Challenges of using phages in the veterinary
world: My learning curve
Arshnee Moodley
Africa Phage Forum Webinar Series
26 May 2021

Who am I?
• South African living in Denmark since 2005
• Associate Professor at the University of Copenhagen
• AMR Team leader at International Livestock Research Institute and the lead
of the CGIAR AMR Hub in Nov 2019,is hosted by ILRI in Nairobi, Kenya
For more information:
www.amr.cgiar.org

Methicillin resistant Staphylococcus
aureus in pigs and dogs
Extended spectrum B-lactamases in
E.coli in pigs
Methicillin resistant staphylococci in
horses

Coincidence…methicillin resistant Staphylococcus
pseudinternmedius
• Commensal found on skin and mucosa in dogs
• Opportunistic pathogen and the most common cause of skin and ear infections
in dogs
• Treatment is typically cephalosporins
• In 2006, we isolated cephalosphorin resistant isolates from dogs with
pyoderma on long term antibiotic therapy (>4 week therapy)
• Strains were susceptible only to gentamicin and mupirocin
• Mechanism of resistance was similar to MRSA (mecA mediated)
• Clonal expansion of dominant multi-drug resistant clone = ST71

MRSP ST71 vs. MSSP
Resistance patterns in MRSP ST71 and MSSP isolates
Author
No.
isolates
Clinda FQs S-TMP Tet Gen
MRSP ST71
Ruscher et al. 2009 76 99% 99% 100% 90% 99%
Perreten et al. 2009 76 97% 100% 100% 68% 70%
Chrobak et al. 2011 23 96% 96% N.D. N.D. 96%
MSSP
Pedersen et al. 2007 201 27% 1% 3% 24% N.D.
Bemis et al. 2009 204 12% 4% 20% 33% 5%
Chrobak et al. 2011 56 27% 2% N.D. N.D. 10%
Rubin et al. 2011 60 13% 0% 5% 34% 0%
Help!

• Chloramphenicol
• 10-40% resistance
• Not available in Denmark and expensive
• Rifampicin
• Only in combination therapy
• Rapid resistance development
• Hepatotoxic
• Critically important antibiotics (CIAs) used in humans
• Linezolid and vancomycin
• Injectable drugs (inconvenient administration route for small animals)
• Not licensed for use in animals
Antibiotic options for systemic therapy

Phages against
MRSP
• Isolated phages from dog faeces using different S.pseud
strains for enrichment
• 7 phages
• all with similar morphology and belonged to
Siphoviridae
• long non-contractile tail, a prominent base-plate and an
icosahedral head
• SN8, SN10, SN11, SN13 were selected for further analyses
• Host range: clear plaques on our MRSP panel BUT variable
lysis patterns on MSSP (clear plaques on 16–28%)
• 40 Kb-similar to each other and S. aureus phage 187
• All phages contained the lysogeny module = temperate phage =
NOT a good treatment option :-(
open arrow =
double-disc
baseplate
single-line =
appendages
attached to
baseplate
Phage
SN8
SN1
0
SN1
1
SN1
3
MSSP
strains HK2 + - + -
HK11 - + + +
HK14 - + + +
HK17 + + + +
HK3 + + + +
Y1 - + + +
S62A
2 + - - +
S55H
7 - + + +
S55H
8 + + + +
S55I1 - + + +
S56C
3 - + + +
S56D
2 + + + +
S56D
3 - + + +
S56F3 + + + +
S56H
7 - + + +
S56H
9 - + + +
S57B
9 - - - -
S57C
4 - - - +
S57C
8 - + + +
S57E7 + + + +
S57E8 - + + +
S60B
9 - + + +
S60C
4 - + + +
S60C
6 + + + +
S60D
3 - + + +
S60D
6 + + + +
S60D
7 + + + +
S60E9 - + + +
S60H
4 - + + +
S60H
7 - + + +
S61A
3 - + + +
S61A
8 - + + +
S61B
7 + + + +
S61D
1 - + - +
S61D
2 - + + +
S61D
8 - + + +
S61E7 + + + +
S61H
2 - + - +
S61H
5 + + + +
S61I9 + + + +
S57E4 - + + -
S61F5 + + + +
S63G
7 + + + +
clear
Turbid
Turbid+
clear

• Phages behaved like lytic phages even on lysogens
• Clear plaques despite containing similar or nearly identical prophages to our
phages
• Recombination between infecting temperate phages & prophages
• Known to happen in E.coli and Lactococcus
• Enable SN8 to overcome the host immunity by cI phage repressor
protein
• Conclusion: future studies to create vir mutants that could be as an
alternative to conventional systemic or topical antibiotic therapy
structural and packaging
modules, replication and
regulation modules, and
lysogeny module

Phages to treat
otitis externa
• Snubi, Rhodesian Ridgeback, first diagnosed with otitis externa in 2011
• “treated daily for 3 months with Aurizon Vet®, a marbofloxacin (antibiotic),
clotrimazol (antifungal) and dexamethasone (steroid) containing ear drop
solution, 300mg of Cefa-Cure® (a first-generation cephalosporin antibiotic) and
8mg Medrol® (corticosteroid).
• A year later it presents again- chronic infection, after 5 years battling, the owner
considers euthanasia due to the reduced quality of life.

- Marza et al. (2006), phages to treat a St. Bernard dog with chronic bilateral otitis
externa caused by P. aeruginosa
- Hawkins et al. (2010) used a cocktail containing 6 bacteriophages to treat P. aeruginosa
otitis in 10 dogs
- This same phage cocktail was tested in a Phase I/II clinical trial in 24 human patients
with chronic otitis caused by an antibiotic resistant P. aeruginosa (Wright et al., 2009)

• No phage preparations registered for use in animals or humans in Denmark
• Safety trial of a phage cocktail against P. aeruginosa, exhibited no adverse
effects in a human safety trial in burn victims (Rose et al. 2014).
• Contact JP Pirnay who put me in contact with M Merabishvili
• Isolated Snubi’s Pseudomonas, sent it to Belgium to be tested against the
cocktail →Only one phage active
• Experimental study we proceed with mono-therapy
• Was approved by Rådet for Dyreforsøg to use bacteriophages as an experimental
last resort treatment for chronic P. aeruginosa ear infection
• 0.5mL dose containing 1 x 107 PFU/mL in PBS, administered directly into
each ear and massaged to promote deep penetration, and include Easotic®
Within 6 days, 1-2 log reduction, but
never reached zero
Better clinical outcome: wound healing,
skin renegeration and positive
improvement in Snubi’s behaviour

Phage lysins against methicillin
resistant S. pseudintermedius
• Post-Doc project for $0.5M
• Phages use a 2-component lysis
system to escape from cells
• Endolysin
• monomeric enzyme targeting the
cell wall
• do not contain signal sequences
and therefore accumulate in the
cytosol
• Holin
• Small hydrophobic intergral
membrane protein that
permeabilize/depolorized the
cytoplasmic membrane
http://parts.igem.org/Part:BBa_K1378032

Gram-Positive Phage Lysins
Modified from Fischetti (2008)
http://www.rockefeller.edu/vaf/e
ms_Strep_after_lysin.php

Enzybiotics
• Promising in vitro and animal in vivo data illustrating the efficacy:
• Bacillus anthracis
• Clostridium perfringens
• Listeria monocytogenes
• Staphylococcus aureus including MRSA
• Streptococcus pneumoniae
• Streptococcus agalactiae
• Streptococcus pyogenes
• Streptococcus suis
• Acinetobacter baumannii *
• Stand alone therapy or in combination with antibiotics
• Observed synergistic actions when combined with antibiotics
• ContraFect has Excebacase (CF-301) for systemic treatment of
bacteremia and endocarditis caused by S. aureus
• In Phase II human clinical trails (IV CF-301 vs. Placebo), Completed in early
2019, awaiting phase III

Lysins against MRSP
• MRSP are frequently resistant to most or even all other antibiotic
classes used in veterinary medicine
• MRSP primarily causes skin and ear infections
• Topical application of lysins
• Two lysins under investigation

1. ASM2
• N-Acetylmuramyl-l-alanine amidase
• Endolysin from prophage in MRSP
ST71 strain E140
• Successfully cloned and expressed
but endless problems purifying
• Some lysins have poor solubility
35kDa
50kDa
ind ind ind ind

2. PlySS2 (Gilmer et al. 2013)
• Aminopeptidase derived from a Streptococcus suis phage
• broad lytic activity against many different streptococal species incl. S. suis, S.
pneumoniae, S. pyogenes, Grp B Strep, Listeria, MRSA, VISA
• Stable at 50°C for 30 min, 37°C for >24 hrs, and 4°C for 15 days
• In vitro at 128 μg/ml, reduced MRSA by 5 logs with 1hr treatment
• A single, 2mg dose of PlySs2 protected 92% (22/24) of the mice in a bacteremia

PlySS2 against MRSP E140
Absorbance
at
OD
600
Time (minutes)
32µg/mL
64µg/mL
3 log reduction within 12
minutes with a single dose

Time kill Assay with lower concentrations of PlySS2
and E140 (MIC = 64μg/mL)
Approx. 4 log reduction after 40min with a single dose of 4ug/mL
1000
10000
100000
1000000
10000000
100000000
1E+09
0 10 20 30 40 50 60
CFU/mL
Time after treatment (min)
0ug/ml
0.5ug/ml
1ug/ml
2ug/ml
4ug/ml

100
1000
10000
100000
1000000
10000000
100000000
0 10 20 30 40 50 60 70 80 90 100 110 120
CFU/ml
Time (min)
0ug/ml
2ug/ml
4ug/ml
Add a second dose after 60min -> further 1log reduction
Two doses of PlySS2

• ASM2 purification problems
• Expression in a Gram-positive system
• Engineer a recombinant lysin
• PlySS2 would be to test efficacy in dogs with skin
infections
• Clinical trial at University of Tennessee
• ”Natural infections”
• Regulation and study design was challenging
• Vs. antibiotic
• Cannot deny patient care
• Costly experiment

Pro’s and con’s of lysins
Pro
• Bypass the use of whole
phages
• Rapid cell lysis
• No observable resistance
• Synergy with antibiotics
• Increase specificity by
domain swapping
• Non toxic
Con
• Binding affinity is high
• Potential immunogenicity
(decrease lysin activity)
• Production costs are high

Antibiophage = 1.443.661€
23
Funded by
Abiyad Baig
Paul Barrow
Robert Atterbury
Adriano Gigante
Jeroen Wagemans
Rob Lavigne
Angelina Trotereau
Catherine Schouler
Arshnee Moodley
Dziuginta Jakociune
Jens Peter Nielsen
Søren Saxmose Nielsen
Ida Thøfner
Michela Gambino

Target: Avian pathogenic E. coli
24
Courtesy of Ida Thøfner
APEC
Leading causes of mortality and morbidity
• Reduces egg production
• Reduces body mass
• Death
Poulvac®
Antibiotic therapy
Poor efficacy and only against O78
Resistance – public health, overlap of
APEC and hu_ExPEC
Avian colibacillosis = wide
range of localized and
systemic infections
Different serogroups
• O1, O2, O78
• heterogeneous

Phages
APEC
13 phages
confirmed
by
traditional
plating

Testing dual combinations with the 13 phages
• 13 phages: 1 x Siphoviridae and 12 x Myoviridae
• Dual combinations (12 combinations) against Cp6salp
05/10/2021 26
7
1+7
Pos Control
Phage 1
Cp6salp_O1

Testing dual combinations with the 13 phages
• 13 phages: 1 x Siphoviridae and 12 x Myoviridae
• Dual combinations (12 combinations) against Ben5202
05/10/2021 27
7
1+7
Pos Control
Phage 1
Ben5202_O2

Triple combinations with the 13 phages
• 66 combinations with Phage 1 as the baseline against Cp6Salp
28

Some cases 
05/10/2021 29

• 8 combinations that consistently produced a flat
growth line against Cp6Salp, we test against Ben5202
05/10/2021 30

Chicken Embryo Lethality Assay
• A model to assess bacterial virulence and now phage efficacy
• Chicken amniotic fluid contains antibacterials = defence
• Avian embryos are not regulated by the same legislations as
animal experiments
• Use 11-day old fertilized eggs
05/10/2021 31
Air sac
injection site
Embryo
dead live

32
D1 D2 D3 D4 D5 D6
DPBS 10 10 10 10 10 10
Ceftiofur 1µg/ml 10 10 10 10 10 10
BEN5217 6 6 5 4 1 0
BEN5217 + ceftiofur 1µg/ml 10 10 10 10 10 10
BEN5217 + 1+2+4 9 9 9 8 8 8
BEN5217 + 1+3+4 10 10 10 10 10 9
BEN5217 + 1+4+8 9 9 9 8 8 8
BEN5217 + 1+4+12 10 10 10 10 10 10
BEN5217 + 1+3+9 9 8 5 4 2 1
BEN5217 + 1+3+10 6 5 4 3 3 1
Embryo survival
MOI =100
Interesting observation: presence of phage 4 seems important in the cocktail
- Test various MOIs
- Con- no immune response

Experimental Salpingitis Infection
• Test 1:phage cocktail(1,3,4)
• Test 2:phage cocktail (1,4,12)
• Challenge is Cp6salp
After48h: Dissection, pathology scoring, sampling for microbiology (CFU and PFU):
- Ovary, Blood, Mucus from Salphinix, Spleen

I didn’t do this by myself!
• Post- Doc Dziuginta Jakociune
• Several Masters Student: Sofia, Maja, Amelia, Emelia, Mark
• Two technicians: Tanja and Manja
• Co-supervise three PhD students on phages
• Irene Mutuku (2013-2016). University of Ghent, BE. Thesis: “Phage
therapy against avian pathogenic Escherichia coli”
• Muriel Breteau (2012-2016). University of Warwick, UK. Thesis:
“Phage therapy against methicillin-resistant Staphylococcus
pseudintermedius
• Patricia Sørensen (2018-2021). University of Ghent, BE. Thesis:
“Mechanisms of phage resistance in Escherichia coli”

Collaborators

THANK YOU