Bacteriophage application for elimination of E coli for elimination of ...

Bacteriophage application
for elimination of E. coli
O157:H7 in ground beef
Jennyka Hallewell

• Food and waterborne zoonotic
pathogen
• Initially isolated from Michigan
ground beef outbreak in 1982
• Most commonly isolated serotype
in North America
E. coli O157:H7
• Hemorrhagic colitis, Hemolytic
Uremic Syndrome (HUS), death
• Everyone is susceptible (!)
although young and elderly are
high risk

E. coli O157:H7 in the feedlot
• Cattle are natural reservoirs of
E. coli O157:H7 and harbour in
feces
• Asymptomatic carriers:
transient and intermittent (10-
100 cfu/g)
• Super shedders (10,000 cfu/g)
• Season, age, weaning,
transport, stress, diet
• The effect of feeding Distillers
The effect of feeding Distillers
grains to cattle
Environment

Distillers’ Dried Grain with
• By-product off the bioethanol
industry
• DDGS has higher protein, fat and
fibre content than complete grains
and is a valuable feed source for
cattle
solubles (DDGS)
• Corn DDGS (CDDGS) has been
linked to bovine shedding of E.
coli O157:H7
• In Canada, DDGS is made
primarily from wheat (WDDGS)
• No studies on WDDGS and fecal
shedding of E. coli O157:H7

Effect of DDGS on E. coli O157:H7
in cattle
A. Commercial feedlot study
-Cattle fed finishing diets containing 22.5% CDDGS, WDDGS or CTRL
(n=6,817)
-Collected fecal pats, pH, hide swabs and screened for naturally occurring
E. coli O157:H7
B. Fecal Persistence study
-Inoculated feces with nalidixic resistance E. coli O157:H7 (Nal-R) from cattle
fed 22.5% CDDGS, WDDGS or CTRL
C. Challenge Study
-Inoculated steers with Nal-R E. coli O157:H7 (n =32)
-Cattle fed 40% CDDGS, WDDGS, CWDDGS or CTRL
-Collected fecal grabs, pH and screened for Nal-R E. coli O157:H7

Effect of DDGS on E. coli
O157:H7 in cattle
• Cattle fed CDDGS or WDDGS did not increase fecal shedding or persistence
of E. coli O157:H7 in feces
• Cattle adapting to finishing diets had enhanced persistence of E. coli O157:H7
in feces compared to cattle adapted to the diets for more than two weeks.
• Many factors may be responsible for the variability of shedding of E. coli
O157:H7
• Publications
Hallewell,J., McAllister, T.A., Thomas, J., Booker, C. W., Hannon, S., Jim, G. K., Burciaga-Robles, L.O.,
May, M. L., Peterson, R. E., Flaig, C., Hussey, E. M., and K. Stanford. 2012. An evaluation of the effects of
wheat or corn distillers dried grain with solubles on feedlot performance and fecal shedding and
persistence of Escherichia coli O157:H7. JAS. Accepted.
Hallewell, J., Barberi, L.R. Thomas, J., Stanford, K. and T. A. McAllister. 2012. Fecal shedding of
Escherichia coli O157:H7 in inoculated cattle fed corn or wheat distillers’ dried grain with solubles. Journal
of Food Protection. Submitted.

E. coli O157:H7 in ground beef
• Contaminated ground beef has been implicated
as a source of multiple E. coli O157:H7
outbreaks
• Low infectious dose (~10 cells)
• Large scale production
• Eradication of E. coli O157:H7 at critical control
points (ie. Grinding and mixing augers) can
prevent mass contamination and massive
recalls
• Bacteriophage application may provide a
Bacteriophage application may provide a
natural alternative for reduction of pathogens in
meat

Bacteriophage Application
• Bacteriophages (phages) are natural
predators of bacteria including E. coli
O157:H7
• Viruses (lytic) target specific receptors on
the bacteria, use host machinery to
generate new phage progeny and lyse
host cell releasing new progeny
• Self-replicating, self-limiting, very specific
to targets
• FDA in United States approved phage
preparations p for eradication of Listeria
monocytogenes
• Phage cocktails can increase specificity
and reduce likelihood of resistance
• Phage are ubiquitous in the environment

Isolation of Endemic phage from the
environment
• 42 phage isolates were isolated from cattle
feces (SS and CTRL) at the Lethbridge
research center between July and August
2011.
• Phage isolates were screened for lytic
activity and purified using E. coli O157:H7
host strains.
• Identified highly lytic phage
• Determined Multiplicity of Infection (MOI)
• MOI = plaque forming units (pfu) phage
colony forming units (cfu) E. coli
• Increase titer by infection (propogation)

Multiplicity of Infection (MOI)
1400
1200
1000
Titer
pfu/ml (10E E6)
800
600
400
200
0
MOI
Animal 20 Animal 651 Animal 176 Animal 219 Animal 222 Animal 299

Further characterization of phages
• 20/42 phage isolates were considered highly lytic to
E. coli O157:H7
• Multiplicity of Infection (MOI) is unique for each phage
• Contact t concentration ti of phages with target t is critical
• Allows us to target low numbers of E. coli O157:H7
found in meat (highly lytic)
• Pulsed Field Gel Electrophoresis (PFGE) (genome
sizing, restriction patterns) to type naturally-occurring
phage and E. coli O157:H7 (SS and CTRL animals).

Application of phages to meat for reduction
of E. coli O157:H7
• Highly virulent phages cocktails will be applied to ground beef to reduce E. coli
O157:H7.
• Ground beef samples will be inoculated with Nal-R E. coli O157:H7 and
screened for E. coli O157:H7 and phage.
• The effects of mixing, temperature, and level of contamination will be
investigated.
• Results from this study will allow us to assess the possibility of phage
application at critical control points such during processing for eradication of E.
coli O157:H7 post-slaughter
• Characterization of naturally occurring phage in the environment will contribute
to limited knowledge of vast populations of phages and accelerate progress to
many new exciting applications!
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Acknowledgements
T. A. McAllister, R. Barbieri, K. Munns, L. Selinger
Agriculture and Agri-Food Canada
K. Stanford, C. Agopsowicz, Y. Han, J. Peters,
H. Zhahiroddini, G. Wallins
Alberta Agriculture and Rural Development
C.W. Booker, S. Hannon, G. Jim, L. Burciaga-Robles Robles, M.L. May,
R.E. Peterson, C. Flaig, E.M. Hussey
Feedlot Health Management Services
J. Thomas, B. Selinger, S. Wetmore
University of Lethbridge

Thank You!