FINAL REPORT B12008 Persistence of Mycobacterium bovis and Verocytotoxin-producing Escherichia coli (VTEC) in UK-made raw-milk cheeses Project Manager: Dr P. A. Voysey Project Teams 1. Work carried out on VTEC P. A. Voysey, R. A. Green, C. J. Baylis, K. J. Bridgwater and P. Anslow Microbiology Department Campden BRI Chipping Campden Gloucestershire GL55 6LD 2. Work carried out on M. bovis R. W. J. Forgrave, J. Donaghy and M. T. Rowe Food Microbiology Department Queens University of Belfast (QUB) / Agri-Food and Biosciences Institute (AFBI) Newforge Lane Belfast BT9 5PX Start date: 1 September 2007 End date: 31 August 2010 (extended to 30 June 2011) Page 1 of 298 CONTENTS Title page Executive summary Lay person’s summary Project Objectives Introduction to the project Project team Acknowledgements Project seminar Layout of this project report PART 1 WORK CARRIED OUT AT CAMPDEN BRI ON VTEC 1. Introduction The scientific problem State of the art in this area Anticipated outcomes 2. Information on UK raw milk cheeses Contents 1. Introduction 2. Cheese types 2.1 Hard cheese 2.2 Semi-hard cheese 2.3 Soft cheese 2.4 Blue-veined cheese 3. UK produced raw milk cheeses 3.1 Raw milk cheeses produced and on sale in the UK 4. Cheesemaking 5. Characteristics of cheeses 5.1 pH and Titratable Acidity 5.2 Salt content 5.3 Water activity 5.4 Moisture content 5.5 Background microbiological flora 5.6 Starter cultures used in cheese manufacture 5.7 Maturation conditions and duration 6. Microbiological risks associated with cheeses 7. Conclusions 3. Raw milk and raw milk cheeses as vehicles for infection by Verocytotoxin-producing Escherichia coli Abstract Introduction Verocytotoxin-producing E. coli Dairy animals as a source of VTEC Outbreaks associated with raw milk and associated cheese Raw milk Page 2 of 298 Cheese Prevalence of VTEC in raw milk and dairy products Diversity of serotypes and virulence factors Survival of VTEC during cheese manufacture Methods for the detection and isolation of VTEC in cheese Conclusions 4. Experimental plan 5. Methodology for isolating and detecting VTEC in cheese 5.1 Introduction Study 1 Growth of VTEC O157 and O26 strains in pure culture. Study 2 Growth and colony morphology of strains of competitor organisms on selective media after enrichment. Study 3 Evaluation of enumeration methods and immune-magnetic separation procedure for VTEC O157 and O26 in cheese. 6. Caerphilly cheese production protocol and experimental results Contents 1. Introduction 2. Equipment and ingredients 3. Method 4. Cheesemaking results 5. Sampling of cheeses inoculated with VTEC 6. VTEC persistence in cheese – results 7. End of life analysis 8. Discussion of results 7. Conclusions and recommendations 8. Suggestions for future work 9. References Appendix 1 Poster PART 2 WORK CARRIED OUT AT QUEENS UNIVERSITY BELFAST ON M. bovis 1. Introduction 2. Current evidence that M. bovis survives in cheese 3. Project objectives 4. Approach adopted 5. Experimental plan Page 3 of 298 6. Strains chosen and preparation of inoculum 6.1. Nomenclature and description of M. bovis isolates 6.1.1. M. bovis AF2122/97 6.1.2. M. bovis VNTR0024(SB0130) 6.1.3. M. bovis VNTR001(SB0140) 6.2. Preparation of inoculum 7. Methods 8. Results and discussion 8.1. Evaluation of M. bovis selective media for use with cheese 8.2. Cheddar cheese manufacture 8.3. Caerphilly cheese manufacture 8.4. Statistical analysis of high-inoculum Cheddar and Caerphilly 8.5. Low-inoculum cheese manufacture 8.6. Statistical analysis of low- inoculum Cheddar and Caerphilly 9. Overall discussion and conclusion 10. References Appendix 1 1.1 Method: preparation of media. 1.2 Method: preparation of inoculated Cheddar cheese. 1.3 Method: preparation of inoculated Caerphilly cheese. 1.4 Method: enumeration of Mycobacterium bovis from cheese. Appendix 2 Statistical analysis of selective media: Cheddar and Caerphilly homogenates. Appendix 3 Statistical analysis of selective media: Cheddar M. bovis AF2122 homogenates. Appendix 4 Inactivation curves generated by extended sampling of Caerphilly: up to eight months. Appendix 5 Statistical analysis of high- and low-inoculum Cheddar and Caerphilly. Appendix 6 Statistical analysis of high- and low-inoculum Cheddar and Caerphilly: Fisher’s LSD test. Appendix 7 7.1 Poster: European Society of Mycobacteriology summer conference ESM 2008. 7.2 Poster: European symposium of the International Association of Food Protection IAFP 2009. 7.3 Poster: Society for Applied Microbiology summer conference SFAM 2011. Page 4 of 298 EXCECUTIVE SUMMARY WORK ON VTEC Verocytotoxin-producing E. coli (VTEC) is a human pathogen which has occasionally been reported associated with raw milk and the cheese made from it. In the UK though especially, this is a rare event. Elsewhere in the world, a number of serotypes of VTEC including E. coli O157:H7 and E. coli O26 have been found associated with these foodstuffs. Previously reported work has shown that VTEC strains can survive the various stages of the cheesemaking process and that raw milk and raw milk cheeses are a potential vehicle for VTEC infections. Aims The aim of the project was to investigate the behaviour of relevant VTEC serotypes (E. coli O157:H7 and E. coli O26) through the manufacture of raw milk cheeses as made in the UK. From the information gleaned, authoritative advice can be issued on the risk associated with these hazards should cheeses be found to be contaminated. Objectives As far as VTEC work was concerned, the objectives of the project were firstly to carry out a literature review of relevant aspects including: information on VTEC in relation to cheese (outbreaks and methodology); and the microbiology of raw milk and starter cultures. A second objective was to set up a Steering Group of interested parties to advise on the work carried out in the project. The Steering Group would need to meet as required, but at least every 6 months of the project duration. In order to inform work on the project, it was necessary to determine the range of raw milk cheeses made in the UK, and their characteristics including: pH; salt content; water activity; background microflora; starter cultures used; maturation conditions and duration. It was felt cost-effective to use the write-up of project B12006 (Banks 2006) for this. The next objective was to secure equipment and devise protocols for the production of cheeses under laboratory conditions. As part of this objective it was necessary to develop a protocol for the inoculation of the Hazard Group 3 bacterium E. coli O157:H7 and other VTEC. Once VTECs had been inoculated into the cheeses made, the next objective was to identify the most appropriate methods to be used for isolation and enumeration of these pathogens. The intention was that once these methods had been identified, they could be used to detect VTEC in uninoculated raw milk cheeses also. A crucial part of the experimental work was to ensure that the cheese being made in the laboratory had the same characteristics as cheeses being made commercially. To this end, the microbiological content, as well as the pH, salt content and water activity of the cheese used in the inoculation studies were determined. Once the practical work had been completed, the final objectives of the project were: to produce a final report with conclusions on the survivability of VTEC in different cheese- making processes; to produce peer-reviewed publications on the work; and to hold a dissemination event such that the findings of the project could be discussed by interested parties. Page 5 of 298 Approaches A large range of cheese types are made in the UK from pasteurised and unpasteurised milks. It was decided in collaboration with the project Steering Group, that Caerphilly and Camembert type cheeses were the most appropriate for study with VTEC. These are semi- hard and soft cheeses respectively. Caerphilly cheese was chosen because there have been a number of outbreaks of VTEC illness associated with this cheese type (Baylis 2009). It was also decided that the team at QUB could work with this cheese type to look at the behaviour of M. bovis. With both teams looking at the behaviour of their respective target microorganisms in the same cheese type, it would be possible for some direct comparisons to be made between the two pathogens being studied. The first challenge for the project team was to learn how to make cheese as would be done commercially. To this end, a technician from Campden BRI attended a cheesemaking course at Reaseheath College. A 10L vat was purchased and modified so that it could be fitted into a safety cabinet in a containment level 3 microbiology laboratory. (VTEC require handling in a containment level 3 laboratory.) The cheesemaking technique was then modified from a commercial scale to the laboratory scale, but such that cheese with the appropriate attributes (pH, salt, etc.) was produced. A number of VTEC isolates (O157 and O26) were obtained from various sources. These included isolates from dairy foods and some were outbreak strains. Three of these strains were chosen for inoculation work in the project. The next challenge was to identify/develop a method which was most suitable for the detection and isolation of VTEC in fatty foods such as milk and cheese. The three chosen VTEC strains were inoculated in turn into milk from which Caerphilly cheeses were made. The milk was purchased from a local farm. The chemical characteristics and microbiological content of the milk and the cheese was measured. The behaviour of the VTEC strains during the cheesemaking process and maturation (for up to 5 months) of the cheeses was monitored. An interpretation was then made on how these findings could be used to Key findings Unfortunately, due to technical and project constraints, it was only possible to work on semi- hard (Caerphilly) cheese in this project.