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• The zoonotic Multi-Drug Resistant (MDR) non-typhoidal
Salmonella (NTS) enterica serovar Enteritidis is one of the
major causes of foodborne infections worldwide.
• Current methods of controlling Salmonella infections at the
farm level include the use of antibiotics, particularly in poultry
farming.
• An estimated 75% of antibiotics administered to poultry are
released in the environment and contribute to the emergence
of antimicrobial resistance (AMR).
• Bacteriophages are a potential alternative to fight MDR NTS.
Phages stable at low pH and high temperatures would render
them more suitable for the control of Salmonella in poultry as
they have a higher chance to survive the harsh gastrointestinal
environment
INTRODUCTION
OBJECTIVES
• Thermal and pH Stability
• Rate of phage release from SV
• Effect of SV on phage survival
MATERIALS AND METHODS
• All 13 phages were relatedly stable from pH 4 to 12 after 24
hours of incubation with an average titre of 8.1 x109 PFU/ml,
while they all lost their viability within 3 hours at pH 2-3.
• The thirteen phages were relatively stables from temperatures
ranging from 25℃ to 42℃ after 12 hours of incubation but
started losing their viability at 50℃.
• All three Novel Silica Vesicles demonstrated a low but longer
rate of phage release upon adsorption for 96 hours.
• Preliminary data indicate that SV 140 C18 nanoparticles shown
the ability to protect phages longer, with an average titter of
6.4 x 106 PFU/ml at 60 minutes compared to SV 100 (12.6 X 104
PFU/ml) and SV 140 (6.3 X104 PFU/ml).
• In contrast, free phages in SGF had an average concentration
of 3.7 x103 PFU/ml after 60 minutes of incubation.
RESULTS
CONCLUSION
• Most phages are relatively stable at 4-9 pH and 25-37℃.
Varying phage stability at different pH and temperatures
over time indicate varying physiological characteristics of
phage
• Phages more stable at low pH and high temperature to
be used as potential candidate phages for the in vivo
cocktail
• SV demonstrated the ability to protect phages from acidic
environment posing as a suitable delivery tool for phages
in the gastrointestinal tract
• This study tested the thermal and pH stability of 13 different S.
Enteritidis specific phages, previously selected from a cohort of
phages based on Restriction Fragment Length Polymorphism
(RFLP) patterns.
• Three novel silica vesicles (SV 100, SV 140, and SV 100 C18)
were used to test whether they increased the survival of
phages in simulated gastric fluid (SGF)
1Animal and Human Health Program, International Livestock Research Institute (ILRI), Nairobi, Kenya
Amos L. Mhone1, Angela Makumi1, Linda Guantai1, Josiah Odaba1, Anna Lacasta1, Nicholas Svitek1
Stability of novel non-typhoidal Salmonella phages in simulated gastric fluid and in vitro efficacy of
silica vesicle to protect phages
0 15 30 45 60
2
4
6
8
10
12
14
Time (Minutes)
Phage
Titre
(Log
10
(PFU/ml)
SGF & Phage Only
SV 140
SV 140-C18
SV 100
Phage only
0 12 24 36 48 60 72 84 96
0
1
2
3
4
5
6
7
8
9
10
Time
Phage
dilution
log
10
SV 100
SV-140
SV-140 C18
CONTROL
p H
P
h
a
g
e
T
i
t
r
e
(
L
o
g
1
0
(
P
F
U
/m
l)
1
2
3
4
5
6
7
8
9
1
2
0
1
2
3
4
5
6
7
8
9
O v e ra ll S ta b ility o f S .E n te ritid is p h a g e s o n d iffe re n t p H V a lu e s a fte r 1 2 H rs
Figure 2: Overall Stability of phages at different pH values after 12 Hours
O v e ra ll s ta b ility o f S . E n te ritid is P h a g e s in d iffe re n t te m p e ra tu re v a lu e s
T e m p e ra tu re
P
h
a
g
e
T
i
t
r
e
(
L
o
g
1
0
(
P
F
U
/m
l)
2
5
3
0
3
7
4
2
5
0
6
0
0
1
2
3
4
5
6
7
8
9
Figure 1: Overall Stability of phages at different temperatures after 12 Hours
P h a g e ID
P
h
a
g
e
T
i
t
r
e
(
L
o
g
1
0
(
P
F
U
/m
l)
S
a
l
1
6
(1
7
6
)
S
a
l
1
6
(4
6
0
)
S
a
l
1
6
(4
7
8
)
S
a
l
1
6
(4
8
3
)
S
a
l
1
6
(5
7
5
)
S
a
l
7
3
(1
9
0
)
S
a
l
7
3
(5
7
5
)
S
a
l
1
7
7
(1
7
6
)
S
a
l
1
7
7
(1
9
0
)
S
a
l
1
7
7
(4
5
9
)
S
a
l
1
7
7
(
4
6
8
)
S
a
l
1
7
7
(5
7
5
)
S
a
l
3
1
2
(4
6
0
)
4
5
6
7
8
9
0 M in u te s
3 0 M in u te s
1 H o u r
2 H o u rs
3 H o u rs
K e y
Figure 3: Stability of phages at pH 3 for 3 Hours
0 15 30 45 60
2
4
6
8
10
12
14
AverageperfomanceofSVNanoparticleswithphages
Time(Minutes)
P
h
a
g
e
T
it
r
e
(
L
o
g
1
0
(PFU
/m
l)
SimulatedGatricFluidwith
PhageOnly
SV140
SV140-C18
SV100
Phageonly
Figure 5: Effect of SV on phage survival
Figure 6: Rate of phage Release from SV
Phages 2021 (Oxford), Virtual, 7-8 September 2021
FUTURE WORK
• Evaluate the stability of these phages in vivo in
chickens with and without SV.
• Effect of different pH and temperature values on
phage-bacterial lysis
• The effect of SGF and SIF on phage-bacterial lysis
P h a g e ID
P
h
a
g
e
T
i
t
r
e
(
L
o
g
1
0
(
P
F
U
/m
l)
S
a
l
1
6
(
1
7
6
)
S
a
l
1
6
(
4
6
0
)
S
a
l
1
6
(
4
7
8
)
S
a
l
1
6
(
4
8
3
)
S
a
l
1
6
(
5
7
5
)
S
a
l
7
3
(
1
9
0
)
S
a
l
7
3
(
5
7
5
)
S
a
l
1
7
7
(
1
7
6
)
S
a
l
1
7
7
(
1
9
0
)
S
a
l
1
7
7
(
4
5
9
)
S
a
l
1
7
7
(
4
6
8
)
S
a
l
1
7
7
(
5
7
5
)
S
a
l
3
1
2
(
4
6
0
)
7 .0
7 .5
8 .0
8 .5
9 .0
0 M in u te s
3 0 M in u te s
1 H o u r
2 H o u rs
3 H o u rs
K e y
Figure 4: Stability of phages at 42℃ for 3 Hours
This document is licensed for use under the Creative Commons Attribution 4.0 International Licence.
September 2021