Phage as a food safety tool 2017 Mar

Dr Cath Rees School of Biosciences

IAFP MEETING 2017 1 (phage) • Bacteriophage are that specifically infect • First described by Felix d’Herelle (1917) &

Frederick Twort (1915) Mar 2017 Mar

• Both noted that these unknown agents had the ability to “eat” bacterial cells • Bacteriophage = “bacteria eater” 2 Bacteriophage • Wide range of applications being developed: • in humans • Biocontrol

• Sanitising surfaces 2017 Mar • Sanitising foods • Pathogen reduction prior to slaughter • Rapid detection of pathogens

3 Bacteriophage hosts • Like all viruses they have a limited Host Range • determines the type of cell infected • Have evolved to bind to structures on the surface of correct host cell

• Tail structures help 2017 Mar inject nucleic acid into host

Head

Tail Fibers Base Plate 4

Phage infection Mar 2017 Mar

Image by Dr Elizabeth Kutter, Bacteriophage Ecology Group http://www.mansfield.ohio-state.edu/~sabedon/beg_phage_images.htm 5 Bacteriophage growth • Viruses replicate inside the host cell and produce 50+ phage per infection • Produces enzymes to break open the host once the new viruses are made • Zone of lysis = plaque

1.00E+09 2017 Mar Bacterial 1.00E+08 growth 1.00E+07

1.00E+06 Phage 1.00E+05 Burst 1.00E+04 size = 1.00E+03 100

1.00E+02

NumberofBacteria or Bacteriophage 1.00E+01

1.00E+00 1 2 3 4 5 6 7 8 9 10 No. of Generations/Rounds of Replication 6 Phage Typing • Phage Type (PT) determined from pattern of sensitivity of a bacterial isolate to sets of

specific phage with limited host range Mar 2017 Mar

Result

Incubation Lysis (phage sensitive)

No Lysis (phage resistant) Bacterial Lawn 7 Biocontrol and Resistance 2017 Mar

8 Resistance and Co-evolution • Spontaneous mutations rate in any bacterial population is approx. 1 in 106 • Phage replication also produces variants

• Approx. 102 phage per infected

host 2017 Mar • Therefore only 104 infections required to generate a variant • Faster generation of variants than host cell population New viral particles

9 Resistance and Co-evolution • In the “natural” world numbers of an individual phage are low • Lots of unchallenged/uninfected cells exist • Many phage receptors are essential so mutations result in a fitness cost

• host cells resistant to infection are less likely to multiply in Mar 2017 Mar the presence of competitors

10 Resistance and Co-evolution • In the “natural” world numbers of an individual phage are low • Lots of unchallenged/uninfected cells exist • Many phage receptors are essential so mutations result in a fitness cost

• host cells resistant to infection are less likely to multiply in Mar 2017 Mar the presence of competitors • Without a selective advantage, the few resistant cells are likely to be out competed by the large number of “fitter” uninfected sensitive cells

11 Resistance and Co-evolution

• However when we apply high levels of phage to an

environment the situation changes Mar 2017 Mar

• Phage kill sensitive cells, but a resistant variant survives • In the absence of competition a new population of resistant cells will predominate

12 Resistance and Co-evolution • …but lots of phage variants are also produced • Phage variant that can infect a resistant cells will kill resistant

population Mar 2017 Mar

• …only a new resistant cell variant survives • Process results in Co-evolution of both bacteria and phage

13 Resistance and Co-evolution • If remaining population is less fit this could be a benefit • BUT mutations can arise that alter receptors without loss of function • Challenging bacterial populations with large numbers of phage drives a faster mutation rate in 2017 Mar receptors • Results in cells with altered surface properties/may better evade the host immune response • Co-evolution could be a benefit OR a threat

14 Applications in Food Industry • Sanitising surfaces • Use of phage to target specific pathogens within the food processing environment • Co-evolution predicted to occur • Long term use may result in resistant cell population

• Sanitising foods 2017 Mar • Application of phage to product surface to control growth during storage/maturation • Co-evolution predicted to occur • BUT product is continually removed from environment • therefore less likely to result in resistant strains in factory

15 Applications in Food Industry • Pathogen reduction prior to slaughter/product release • Phage applied immediately prior to animals/products being removed from production site • Phage and bacteria removed from production facilities 2017 Mar • Less likely to see development of resistance in production site

16 Examples of Commercial Products

• Listex • Salmonelex • Ecoshield • ESR (NZ) • Biolyse 2017 Mar

17 Pathogen detection 2017 Mar

18 Why use bacteriophage to detect bacteria? • Culture methods are the “Gold Standard” of • Results are retrospective • Culture is not always specific

• confirmatory tests required 2017 Mar • Not all organisms are easily cultured • M. leprae : mouse foot pad, nine banded armadillos • Not all rapid tests detect viable organisms • Antibody-based tests • PCR-based tests 19 The challenge for food analysis

• Microbiological analysis performed for 2 reasons 1. Determining microbial load • Quality of product • Confirmation of CCP/hygienic manufacture • Determining microbial load requires enumeration from a non-homogenous sample 2017 Mar

2. Demonstrating that levels of pathogens are below acceptable limits • e.g. Absence of specific organisms from 25 g sample

• Proving absence of a single cell normally requires 20 enrichment by growth to detectable levels 20 GM Reporter Phage • Reporter genes introduced into phage • not expressed before infection • Infection of host indicated by production of signal • Only host cells will allow infection

• No need for purification of target cells 2017 Mar

Tanji et al., (2004) J. Biotechnol 114:11–20 21 Reporter Phage • First rapid phage-based detection tests described in 1987 • lux reporter genes cloned into phage  vector for detection of E. coli

• Since then many different reporter phage developed Mar 2017 Mar

 Lux  Gfp  Fluc  LacZ  RLuc  CelB  Ina 22 Detection of Listeria monocytogenes using a lux reporter bacteriophage

500 bioluminescence (RLU)

400

300 Mar 2017 Mar

200

100

0 100 500 1000 2500 5000 10000

Loessner et al., (1996) Appl. Environ. Microbiol. 62:1133–1140 23 Commercial reagents now available • Phage-based detection methods have been developed

• Sample 6 (http://sample6.com/) Mar 2017 Mar

24 Why so little commercial development?

• High cost of development of each reagent • Detection events require specialised equipment • GM organisms fell out of favour, especially in

food industry 2017 Mar • Requirement for enrichment (LOD>1cell) • No cultures for genotyping for trace-back studies

25 Detecting of Bacteriophage growth

Assays simply look for an increase in phage number or other evidence of phage growth to indicate presence of host cell • No modification of phage required

• simplifies development and no GMO issue Mar 2017 Mar • Phage growth far faster than host cell growth • Provides required time advantage • Amplifies signal • Variety of end-point detection methods can be used

• e.g.lateral -flow devices 26 Pathogen detection: Mycobacteria 2017 Mar

27 Phage milk test for Mycobacteria

65 min Mar 2017 Mar

20 min 18 h 24 h Enumeration Molecular • Rapid and Sensitive Identification • Only identifies viable cells • PCR can be modified to identify pathogen of choice 28 M. paratuberculosis

• Link between MAP and Crohn’s disease was made because of similarities between aetiology of Johne’s disease and Crohn’s disease • Still no conclusive evidence that MAP is causal agent 2017 Mar

• Meta-analyses suggests that there is an association between MAP and Crohn’s • Food regulators have recommended MAP is eliminated from the food chain • e.g. ACMSF UK 29 Milk as a source of human exposure

• UK study showed that 1.8 % of retail pasteurised milk contained viable MAP • Grant et al., 2002 Appl. Env. Micro. 68, 2428-2435. MAP • US study found 2.8 % of retail whole milk from 3 states

• Ellingson et al., 2005 J. Food Prot. 68, 966-972. 2017 Mar COWS • Czech Republic study isolated MAP from 1.6 % samples pasteurised retail milk • Ayele et al., 2005 Appl. Env. Micro. 71, 1210-1214 • Argentina isolated MAP from 2.8% of samples MILK • Paolicchi et al., 2012 Brazil. J Microbiol. 43, 1034-37

Very good evidence that MAP is present in retail milk 30 Development of MAP Detection methods • Milk • Stanley et al., (2007) Appl Env Micro, 73: 1851–1857 • Botsaris et al. (2013) Int. J. Food Microbiol. 164: 76-80

• Cheese 2017 Mar • Botsaris et al. (2010) Int. J. Food Micro, 141: S87–S90

• Powdered Infant formula

• Botsaris et al. (2016) Int J Food Micro 216: 91-94 31

31 Survey of retail pasteurised milk 368 semi skimmed (1.7 % fat) 8.0 milk samples

6.0

positive positive -

4.0 MAP

samples 2.0

0.0 Percentage Percentage 1 - 2 3 - 9 > 10 2017 Mar Plaque Number/ 50 ml • Overall 10.3 % contained viable MAP by Phage-PCR • 1.1 % potentially detectable by culture • 3.5 % potentially detectable by PCR • 6.8 % not detectable by other methods • Provides new tool to improve milk quality 32 Newest Application • Detection of Bovine TB in raw milk • Specific application for artisan cheese producers • Raw milk used so M. bovis is not destroyed by pasteurisation

Annual Annual

TB test TB test Mar 2017 Mar

Maturing product is safe 33 Newest Application • Detection of Bovine TB in raw milk • Specific application for artisan cheese producers • Raw milk used so M. bovis is not destroyed by pasteurisation

When did the cows get TB? Mar 2017 Mar

Failed Annual TB test

• Threat to producers if bovine TB detected • New source of milk required • Maturing product at risk 34 Newest Application • ACMSF guidelines set safe levels of M. bovis in milk as maximum of 102 at single ingestion • ACM/1047a RISK ASSESSMENT: The possible health risks to consumers associated with M. bovis and unpasteurised milk and milk products

• Phage milk test used to determine detectable 2017 Mar levels • Do NOT have to prove absence • Testing milk before annual TB test acts as a QA marker in case of TB breakdown • No new product can be made, but existing product protected 35 Conclusions

• Phage biocontrol agents becoming established in the food industry • Potential to target specific pathogens of concern

• Applications in product already demonstrated 2017 Mar • Issues of surrounding long terms environmental applications remain • Phage-based detection methods now being applied to some real world applications

36 Acknowledgements Food Sciences • Dr Ben Swift • Dr George Botsaris • Zara Gerrard • Vuong Le

. Dr Jon Huxley: UoN 2017 Mar . Prof. Mike Hutchings SRUC . Specialist Cheese Makers Association

Sutton Bonington Campus 37