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

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

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 ?

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

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

Absence of known stx genes 1 2 1 kb ehxA 534 eae 384 stx2 255 stx1 180

Effect on growing STEC O157

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

Incubation time and control on meat

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

On cooling meat

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)

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”

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