform this review. The 237

authors thank Raymond Schuch (ContraFect) for his thoughtful review of this manuscript. 238

DISCLOSURES 239

RP reports grants from ContraFect, CD Diagnostics, Merck, Hutchison Biofilm Medical 240

Solutions, Accelerate Diagnostics, and Shionogi (monies are paid to Mayo Clinic), serving as a 241

consultant to Curetis, Specific Technologies, Next Gen Diagnostics, Selux Dx, GenMark 242

Diagnostics, PathoQuest, and Qvella (monies are paid to Mayo Clinic), patents on Bordetella 243

pertussis/parapertussis PCR and an anti-biofilm substance issued, a patent on a device/method 244

for sonication with royalties paid by Samsung to Mayo Clinic, receiving editor’s stipends from 245

ASM and IDSA, and receiving honoraria from the NBME, Up-to-Date, and the Infectious 246

Diseases Board Review Course. KMC: None to report. 247

FUNDING 248

RP is supported by UM1 AI104681-01, R01 AR056647, and R21 AI125870. 249

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REFERENCES 250

1. Riley PA. 2006. Phages: their role in bacterial pathogenesis and biotechnology. J Clin 251 Path 59:1003-1004. 252

2. Górski A, Międzybrodzki R, Łobocka M, Głowacka-Rutkowska A, Bednarek A, Borysowski 253 J, Jończyk-Matysiak E, Łusiak-Szelachowska M, Weber-Dąbrowska B, Bagińska N, 254 Letkiewicz S, Dąbrowska K, Scheres J. 2018. Phage therapy: what have we learned? 255 Viruses 10:288. 256

3. Kortright KE, Chan BK, Koff JL, Turner PE. 2019. Phage therapy: a renewed approach to 257 combat antibiotic-resistant bacteria. Cell Host Microbe 25:219-232. 258

4. Brussow H. 2012. What is needed for phage therapy to become a reality in Western 259 medicine? Virology 434:138-42. 260

5. Harada LK, Silva EC, Campos WF, Del Fiol FS, Vila M, Dabrowska K, Krylov VN, Balcao VM. 261 2018. Biotechnological applications of bacteriophages: state of the art. Microbiol Res 262 212-213:38-58. 263

6. Hawkins C, Harper D, Burch D, Anggard E, Soothill J. 2010. Topical treatment of 264 Pseudomonas aeruginosa otitis of dogs with a bacteriophage mixture: a before/after 265 clinical trial. Vet Microbiol 146:309-13. 266

7. Guttman B, Raya R, Kutter E. 2005. Basic phage biology, p 29-66. In Kutter E, 267 Sulakvelidze A (ed), Bacteriophages: biology and applications 268 doi:10.1201/9780203491751.ch3. CRC Press, Boca Raton, FL. 269

8. Pantucek R, Rosypalova A, Doskar J, Kailerova J, Ruzickova V, Borecka P, Snopkova S, 270 Horvath R, Gotz F, Rosypal S. 1998. The polyvalent staphylococcal phage phi 812: its 271 host-range mutants and related phages. Virology 246:241-52. 272

9. Comeau AM, Hatfull GF, Krisch HM, Lindell D, Mann NH, Prangishvili D. 2008. Exploring 273 the prokaryotic virosphere. Res Microbiol 159:306-13. 274

10. Van Twest R, Kropinski AM. 2009. Bacteriophage enrichment from water and soil. In 275 Clokie MRJ, Kropinski AM (ed), Bacteriophages: isolation, characterization, and 276 interactions, vol 1. Humana Press. 277

11. Ackermann H-W. 2004. Bacteriophage classification, p 67-89. In Kutter E, Sulakvelidze A 278 (ed), Bacteriophages: biology and applications. CRC Press. 279

12. Lyon J. 2017. Phage therapy's role in combating antibiotic-resistant pathogens. JAMA 280 318:1746-1748. 281

13. Hobbs Z, Abedon ST. 2016. Diversity of phage infection types and associated 282 terminology: the problem with 'lytic or lysogenic'. FEMS Microbiol Lett 363. 283

14. Howard-Varona C, Hargreaves KR, Abedon ST, Sullivan MB. 2017. Lysogeny in nature: 284 mechanisms, impact and ecology of temperate phages. ISME J 11:1511. 285

15. Sweere JM, Van Belleghem JD, Ishak H, Bach MS, Popescu M, Sunkari V, Kaber G, 286 Manasherob R, Suh GA, Cao X, de Vries CR, Lam DN, Marshall PL, Birukova M, 287 Katznelson E, Lazzareschi DV, Balaji S, Keswani SG, Hawn TR, Secor PR, Bollyky PL. 2019. 288 Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection. 289 Science 363:eaat9691. 290

on May 26, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

14

16. Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B, Delattre AS, Lavigne R. 2012. 291 Learning from bacteriophages - advantages and limitations of phage and phage-encoded 292 protein applications. Curr Protein Pept Sc 13:699-722. 293

17. Fan X, Li W, Zheng F, Xie J. 2013. Bacteriophage inspired antibiotics discovery against 294 infection involved biofilm. Crit Rev Eukaryot Gene Expr 23:317-326. 295

18. Maciejewska B, Olszak T, Drulis-Kawa Z. 2018. Applications of bacteriophages versus 296 phage enzymes to combat and cure bacterial infections: an ambitious and also a realistic 297 application? Appl Microbiol Biotechnol 102:2563-2581. 298

19. Parisien A, Allain B, Zhang J, Mandeville R, Lan CQ. 2008. Novel alternatives to 299 antibiotics: bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides. J 300 Appl Microbiol 104:1-13. 301

20. Vollmer W, Joris B, Charlier P, Foster S. 2008. Bacterial peptidoglycan (murein) 302 hydrolases. FEMS Micro Rev 32:259-286. 303

21. Kim N-H, Park WB, Cho JE, Choi YJ, Choi SJ, Jun SY, Kang CK, Song K-H, Choe PG, Bang J-304 H, Kim ES, Park SW, Kim N-J, Oh M-d, Kim HB. 2018. Effects of phage endolysin SAL200 305 combined with antibiotics on Staphylococcus aureus infection. Antimicrob Agents 306 Chemother 62:e00731-18. 307

22. Schuch R, Khan BK, Raz A, Rotolo JA, Wittekind M. 2017. Bacteriophage lysin CF-301, a 308 potent antistaphylococcal biofilm agent. Antimicrob Agents Chemother 61. 309

23. Gilmer DB, Schmitz JE, Euler CW, Fischetti VA. 2013. Novel bacteriophage lysin with 310 broad lytic activity protects against mixed infection by Streptococcus pyogenes and 311 methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 57:2743-312 2750. 313

24. Jun SY, Jung GM, Son J-S, Yoon SJ, Choi Y-J, Kang SH. 2011. Comparison of the 314 antibacterial properties of phage endolysins SAL-1 and LysK. Antimicrob Agents 315 Chemother 55:1764-1767. 316

25. Lood R, Molina H, Fischetti VA. 2017. Determining bacteriophage endopeptidase activity 317 using either fluorophore-quencher labeled peptides combined with liquid 318 chromatography-mass spectrometry (LC-MS) or Forster resonance energy transfer 319 (FRET) assays. PLoS One 12:e0173919. 320

26. Kvachadze L, Balarjishvili N, Meskhi T, Tevdoradze E, Skhirtladze N, Pataridze T, Adamia 321 R, Topuria T, Kutter E, Rohde C, Kutateladze M. 2011. Evaluation of lytic activity of 322 staphylococcal bacteriophage Sb-1 against freshly isolated clinical pathogens. Microb 323 Biotechnol 4:643-650. 324

27. Pirnay JP, Verbeken G, Ceyssens PJ, Huys I, De Vos D, Ameloot C, Fauconnier A. 2018. 325 The magistral phage. Viruses 10. 326

28. Drab M. 2018. Phage aggregation-dispersion by ions: striving beyond antibacterial 327 therapy. Trends Biotechnol 36:875-881. 328

29. Bonilla N, Rojas MI, Netto Flores Cruz G, Hung SH, Rohwer F, Barr JJ. 2016. Phage on tap-329 a quick and efficient protocol for the preparation of bacteriophage laboratory stocks. 330 PeerJ 4:e2261. 331

30. Balouiri M, Sadiki M, Ibnsouda SK. 2016. Methods for in vitro evaluating antimicrobial 332 activity: a review. J Pharm Anal 6:71-79. 333

on May 26, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

15

31. Abedon ST. 2016. Phage therapy dosing: the problem(s) with multiplicity of infection 334 (MOI). Bacteriophage 6:e1220348-e1220348. 335

32. Oh J, Sauve KL, Wittekind M, Schuch R. Development of an antimicrobial susceptibility 336 test (AST) for the antistaphylococcal lysin CF-301, p. In (ed), 337

33. Schuch R. 2016. Request for endorsement of a modification to the standard CLSI broth 338 microdilution methodology, abstr Methods Development and Standardization Working 339 Group, Clinical Laboratory Science Institute, Wayne, PA., 340

34. Zimmer B, Hardy D, Sei K, Brasso B, Hymphries R, Koeth L, Shawar R. Methods 341 development and standardization, p. In (CLSI) CaLSI (ed), 342

35. Xie Y, Wahab L, Gill JJ. 2018. Development and validation of a microtiter plate-based 343 assay for determination of bacteriophage host range and virulence. Viruses 10. 344

36. Leung SSY, Parumasivam T, Nguyen A, Gengenbach T, Carter EA, Carrigy NB, Wang H, 345 Vehring R, Finlay WH, Morales S, Britton WJ, Kutter E, Chan HK. 2018. Effect of storage 346 temperature on the stability of spray dried bacteriophage powders. Eur J Pharm 347 Biopharm 127:213-222. 348

37. Clark WA. 1962. Comparison of several methods for preserving bacteriophages. Appl 349 Microbiol 10:466-471. 350

38. Clark WA, Geary D. 1973. Proceedings: preservation of bacteriophages by freezing and 351 freeze-drying. Cryobiology 10:351-60. 352

39. Zhang Y, Peng X, Zhang H, Watts AB, Ghosh D. 2018. Manufacturing and ambient 353 stability of shelf freeze dried bacteriophage powder formulations. Int J Pharm 542:1-7. 354

40. Gonzalez-Menendez E, Fernandez L, Gutierrez D, Rodriguez A, Martinez B, Garcia P. 355 2018. Comparative analysis of different preservation techniques for the storage of 356 Staphylococcus phages aimed for the industrial development of phage-based 357 antimicrobial products. PLoS One 13:e0205728. 358

41. Fortier LC, Moineau S. 2009. Phage production and maintenance of stocks, including 359 expected stock lifetimes. In Clokie MRJ, Kropinski AM (ed), Bacteriophages: methods 360 and protocols, vol 1: Isolation, characterization, and interactions. Humana Press. 361

42. Patey O, McCallin S, Mazure H, Liddle M, Smithyman A, Dublanchet A. 2018. Clinical 362 indications and compassionate use of phage therapy: personal experience and literature 363 review with a focus on osteoarticular infections. Viruses 11. 364

43. Harper DR. 2018. Criteria for selecting suitable infectious diseases for phage therapy. 365 Viruses 10. 366

44. Cooper CJ, Khan Mirzaei M, Nilsson AS. 2016. Adapting drug approval pathways for 367 bacteriophage-based therapeutics. Front Microbiol 7:1209. 368

45. Administration UFaD. 29 Jun 2018 2018. The history of FDA's fight for consumer 369 protection and public health. https://www.fda.gov/AboutFDA/History/default.htm. 370 Accessed 19 Apr 2019. 371

46. Nagel TE. 2018. Delivering phage products to combat antibiotic resistance in developing 372 countries: lessons learned from the HIV/AIDS epidemic in Africa. Viruses 10:345. 373

47. Górski A, Międzybrodzki R, Weber-Dąbrowska B, Fortuna W, Letkiewicz S, Rogóż P, 374 Jończyk-Matysiak E, Dąbrowska K, Majewska J, Borysowski J. 2016. Phage therapy: 375 combating infections with potential for evolving from merely a treatment for 376 complications to targeting diseases. Front Microbiol 7:1515-1515. 377

on May 26, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

16

48. Pirnay J-P, Verbeken G, Ceyssens P-J, Huys I, De Vos D, Ameloot C, Fauconnier A. 2018. 378 The magistral phage. Viruses 10:64. 379

49. Huys I, Pirnay JP, Lavigne R, Jennes S, De Vos D, Casteels M, Verbeken G. 2013. Paving a 380 regulatory pathway for phage therapy: Europe should muster the resources to 381 financially, technically and legally support the introduction of phage therapy. EMBO Rep 382 14:951-954. 383

50. Verbeken G, De Vos D, Vaneechoutte M, Merabishvils M, Zizi M, Pirnay JP. 2007. 384 European regulatory conundrum of phage therapy. Future Microbiol 2:485-491. 385

51. Schooley RT, Biswas B, Gill JJ, Hernandez-Morales A, Lancaster J, Lessor L, Barr JJ, Reed 386 SL, Rohwer F, Benler S, Segall AM, Taplitz R, Smith DM, Kerr K, Kumaraswamy M, Nizet 387 V, Lin L, McCauley MD, Strathdee SA, Benson CA, Pope RK, Leroux BM, Picel AC, 388 Mateczun AJ, Cilwa KE, Regeimbal JM, Estrella LA, Wolfe DM, Henry MS, Quinones J, 389 Salka S, Bishop-Lilly KA, Young R, Hamilton T. 2017. Development and use of 390 personalized bacteriophage-based therapeutic cocktails to treat a patient with a 391 disseminated resistant Acinetobacter baumannii infection. Antimicrob Agents 392 Chemother 61. 393

52. LaVergne S, Hamilton T, Biswas B, Kumaraswamy M, Schooley RT, Wooten D. 2018. 394 Phage therapy for a multidrug-resistant Acinetobacter baumannii craniectomy site 395 infection. Open Forum Infect Dis 5:ofy064-ofy064. 396

53. Letkiewicz S, Miedzybrodzki R, Fortuna W, Weber-Dabrowska B, Gorski A. 2009. 397 Eradication of Enterococcus faecalis by phage therapy in chronic bacterial prostatitis--398 case report. Folia Microbiol 54:457-461. 399

54. Letkiewicz S, Miedzybrodzki R, Klak M, Jonczyk E, Weber-Dabrowska B, Gorski A. 2010. 400 The perspectives of the application of phage therapy in chronic bacterial prostatitis. 401 FEMS Immunol Med Microbiol 60:99-112. 402

55. Lusiak-Szelachowska M, Zaczek M, Weber-Dabrowska B, Miedzybrodzki R, Letkiewicz S, 403 Fortuna W, Rogoz P, Szufnarowski K, Jonczyk-Matysiak E, Olchawa E, Walaszek KM, 404 Gorski A. 2017. Antiphage activity of sera during phage therapy in relation to its 405 outcome. Future Microbiol 12:109-117. 406

56. Chan BK, Turner PE, Narayan D, Kim S, Mojibian HR, Elefteriades JA. 2018. Phage 407 treatment of an aortic graft infected with Pseudomonas aeruginosa. Evol Med Public 408 Health 2018:60-66. 409

57. Jennes S, Merabishvili M, Soentjens P, Pang KW, Rose T, Keersebilck E, Soete O, François 410 P-M, Teodorescu S, Verween G, Verbeken G, De Vos D, Pirnay J-P. 2017. Use of 411 bacteriophages in the treatment of colistin-only-sensitive Pseudomonas aeruginosa 412 septicaemia in a patient with acute kidney injury-a case report. Crit Care 21:129-129. 413

58. Khawaldeh A, Morales S, Dillon B, Alavidze Z, Ginn AN, Thomas L, Chapman SJ, 414 Dublanchet A, Smithyman A, Iredell JR. 2011. Bacteriophage therapy for refractory 415 Pseudomonas aeruginosa urinary tract infection. J Med Microbiol 60:1697-1700. 416

59. Duplessis C, Wolfe D, Quinones J, Estrella L, Henry M, Hamilton T, Biswas B, Hanisch B, 417 Perkins M. 2018. Refractory Pseudomonas bacteremia in a 2-year-old sterilized by 418 bacteriophage therapy. J Pediatric Infect Dis Soc 7:253-256. 419

60. Fadlallah A, Chelala E, Legeais JM. 2015. Corneal infection therapy with topical 420 bacteriophage administration. Open Ophthalmol J 9:167-168. 421

on May 26, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

17

61. Fish R, Kutter E, Wheat G, Blasdel B, Kutateladze M, Kuhl S. 2016. Bacteriophage 422 treatment of intransigent diabetic toe ulcers: a case series. J Wound Care 25:S27-S33. 423

62. Rhoads DD, Wolcott RD, Kuskowski MA, Wolcott BM, Ward LS, Sulakvelidze A. 2009. 424 Bacteriophage therapy of venous leg ulcers in humans: results of a phase I safety trial. J 425 Wound Care 18:237-8, 240-3. 426

63. Vandenheuvel D, Lavigne R, Brussow H. 2015. Bacteriophage therapy: advances in 427 formulation strategies and human clinical trials. Ann Rev Virol 2:599-618. 428

64. Wright A, Hawkins CH, Anggard EE, Harper DR. 2009. A controlled clinical trial of a 429 therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant 430 Pseudomonas aeruginosa; A preliminary report of efficacy. Clin Otolaryngol 34:349-357. 431

65. Fowler J, Vance G., Das A, Lipka J, Schuch R, Cassino C. Exebacase (lysin CF-301) 432 improved clinical responder rates in methicillin resistant Staphylococcus aureus (MRSA) 433 bacteremia including endocarditis compared to standard of care antibiotics (SOC) alone 434 in a first-in-patient phase II study. 435 https://d1io3yog0oux5.cloudfront.net/_53ec10a3e327f1e291fe5c77b7504a7a/contrafe436 ct/db/226/1306/pdf/ECCMID+ORAL+Ph2_FINAL.pdf. Accessed 30 Apr 2019. 437

438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466

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467 Table 1a. 468

Bacterium Clinical

Presentation

Phage Administration

of Phage

Outcome Reference

Acinetobacter

baumannii

68 year old man,

necrotizing

pancreatitis

ΦPC cocktail

(AC4, C1P12,

C2P21, C2P24);

ΦIV cocktail (AB-

Navy1, AB-Navy4,

AB-Navy71, AB-

Navy97); ΦIVB

cocktail (AB-

Navy71,

AbTP3Φ1)

Intravenous and intracavitary

administration of

approximately 109 pfu/dose

alongside multiple antibiotics

Recovery (51)

77 year old man,

craniectomy site

infection

1 proprietary phage Intravenous administration of

2.14x107 pfu/mL in 4 mL

lactated Ringer’s every 2

hours for 8 days alongside

colistin, rifampin and

azithromycin

Withdrawal of

treatment and

death

(52)

Enterococcus

faecalis

3 men (32-45

years old), chronic

bacterial

prostatitis

Assorted lytic

phages (from the

Institute of

Immunology and

Experimental

Therapy of the

Polish Academy of

Sciences) screened

against prostatic

secretions isolates

Twice daily intrarectal

administration of 10 mL of

4.5x107 to 2.8x108 pfu/mL

for 28-33 days

Recovery (53, 54)

Multiple

antibiotic

resistant

bacteria

62 patients with

genitourinary tract

infection,

prostatitis, bone

infection, upper

and lower

respiratory tract

infections, or skin

and soft tissue

infections

MS-1 cocktail,

OPMS-1 cocktail,

monovalent

Staphylococcus

aureus phages

(unspecified),

monovalent

Enterococcus

faecalis phages

(unspecified),

monovalent Gram-

negative phages

(unspecified)

Oral, oral and local,

intrarectal or local

bacteriophage (106-109

pfu/mL) twice or three times

daily for 12 weeks or more at

dosing volumes of 10 mL for

oral and intrarectal

administration (volume

unspecified for local

administration)

Favorable

response in 40-

55% patients

(55)

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Pseudomonas

aeruginosa

76 year old man,

fistulated aortic

graft

OMKO1 10 mL 10x107 pfu/mL

intrafistular phage and

ceftazidime

Recovery (56)

61 year old man,

pressure sores and

septicemia

BFC1 cocktail

(PNM, 14/1, ISP)

Topical (50 mL 109 pfu/mL

q8h) and intravenous (50 µL

109 pfu/mL/6 h) for 10 days

Blood cultures

negative but local

infection

persisted and

patient died 4

months later

(57)

67 year old

woman, urinary

tract infection

Pyophage 051007

(proprietary 6-

phage cocktail)

Bladder irrigation (2x107 pfu)

twice daily for 10 days with

concurrent colistin and

subsequent meropenem

Recovery (58)

2 year old boy,

bacteremia

Proprietary 2-phage

cocktail

Intravenous administration of

3.5x105 pfu every 6 hours for

3 days alongside meropenem,

tobramycin, aztreonam,

colistin, and polymyxin B

Recovery (59)

Staphylococcus

aureus

65 year old

woman, bacterial

keratitis

SATA-8505 Intravenous, intranasal,

intraocular administration

over 4 weeks (dose

unspecified)

Recovery (60)

6 men (44-92 year

olds), diabetic

foot ulcers

Sb-1 Weekly tissue debridement

and topical phage treatment

(0.1-0.5 mL of 107-108

pfu/mL stock)

Recovery (61)

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Table 1b. 469

Trial

Start

Trial

Registrati

on

Number

Ref. Phage/Lysin

Formulation

(Investigator

/Sponsor)

Bacteria

Targeted

Treatment

Indication

Phase

Outcome

2006 NCT00663

091

(62) WPP-201 cocktail

(Southwest Regional

Wound Care Center)

Escherichia

coli,

Pseudomona

s

aeruginosa,

Staphylococ

cus aureus

Infected

venous leg

ulcers

I No adverse

effects observed

2009 NCT00937

274

(63) T4 coliphage

cocktail: AB2, 4, 6,

11, 46, 50, 55, JS34,

37, 98, D1.4 (Nestlé)

E. coli Dysentery I/II No adverse

effects or

benefit noted;

study

discontinued

prematurely

2009 EudraCT

2004-

001691-39

(64) Biophage-PA

(Biocontrol Ltd)

P.

aeruginosa

Otitis externa I/II No adverse

effects noted; at

42 days post-

treatment,

phage-treated

cohort exhibited

reduced median

bacterial

abundance

compared to

placebo group

2015 NCT02116

010

(62) PhagoBurn

(Pherecydes Pharma)

P.

aeruginosa,

E. coli

Infected burn

wounds

I/II No adverse

effects noted;

time to

“sustained

bacterial

reduction”

longer in phage

treatment arm;

study

prematurely

discontinued

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2017 NCT03089

697

N/A SAL200

(Tonabacase)

(iNtRON Biotech)

S. aureus S. aureus

bacteremia

II Ongoing

2017

NCT03163

446

(65) Exebacase (CF-301)

(ContraFect Corp)

S. aureus Bacteremia,

including

endocarditis

(single

intravenous

dose added to

standard of

care

antibiotics)

II Well tolerated;

higher clinical

response rate

compared to

standard of care

antibiotics alone

in methicillin-

resistant but not

methicillin-

susceptible S.

aureus

subgroups

2017 NCT03140

085

N/A Pyophage

(Tzulakidze National

Center of Urology)

Enterococcu

s species, E.

coli, Proteus

mirabilis, sta

phylococci,

streptococci,

P.

aeruginosa

Urinary tract

infections

II/III Results pending

(personal

communication,

Thomas

Kessler)

2019 NCT03808

103

N/A EcoActive

(Intralytix, Inc.)

Adherent

invasive E.

coli (AIEC)

Exacerbation

of

inflammation

in Crohn

disease

secondary to

AIEC

I/II Recruitment

ongoing

2019 NCT02664

740

N/A PhagoPied

(Pherecydes Pharma)

S. aureus Diabetic

wounds

I/II Not yet

recruiting

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FIGURE LEGENDS 470

Figure 1. Double overlay plaque assay. A combination of amplified phage and host bacterium 471

in cooled, molten agar supplemented with divalent cations (e.g., CaCl2, MgSO4) is poured over 472

solid agar and the medium incubated overnight. Quantifiable clearings (“plaques”) in the 473

bacterial lawn indicate the presence of infectious phage. At least two different plaque 474

morphologies are observed in the example photograph illustrating an experiment involving co-475

culture of Salmonella enterica serotype Typhimurium and phages obtained from municipal 476

sewage. 477

Figure 2. Bacteriophage life cycle. 478

Table 1a. Phage and phage lysin therapy in human clinical trials from January 2007 to 479

March 2019. Abbreviation: pfu, plaque-forming unit. 480

Table 1b. Human case reports and case series involving phage and phage lysin therapy 481 published between January 2007 and March 2019. Abbreviations: SSTI, skin and soft tissue 482 infection. 483

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