Phage as a food safety tool Mar 2017 Dr Cath Rees School of Biosciences IAFP MEETING 2017 1 Bacteriophage (phage) • Bacteriophage are viruses that specifically infect bacteria • First described by Felix d’Herelle (1917) & Frederick Twort (1915) Mar 2017 • Both noted that these unknown agents had the ability to “eat” bacterial cells • Bacteriophage = “bacteria eater” 2 Bacteriophage • Wide range of applications being developed: • Phage Therapy in humans • Biocontrol • Sanitising surfaces Mar 2017 • 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 virus Mar 2017 inject nucleic acid into host Head Tail Fibers Base Plate 4 Phage infection Mar 2017 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 Mar 2017 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 Numberof Bacteria orBacteriophage 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 Result Incubation Lysis (phage sensitive) No Lysis (phage resistant) Bacterial Lawn 7 Biocontrol and Resistance Mar 2017 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 Mar 2017 • 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 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 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 • 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 • …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 Mar 2017 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 Mar 2017 • 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 Mar 2017 • Less likely to see development of resistance in production site 16 Examples of Commercial Products • Listex • Salmonelex • Ecoshield • ESR (NZ) • Biolyse Mar 2017 17 Pathogen detection Mar 2017 18 Why use bacteriophage to detect bacteria? • Culture methods are the “Gold Standard” of microbiology • Results are retrospective • Culture is not always specific • confirmatory tests required Mar 2017 • 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 Mar 2017 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 Mar 2017 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 Lux Gfp Fluc LacZ RLuc CelB Ina 22 Detection of Listeria monocytogenes using a lux reporter bacteriophage 500 bioluminescence (RLU) 400 300 Mar 2017 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 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 Mar 2017 • 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 • 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 Mar 2017 27 Phage milk test for Mycobacteria 65 min Mar 2017 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 Mar 2017 • 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. Mar 2017 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 Mar 2017 • 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 Mar 2017 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 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