1. Phage “Angela” and the control of E. coli O157:H7 on meat
J. Andrew Hudson, Craig Billington, Angela Cornelius, Lucia Rivas, Stephen On, Nicola King, Salim Ismail

2. The state of play Phages are increasingly being
recognised as an effective control
There are a few commercial products
They are achieving recognition as
GRAS
Organic
Kosher
Processing aids

3. The challenge of E. coli O157:H7
O157:H7 is considered an adulterant in some kinds of meat exported to the US
The US market is very important to the NZ economy
Acceptable and effective controls don’t exist
Could we develop a phage cocktail that would inactivate E. coli O157:H7 on fresh meat ?

4. Phage isolation Sample (e.g. screened sewage)
Centrifuge and filter “sterilise”
Add 0.1 ml sample to 0.1 ml host cells in soft agar
Pour on to standard agar plate and incubate
Check for plaques

6. Host range of phage “Angela”
The range of taxa supporting
phage replication
“Goldilocks” host range desirable
Angela infects:
28/30 (93.3%) of E. coli O157 isolates
1/30 (3.3%) other E. coli serotypes
0/13 other foodborne species
Photos by UoOtago EM Unit

7. Absence of known stx genes1 2
1 kb
ehxA 534
eae 384
stx2 255
stx1 180

8. Effect on growing STEC O157

9. A case study in pathogen control
Experiment on 2 x 2 cm pieces of beef (x3)
Add cells, allow attachment, add phages, incubate (37°C x 1 h), count survivors
Host cells
After attachment 1.41 x 104/piece
Alone 5.36 x 104/piece
With phages 4.3 x 101/piece (1 colony on 9 plates)
>99.9% (3 log10) reduction
Phages
Alone 2.64 x 106/piece
With host cells 2.40 x 106/piece

10. Incubation time and control on meat

11. Effect of phage concentration
Mean phages added (PFU/piece)
Final host concentration (mean (± SE)a CFU/piece)
Mean log10 change compared to inoculum
P value compared to inoculum
3 x 108
<1 (± 0) x 101
>-2.7
<0.01
3 x 107
3.5 (± 1.9) x 102
-1.2
3 x 106
1.1 (± 0.6) x 103
-0.8
3 x 105
6.3 (± 1.8) x 103 NS
1.1 (± 0.4) x 104
+0.2
1 hour 37°C

12. On cooling meat

13. On cooling meat 2 Phage concentration (mean PFU/sample)
Cell concentration pre-cooling
(mean CFU (± SE) /sample)
Cell concentration post cooling
Mean log10 change (P value) in host numbers compared to:
Inoculum
Phage-free control
Chilling Simulation: Conventional carcass
3.2 x 107
3.1 (± 1.6) x 103
2.6 (± 1.7) x 101
-2.1 (<0.01)
-1.6 (<0.01)
3.0 x 105
1.0 (± 0.2) x 103
2.6 (± 1.4) x 102
-0.6 (<0.01)
-0.8 (<0.05)
4.4 x 103
3.3 (± 0.1) x 103
3.6 (± 0.8) x 103
0 (NS)
-1.0 (NS)
Chilling Simulation: Hot boned boxed beef
7.4 x 106
4.3 (± 0.2) x 103
6.6 (± 2.7) x 102
-0.8 (<0.01)
-1.4 (NS)
4.7 x 105
5.9 (± 1.0) x 103
1.1 (± 0.6) x 104
0.3 (NS)

14. Prospects and problems
More than 105 phages/cm2 needed, better control at higher concentrations
Reasonable control under industry TT curves
Phage replication not important
Good host range
…..but not good enough
Regulations
Shifting goal posts: the “super seven”

15. Acknowledgements
The meat industry (Meat and Wool New Zealand (now Beef+Lamb NZ), MIA)
New Zealand Foundation for Research, Science and Technology (now the Ministry of Business, Innovation and Employment). Contracts CO3X0201 and CO3X0701
Rhys Jones of AgResearch for beef cooling data
E. coli isolates were provided by ESR’s ERL
Electron Microscope Unit of the University of Otago for the electron micrographs