Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Occurrence of Staphylococcus aureus in a commercial poultry plant and poultry farm A Thesis submitted in partial fulfilment of the requirement for the degree of Master of Food Technology Massey University Albany, New Zealand Cheng QIAN April, 2015 Cheng QIAN ABSTRACT I ABSTRACT Poultry products are popular due to their healthier image compared to red meats. However, the products are susceptible to contamination by many spoilage microorganisms and pathogens, including Staphylococcus aureus, Campylobacter spp., Clostridium perfringens, Yesinia enterocolitica, Pseudomonas spp. and Escherichia coli. In New Zealand (NZ), foodborne outbreaks caused by S. aureus infections may be uncommon but serious. S. aureus can grow in a wide range of pH, temperature and salt concentrations. Some strains of S. aureus can produce heat-resistant enterotoxins, while others may be methicillin-resistant which can result in hospital-linked and community-linked infections. Raw (fresh) and frozen poultry products have been associated with S. aureus contamination in many countries. The common contamination sources of S. aureus in poultry products have been linked to poor hygiene of food handlers, processing equipment and skins of live chickens. The aim of this project was to identify potential contamination sources of products and processing equipment by S. aureus from a selected processing plant to the farm in Auckland, New Zealand. Poultry meat samples were collected from Final Products, Frozen Mechanically Separated Meat (MSM), Frozen Skin, Frozen Skin-on Breast Fillet (SO BF) (further processing plant), Fresh MSM, Fresh Skin, Fresh SO BF (secondary processing plant). Swab samples were collected from the MSM conveyor, inside the Mechanically Deboning Machine (MDM), the Skinner Conveyor (secondary processing plant), Rubber Fingers in Pluckers (primary processing plant), skin and nostrils of live chicken at the farm. Viable cell counts of S. aureus were enumerated using Petrifilm™ Staph Express Count Plate to determine the contamination level of the samples. Isolates of S. aureus was confirmed by Gram-stain and coagulase-positive test. Six main sampling sites were selected for further investigation which comprised final products, Fresh MSM, Fresh Skin, Fresh SO BF, Rubber Fingers and live chickens. Ten representative S. aureus isolates grown on Petrifilms were randomly selected from samples of each of the six main sampling sites. Polymerase Chain Reaction (PCR) and Multilocus Sequence Typing (MLST) were then used to detect the presence of staphylococcal enterotoxins and identify sequence types of the sixty S. aureus isolates, respectively. eBURST was used to identify the relatedness of the sequence types. Also, the contamination sources of S. aureus in the samples were traced based on the sequence types of the sixty isolates. In the further processing plant, all final product samples (n=36) were contaminated with S. aureus. Frozen MSM had the highest contamination level ranging from 2.00±1.02 to 2.50±0.48 Log10 CFU/g. Similarly, S. aureus in Fresh MSM from the secondary processing plant contained the highest S. aureus cell counts (1.79±0.25 to 2.85±0.51 Log10 CFU/g), followed by Fresh SO BF (1.85±0.56 to 2.33±0.50 Log10 CFU/g) and Fresh Skin (1.72±0.60 to 2.15 [1.67, 3.37] Log10 CFU/g). In primary processing, Rubber Fingers in Plucker 1 had the highest level of S. aureus (2.46±0.50 Log10 CFU/swab). S. aureus counts of chicken skin ranged from 1.00 [0.79, 1.48] to 1.36±0.45 Log10 CFU/swab, while nostrils contained 1.00 [0.85, 1.48] to 1.59±0.70 Log10 CFU/swab. Cell counts of live chicken increased with the age (first, third, sixth week) of the chicken. Eight different types of enterotoxin genes (seg, sei, seh, sek, sel, sem, sen, seo) were identified. Of the 60 S. aureus isolates, 59 were positive for at least two different staphylococcal enterotoxins. Six different sequence types were identified (ST5, ST2594, ST101, ST83, ST398, ST1). Sequence types of isolates that had at least five identical loci were assigned to a single clonal complex (CC). In this study, ST5, ST83 and ST2594 belonged to CC 5 with ST5 being the clonal ancestor. MSM had the highest S. aureus contamination level due to cross-contamination inside the MDM, therefore, a proper hygiene and regular cleaning routine inside the MDM is recommended. The results suggested that the sources of S. aureus contamination in the final poultry products could be Fresh MSM, Fresh Skin, Fresh SO BF (secondary processing), Rubber Fingers in the Pluckers (primary processing) and live chickens at the farm. Chicken skin from live chickens at farm was most likely the origin of contamination of final products and equipment by S. aureus. Since not all the identified strains that colonised on the live chickens were traced back to the final products, further investigations on other potential contamination sources such as gloves and knifes used at the processing plant, feeders and drinkers at the farm are recommended. Cheng QIAN ACKNOWLEDGEMENTS II ACKNOWLEDGEMENTS I would like to thank Tegel Foods Limited for funding the research project. I also would like to thank Roy Biggs, Food Safety and Quality Assurance Manager at Tegel Foods Limited, for offering me such a wonderful opportunity to work on this project. During the course of the project, Roy shared his knowledge in food safety. I also thank Maria Norman, Compliance Coordinator, Tegel Foods Limited and the entire Quality Assurance Team for their support. At Massey University, I would like to convey my sincere thanks to my supervisors Dr Tony Mutukumira and Dr Evelyn Sattlegger for their guidance and passion in the subject matter, thank you for bearing with me in course of my research work. Further, I thank Rachel Liu, Jarod Young and PC Tong for technical support. I also acknowledge Professor Luís Augusto Nero, School of Veterinary Sciences, Universidade Federal de Viçosa, Brazil, Professor Iris Spiliopoulou, Faculty of Medicine, University of Patras, Greece, and BEI Resources, Manassas, Virginia, USA, for providing the reference strains of S. aureus. Cheng QIAN TABLE OF CONTENTS III TABLE OF CONTENTS Abstract.....................................................................................................................................I Acknowledgements..................................................................................................................II List of Tables………………………………………………………………………………VIII List of Figures………………………………………………………………………………..X Abbreviations………………………………………………………………………..…….XIII 1. Introduction .......................................................................................................................... 1 2. Literature Review ................................................................................................................ 5 2.1 Poultry meat .................................................................................................................................. 5 2.2 Poultry processing procedures ...................................................................................................... 6 2.2.1 Slaughtering ........................................................................................................................... 7 2.2.2 Scalding .................................................................................................................................. 8 2.2.3 Defeathering ........................................................................................................................... 9 2.2.4 Evisceration .......................................................................................................................... 10 2.2.5 Chilling ................................................................................................................................ 11 2.2.6 Packaging ............................................................................................................................. 12 2.3 Contamination of raw chicken and chicken products ................................................................. 13 2.3.1 Microbiological contamination ............................................................................................ 13 2.3.2 Contamination sources ......................................................................................................... 15 2.3.3 Decontamination of poultry products .................................................................................. 16 2.4 Characterisation of Staphylococcus aureus ............................................................................... 17 2.4.1 Staphylococcus aureus ......................................................................................................... 17 2.4.1.1 Methicillin-resistant Staphylococcus aureus (MRSA) ....................................... 19 2.4.2 Outbreaks of Staphylococcus aureus ................................................................................... 20 Cheng QIAN TABLE OF CONTENTS IV 2.4.3 Disease and symptom........................................................................................................... 21 2.4.4 Staphylococcal enterotoxins (SEs) ....................................................................................... 21 2.4.4.1 Inactivation of staphylococcal enterotoxins ..................................................... 23 2.5 Isolation of Staphylococcus aureus ............................................................................................. 24 2.5.1 Confirmation tests of suspect S. aureus isolates ................................................................. 25 2.6 Detection of staphylococcal enterotoxins (SEs) ......................................................................... 27 2.6.1 Immunological methods ....................................................................................................... 28 2.6.2 Molecular biology methods ................................................................................................. 29 2.7 Identification the strain of S. aureus ........................................................................................... 31 2.8 Critical sampling locations in poultry processing plants ............................................................ 34 2.8.1 Industrial environment ......................................................................................................... 34 2.8.2 Farm environment ................................................................................................................ 35 2.9 Sampling methods of poultry meat ............................................................................................. 35 3. Materials and Methods ...................................................................................................... 37 3.1 Identification of key processing steps in the plant ...................................................................... 37 3.1.1 Industral level ....................................................................................................................... 37 3.1.2 Farm level ............................................................................................................................ 39 3.2 Collection of swab samples ........................................................................................................ 39 3.2.1 Processing environment ....................................................................................................... 39 3.2.2 Collection of samples at farm level ...................................................................................... 39 3.3 Sampling ..................................................................................................................................... 40 3.3.1 Fresh samples of chicken meat ............................................................................................ 40 3.3.2 Frozen chicken meat ............................................................................................................ 40 3.3.3 Collection of swab samples from processing plant equipment ............................................ 40 3.3.4 Collection of swab samples from live chicken at farm level ............................................... 42 3.4 Enumeration of S. aureus ............................................................................................................ 42 3.4.1 Preparation of dilutions for meat and swab samples ............................................................ 43 Cheng QIAN TABLE OF CONTENTS V 3.4.2 Plating of samples on Petrifilms .......................................................................................... 44 3.5 Confirmation tests for S. aureus isolates .................................................................................... 45 3.5.1 Gram-staining ...................................................................................................................... 46 3.5.2 Coagulate test ....................................................................................................................... 47 3.6 Characterisation of isolates of S. aureus ..................................................................................... 49 3.7 DNA extraction ........................................................................................................................... 50 3.7.1 Recovery of S. aureus isolates ............................................................................................. 50 3.7.2 DNA extraction from S. aureus isolates .............................................................................. 51 3.7.3 Determination of the concentrations of extracted DNA ...................................................... 54 3.8 Identification of enterotoxigenic S. aureus isolates .................................................................... 54 3.8.1 Multiplex PCR protocol ....................................................................................................... 54 3.8.1.1 Preparation of primers ....................................................................................... 54 3.8.1.2 Multiplex PCR reactions .................................................................................... 58 3.8.1.3 Electrophoresis ................................................................................................... 60 3.9 Multilocus sequence typing (MLST) of S. aureus isolates ......................................................... 61 3.9.1 PCR protocol for MLST ...................................................................................................... 61 3.9.1.1 Preparation of primers ....................................................................................... 61 3.9.1.2 PCR reactions .................................................................................................... 62 3.9.1.3 Electrophoresis ................................................................................................... 63 3.9.2 Purification of amplified PCR products ............................................................................... 63 3.9.3 Determination the concentrations of purified PCR products ............................................... 65 3.9.4 Sequencing of the seven house-keeping loci of each S. aureus isolate ................................ 65 4. Statistical analysis .............................................................................................................. 67 5. Results and Discussion ....................................................................................................... 68 5.1 Contamination of S. aureus in further poultry processing plant ................................................. 68 5.1.1 Contamination of S. aureus in final processed products ...................................................... 68 5.1.2 Contamination of S. aureus in three frozen chicken meat ingredients ................................. 70 5.2 Contamination of S. aureus in products in the secondary plant .................................................. 71 Cheng QIAN TABLE OF CONTENTS VI 5.2.1 Fresh MSM (Mechanically Separated Meat) processing ..................................................... 71 5.2.2 Detaching skin processing ................................................................................................... 78 5.2.3 Fresh Skin-On Breast Fillet (SO BF) ................................................................................... 81 5.3 Contamination of S. aureus on Rubber Fingers in the Pluckers ................................................ 82 5.4 Contamination of S. aureus in Nostril and Skin of live chickens at the farm ............................ 84 5.5 Identification of main sampling sites ......................................................................................... 87 5.6 Detection of enterotoxigenic S. aureus isolates ......................................................................... 88 5.7 Multilocus Sequence Typing (MLST) of S. aureus isolates ..................................................... 91 5.8 Clonal complexes of the six sequence types (STs) ..................................................................... 93 5.9 Potential contamination sources of S. aureus ............................................................................ 94 6. Conclusion .......................................................................................................................... 97 Limitations and Recommendations ...................................................................................... 98 References ............................................................................................................................... 99 Appendix .......................................................................................................................................... 115 Appendix A. Preparation of reagents and media ....................................................................... 115 Appendix B. Calculations of primers for enterotoxin detection and MLST .............................. 117 Appendix C. Concentrations of extracted DNA and purified PCR products ............................. 119 Appendix D. Gram-staining and coagulation results of isolated S. aureus colonies ................. 124 Appendix E. Raw data of S. aureus isolation............................................................................. 125 Appendix F. PCR results of staphylococcal enterotoxin detection ............................................ 135 Appendix G. Results of PCR and purified PCR products in terms of MLST of each S. aureus isolates......................................................................................................................................... 140 Appendix H. Sequencing results of the seven house-keeping genes of each S. aureus isolates . 159 Appendix I. Summary of MLST results ..................................................................................... 300 Appendix J. eBURST analysis outputs ....................................................................................... 302 Appendix K. Statistical outputs .................................................................................................. 303 A. Final Products ......................................................................................................... 303 Cheng QIAN TABLE OF CONTENTS VII B. Frozen MSM ............................................................................................................ 308 C. Frozen Skin .............................................................................................................. 312 D. Frozen SO BF .......................................................................................................... 316 E. Fresh MSM .............................................................................................................. 320 F. MDM Conveyor ........................................................................................................ 329 G. MSM carcass ........................................................................................................... 337 H. Inside MDM ............................................................................................................ 344 I. Fresh Skin ................................................................................................................ 348 J. Skinner Conveyor: .................................................................................................... 356 K. Fresh SO BF ............................................................................................................ 365 L. Rubber Fingers ........................................................................................................ 374 M. Nostrils of live chicken: .......................................................................................... 379 N. Skin of live chickens................................................................................................. 384 Cheng QIAN LIST OF TABLES VIII LIST OF TABLES Table 2.1 Dominant nutrient data of chicken breast under different cooking methods in some countries.………………………………………………………………………………………6 Table 2.2 Examples of chemical and physical decontamination methods for poultry meat…17 Table 2.6 Comparison of ELISA-B, SET-RPLA, ELISA-M and ELISA-T kits for the detection of staphylococcal enterotoxins.……………………………………........................28 Table 3.6 The relationship between the codes of S. aureus isolates and source of samples…50 Table 3.8.1 Primer information of enterotoxin genes and expected PCR product size…..….56 Table 3.8.2 Basic information of the reference strains.……………………………………....60 Table 3.9.1 Primer sequences and products size (bp)………………………………………..62 Table 5.1.1 Mean S. aureus counts (Log10 CFU/g) and samples (%) above the standard level (ASL) in each batch of final products……………………….……………………………….70 Table 5.1.2 Mean S. aureus counts (Log10 CFU/g) and samples (%) above the standard level (ASL) in each batch of three frozen ingredients……………………………………………..71 Table 5.2.1 Distribution of S. aureus (Log10 CFU/swab) enumerated from samples of MSM processing.………………………………………………………………………………...….72 Table 5.2.2 Distribution of S. aureus (Log10 CFU/g) enumerated from the detaching skin processing steps……………………………………………………………………………....79 Table 5.2.3 Viable cell counts (Log10 CFU/g) and samples (%) above the standard level (ASL) of S. aureus enumerated from Fresh Skin-On Breast Fillet……..…………………………...82 Table 5.3.1 Viable counts (Log 10 CFU/swab) and samples (%) above the standard level (ASL) of S. aureus enumerated from Rubber Fingers on the three Pluckers….…………………….83 Table 5.4.1 Viable counts and samples (%) above the standard level (ASL) of S. aureus enumerated from the nostrils of live chickens at three different ages……………….……….86 Table 5.6 Distribution of enterotoxigenic genotypes S. aureus isolates.……………...…..…90 Cheng QIAN LIST OF TABLES IX Table 5.7.1 MLST typing of the sixty S. aureus isolates.………………………….….….….92 Table 5.7.2 Sequence types and enterotoxin types of each S. aureus isolate…………….…..93 Table 5.8 eBURST analysis of clonal complex 5 (CC5) .………………………….…….….93 Cheng QIAN LIST OF FIGURES X LIST OF FIGURES Figure 2.1 Schematic processing of poultry………………………..…………………….…...7 Figure 2.2.1 Different effects of traditional scalding and multi-stage scalding methods on controlling of viable cells of microorganisms……………………………….……….….…....9 Figure 2.2.2 Generalised process of opening chicken carcasses……………………………..11 Figure 2.3 Occurrence of microbial counts in retail chicken parts and processed chicken products in Spain.…………………………………………………………………………….14 Figure 2.4.1 Electron micrograph of S. aureus……………………………………………...18 Figure 2.4.2 Structures of some staphylococcus enterotoxins…..………….………………..22 Figure 2.5.1 Schematic procedures of enumeration and confirmation (coagulase test) of S. aureus using plate count method APHA 2001……………………………………………….26 Figure 2.5.2 Schematic procedures of enumeration and confirmation (coagulase test) of S. aureus using plate count method APHA 2001……………………………………………….27 Figure 2.6 Preparation of food samples for PCR analysis….………………………….…….31 Figure 2.7 Overview of procedures for MLST method………………………..…………….33 Figure 3.1.1 Hierarchical relationships of sampling sites in the poultry plant. Arrows indicate the route followed to collect Final Products...………………………………………………..38 Figure 3.3.1 Procedure for collection of swab samples from conveyors on skinner machine, MDM conveyor, inside MDM equipment and Rubber Fingers in Pluckers..……….……….41 Figure 3.4.1 Isolation procedure of S. aureus from chicken meat and swab samples using 3M™ Petrifilm™ Staph Express Count Plate Method………………………………………43 Figure 3.4.2 Petrifilm with typical red-violet colonies after 24±2 h incubation at 37°C…….45 Figure 3.4.3 Incubated colonies with typical pink zones on the Petrifilm after inserting an Express disk…………………………………………………………………………………..45 Cheng QIAN LIST OF FIGURES XI Figure 3.5.1 Typical appearance of Gram-stained S. aureus when examined under ×100 oil- immersion lens……………………………………………………………………………….47 Figure 3.5.2 Typical results of coagulate test for confirming suspect S. aureus colonies: (a) negative, (b) weak positive, (c) positive…………………………………………………..…48 Figure 3.6 Typical selection S. aureus isolates from three samples at one sampling location…………………...…………….…………………………………………………….50 Figure 3.7.2 DNA extraction procedure……………………………………………………...53 Figure 3.8.1 Multiplex PCR results of S. aureus reference strains for each PCR set. ………………………………………………………………………………………...…59 Figure 3.9.1 Amplification results of the seven house-keeping genes in RN4220 (positive control). ………………..…………………………………………………………………….63 Figure 3.9.2 PCR products purification procedure…………………………………………..64 Figure 3.9.3 A prepared 96-well PCR plate for sequencing.………………………………...66 Figure 5.1.1 Mean S. aureus counts (Log10 CFU/g) in final products processed for six different batches..………………………….………………………...……………………….69 Figure 5.1.2 Stack-plot of S. aureus counts (Log10 CFU/g) of three different batches of frozen ingredients…………………………………………………………....………………………70 Figure 5.2.1 Stack-plot of S. aureus counts (Log10 CFU/g, swab) during MSM processing steps…...…………………………………………………………………………………...…72 Figure 5.2.2 Mean S. aureus counts (Log10 CFU/g) of Fresh MSM samples during 6-h of processing n=3 batches and a standard level set by the processing plant…………..………..73 Figure 5.2.3 Mean S. aureus counts (Log10 CFU/swab) of MDM Conveyor during 6-h of processing for three batches and a standard level set by the processing plant...………….…74 Figure 5.2.4 Mean S. aureus counts (Log10 CFU/swab) of Carcass during 6-h of processing for three batches and a standard level set by the processing plant....………………………..75 Cheng QIAN LIST OF FIGURES XII Figure 5.2.5 Mean S. aureus counts (Log10 CFU/swab) inside the MDM during 6-h of processing for three batches and a standard level set by the processing plant.………………76 Figure 5.2.6 Stack-plot of S. aureus counts (Log10 CFU/g, swab) during Skin processing steps for three different batches…………...……………………………………..…………………78 Figure 5.2.7 Mean S. aureus counts (Log10 CFU/g) of Fresh Skin during 6-h of processing for three batches and a standard level set by the processing plant.………………………………79 Figure 5.2.8 Mean S. aureus counts (Log10 CFU/swab) of Skinner Conveyor during 6-h of processing for three batches and a standard level set by the processing plant..…...………....80 Figure 5.2.9 Mean S. aureus counts (Log10 CFU/g) of Fresh Skin-On Breast Fillet during 6-h processing of three different batches and a standard level set by the processing plant.……..81 Figure 5.3.1 Stack-plot of S. aureus counts (Log 10 CFU/swab) of Rubber Fingers on three Pluckers…………………………………………...………………………………………….83 Figure 5.4.1 Viable log cell counts (Log 10 CFU/swab) of S. aureus isolated from the nostrils of live chickens at one week, three weeks and six weeks old and a standard level set by the processing plant………………………………………………………………………………84 Figure 5.4.2 Viable log cell counts (Log10 CFU/swab) of S. aureus enumerated from the skin of live chickens at one week, three weeks and six weeks and a standard level set by the processing plant………………………………………………....……………………………85 Figure 5.5 Main sampling sites in the processing plant (highlighted in red) from further processing to primary processing.……………………………………………………………88 Figure 5.8 Identificatioan of clonal complexes of the S. aureus isolates. Each dot represents a sequence type. ST83, ST5 and ST2594 were clonal complex (cc) 5 with ST5 being the clonal ancestor……………………………………………....………………………………….……94 Figure 5.9 Sequence types of the sixty S. aureus isolates between each source of samples…………………………………………………………………………………….....95 Cheng QIAN LIST OF ABBREVIATIONS XIII LIST OF ABBREVIATIONS AOAC = Association of Official Analytical Chemists ATCC = American Type Culture Collection BFG = Bovine Fibrinogen Agar BHI = Brain Heart Infusion BLAST = Basic Local Alignment Search Tool BPA = Baird-Parker Agar CC = Clonal Complex CCPs = Critical Control Points ClO2 = Chlorine Dioxide DNA = Deoxyribonucleic Acid eBURST = Based Upon Repeat Sequence Types EDTA = Ethylenediaminetetraacetic Acid ELISA = Enzyme-linked Immuno-sorbent Assay ELISA-M = Enzyme-linked Immuno-sorbent Assay Membrane ELISA-T = Enzyme-linked Immuno-sorbent Assay Tube ESR = Environmental Science and Research FSMS = Food Safety Management System HACCP = Hazard Analysis and Critical Control Points HOCI = Hypochlorite ISO = International Organization for Standardization MAP = Modified Atmosphere Packaging MAS = Microbial Assessment Scheme MDCM = Mechanically Deboned Chicken Meat MDM = Mechanically Deboning Machine MRSA = Methicillin-resistant Staphylococcus aureus MSA = Mannitol Salt Agar MSM = Mechanically Separated Meat NaCl = Sodium Chloride PSE = Pale Soft Exudative PSE = Petrifilm Rapid Staph Express Count Cheng QIAN LIST OF ABBREVIATIONS XIV PCR = Polymerase Chain Reaction MLST = Mutillocus Sequencing Typing MPN = Most Probable Number RPFA = Rabbit Plasma Fibrinogen Agar RPLA = Reverse Phase latex agglutination SEs = Staphylococcal Enterotoxins SEA (sea) = Staphylococcal Enterotoxin A SEB(seb) = Staphylococcal Enterotoxin B SEC(sec) = Staphylococcal Enterotoxin C SED(sed) = Staphylococcal Enterotoxin D SEE(see) = Staphylococcal Enterotoxin E SEG(seg) = Staphylococcal Enterotoxin G SEH(seh) = Staphylococcal Enterotoxin H SEI(sei) = Staphylococcal Enterotoxin I SEJ(sej) = Staphylococcal Enterotoxin J SEK(sek) = Staphylococcal Enterotoxin K SEL(sel) = Staphylococcal Enterotoxin L SEM(sem) = Staphylococcal Enterotoxin M SEN(sen) = Staphylococcal Enterotoxin N SEO(seo) = Staphylococcal Enterotoxin O SEP(seo) = Staphylococcal Enterotoxin P SEQ(seq) = Staphylococcal Enterotoxin Q SER(ser) = Staphylococcal Enterotoxin R SET-EIA = Staphylococcal Enterotoxin Enzyme-linked Immuno-sorbent Assay SET-RPLA = Staphylococcal Enterotoxin Reverse Phase Latex Agglutination SEU(seu) = Staphylococcal Enterotoxin U SFP = Staphylococcal Food Poisoning SLVs = Single-locus variants ST = Sequencing Type UK = United Kingdom USA = United States UV = Ultraviolet Cheng QIAN INTRODUCTION 1 1. Introduction The origin of domesticated chickens dates back to thousands of years ago in Southeast Asia (Daghir, 2008; West & Zhou, 1988). Domestication of chickens is a process which humans raise chickens aimed to enhance desired traits, nowadays mostly for commercial trade due to huge demand of poultry meat products (Wiren et al., 2009). Poultry products, such as eggs, fresh chicken and roasted turkey, have become sources of daily meals worldwide (Davies & Board, 1998). In 2007, eggs and chickens processed products were produced by about 145,615 farms in America (EPA, 2013). In New Zealand, poultry production increased from 111,884 to 173,263 tonnes between 2000 and 2012. Chicken products remain the most favourite meat-based meals in New Zealand (PIANZ, 2010). Chicken products are susceptible to contamination by many pathogens or spoilage microorganisms. Farmers raise chickens on litter floors which have higher risks of being contaminated with Enterobacteriaceae, such as Salmonella, Escherichia coli and Yesinia enterocolitica (Davies & Board, 1998). Waldroup (1996) reported the incidence of common pathogens on raw poultry products including Campylobacter spp., Clostridium perfringens, Staphylococcus aureus, Yesinia enterocolitica and Pseudomonas. Campylobacter, Salmonella, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus are human pathogens which means that the contaminations are mainly attributed to poor food handling or cross- contamination during food processing (Davies & Board, 1998). The obsence of S. aureus is an indicator of good hygiene and correct handling practice (Jacxsens et al., 2011) S. aureus strains are Gram-positive cocci and facultative anaerobes (Roberts & Greenwood, 2002). They however, grow better in the presence of adequate oxygen than anaerobic environment (Ministry of Primary Industry, 2001). S. aureus can grow in a wide range of pH (4.2 to 9.3), temperature (7°C to 48.5°C) and salt concentrations up to 15% (Bhatia & Zahoor, 2007; Kérouanton et al., 2007). The optimum temperature for their growth is 37°C. Low water activity is not an inhibiting factor for S. aureus to grow and produce staphylococcal Cheng QIAN INTRODUCTION 2 enterotoxins (SEs) (Ministry of Primary Industry, 2001). Raw (fresh) and frozen poultry have been associated with S. aureus in many countries. In Japan, about 66% of raw chicken meats were contaminated with S. aureus in 2004 (Kitai, Shimizu, Kawano, Sato, Nakano, Kitagawa, et al., 2005). It grows well in processed meat as some normal organisms which may be competitive to S. aureus are killed or inhibited during processing (Ministry of Primary Industry, 2001). Some strains of S. aureus have the ability to produce one or more staphylococcal enterotoxins (SEs), which are the main causes of foodborne illness (Lee, 2006; Matyi et al., 2013; Notermans et al., 1982). Many food poisoning issues caused by consuming contaminated raw poultry meat or poultry products are attributed to SEs (Kérouanton et al., 2007). S. aureus can synthesise SEs at temperature between 10 to 45°C, at pH of 4.8 to 9.0, under a water activity ranging from 0.86 to 0.99 (Ministry of Primary Industry, 2001). There are various SEs that have been reported most of which are stable even under a heat treatment of routine sterilisation in commercial processing (Balaban & Rasooly, 2000; Bhatia & Zahoor, 2007). Also, there has been a great concern about methicillin-resistant S. aureus (MRSA) all over the world as it can cause not only hospital-linked infections but also community-acquired infections (De Boer et al., 2009; Kitai, Shimizu, Kawano, Sato, Nakano, Uji, et al., 2005). MRSA is regarded as the most prevalent nosocomial pathogen which is isolated from human (Lee, 2003; Melter et al., 1999). Animals can be carriers of this pathogen as well. The presence of MRSA strains in some food products has been detected recently, such as meat products including raw chicken meat, turkey, lamb, pig meat (De Boer et al., 2009; Kitai, Shimizu, Kawano, Sato, Nakano, Uji, et al., 2005; Kwon et al., 2006) and dairy products (Normanno et al., 2007). Some isolated MRSA from raw meat were reported to be resistant to some antibiotics (Pesavento et al., 2007), such as aminoglycosides, macrolides, chloramphenicol and tetracycline (Mandel et al., 2000). Therefore, MRSA is considered as a potential causative agent that is responsible for staphylococcal food safety problems. In New Zealand, about 7% of S. aureus isolates were methicillin-resistant (Ministry of Health, 2002). Cheng QIAN INTRODUCTION 3 Food safety issues can result in huge economic losses for the poultry manufacturer as well as issues of affecting their well-developed reputations (Matyi et al., 2013). According to Australia New Zealand Food Standards 1.6.1, the microbiological limit of coagulase-positive Staphylococcus is log 3 CFU/g (ComLaw, 2012). In Australia, 22 people were reported ill due to Staphylococcal food poisoning (SFP) at a buffet in June, 2012 and 20 of them had eaten chicken stir-fry (Pillsbury et al., 2013). New Zealand is one of the developed countries with high reports of the incidence rate for S. aureus infections (Williamson et al., 2014). Between 1989 to 1999, 24 outbreaks of staphylococcal foodborne illness were attributed to the consumption of contaminated yogurt, hot ham sandwiches, chicken salad, hot turkey sandwiches in NZ, causing totally 1762 food poisoning cases (Ministry of Primary Industry, 2001). From April to June, 2014, there was one SFP outbreak comprising 4 cases following the consumption of contaminated food in a restaurant in Taranaki area of Northland in New Zealand (ESR, 2014). It was believed that the sources of contamination by S. aureus were mainly from food workers by direct or indirect contact, live chicken and processing equipment (Davies & Board, 1998; Hayes, 1992; Montville & Matthews, 2008). Controlling cooling temperature and storage temperature can prevent the propagation of S. aureus in final products (Ministry of Primary Industry, 2001; Pillsbury et al., 2013). Therefore, food handlers must be adequately trained in food safety skills and good food handling hygiene. The processing equipment in poultry plants must be maintained and cleaned properly (ComLaw, 2010). With the popularity of poultry products and the incidences of staphylococcal contaminations, it is critical to ensure microbial safety of the products, food processing environments and equipment (Lindblad et al., 2006). To investigate the contamination of S. aureus in poultry processing plants, it is essential to use reliable analytical methods to identify the enterotoxigenic and strains of S. aureus isolates to determine the potential contamination source (Gibbs, Patterson, & Thompson, 1978; Jacxsens et al., 2009; Jacxsens et al., 2011). The aim of this project was to identify potential contamination sources of poultry products and processing equipment by S. aureus from a selected processing plant to the farm in Auckland, New Zealand. The specific objectives were to: Cheng QIAN INTRODUCTION 4 determine the level of contamination of poultry meat and equipment by S. aureus at selected processing steps in a commercial poultry plant; determine the level of contamination of chicken skins and nostrils of live chickens by S. aureus at a supplying poultry farm; identify the main sampling sites; isolate S. aureus from samples of each of the main sampling sites; determine the presence of staphylococcal enterotoxins (SEs) from S. aureus isolated from main sampling sites using multiplex Polymerase Chain Reaction (PCR) determine the sequence type of the isolated S. aureus using the Multilocus Sequencing Typing (MLST) method identify the clonal complexes of the sequence types using eBURST Cheng QIAN LITERATURE REVIEW 5 2. Literature Review 2.1 Poultry meat Poultry meat had been increasingly welcomed by consumers in America from years ago as its unique properties, such as the consistent composition and a mild flavour. During the processing, the muscle of poultry meat is changed chemically, physically and structurally which determines the overall quality of final processed products (Sams, 2000). Poultry meat quality attributes include colour, aroma, flavour, texture, and microbial levels, however, colour and texture are considered to be the most common indicator of quality attributes for poultry meat which are related to the selection for both raw poultry meat and processed poultry products by manufacturers and consumers (Boylston et al., 2012; Fletcher, 2002). Pre-slaughter of live chickens, processing conditions and microbiological controlling can affect the quality of processed poultry products (Hui et al., 2010; Sams, 2000). The dominant nutrient data of chicken breast with different cooking methods in many countries was analysed by Probst (2008) (Table 2.1). Cheng QIAN LITERATURE REVIEW 6 Table 2.1 Dominant nutrient data of chicken breast under different cooking methods in some countries. Country Australia Australia Finland Australia USA NZ Australia Cooking method Baked Casseroled Raw Raw Raw Raw Stir-fried Water (g) 67.600 66.700 - 75.000 74.760 75.500 68.600 Energy (KJ) 637.000 596.000 617.000 438.000 460.000 453.000 520.000 Total fat (g) 3.900 3.700 6.800 1.600 1.240 2.130 0.900 Total protein(g) 29.000 27.000 21.500 22.300 23.090 22.300 28.600 Ash (g) 1.200 1.000 - 1.100 1.020 - 1.200 Source: (Probst, 2008). (-) indicates that no data was available from the source. 2.2 Poultry processing procedures From 1950 to 1960, manual operations poultry processing was changed into automatic and mechanical operations which lower the risk in cross-contamination of various microorganisms from human handling (Davies & Board, 1998; Tsola et al., 2008). Although the modernisation and standardisation of processing flow may lead to cross- contamination from the surface of equipment, the total loads of bacteria of end- products are less than that of old traditional manufacturing processing (Fuster-Valls et al., 2008; Tsola et al., 2008). Also, modern processing of poultry meat is much more efficient, slaughtering at least 5000 carcasses per hour (Notermans et al., 1982). The main processing procedures of poultry meat consist of slaughtering, scalding, defeathering, evisceration, washing, chilling and packaging. (Tsola et al., 2008). After slaughtering and bleeding of chickens, they are scalded at temperature around 55°C for about 200s. Before eviscerated automatically, carcasses are defeathered by going through a series of defeathering machines. Spray washing normally with chlorinated water then is conducted before final chilling and packaging of chicken portions (Mead et al., 1993). More information about each stage is discussed below and Figure 2.1 illustrates a schematic processing of poultry. Cheng QIAN LITERATURE REVIEW 7 Slaughtering Scalding Defeathering Evisceration Washing Chilling & Packaging Receiving Figure 2.1 Schematic processing of poultry (Davies & Board, 1998; Tsola et al., 2008). 2.2.1 Slaughtering Slaughtering of chickens includes hanging, stunning, killing and bleeding (Davies & Board, 1998). Before birds are slaughtered and bled out, stunning is usually applied to avoid the feeling of pain during bleeding, to minimise distress, to enable the bleeding of birds easily and accurately and also to prevent convulsions (Gregory, 1995). Raj (2006) used stunning methods, such as electrical water bath stunning and gas stunning to stun the birds. Chickens are hung upside down which involves their feet in shackles both during stunning and bleeding. They are bled out for at least 90s by cutting neck before going to next stage in the UK (Gregory & Wotton, 1986; Sparrey & Kettlewell, 1994). Cheng QIAN LITERATURE REVIEW 8 2.2.2 Scalding Temperature and time for chickens to be immersed in scalding liquid are the two important factors which manufacturers need to decide, because if the chicken is scalded at a high temperature for a long time, the skin would be overheated (Covell III, 1990). Mead et al. (1993) did a microbiological survey of five poultry processing plants in the UK, however, the temperature and time of scalding were all different from each other, ranging from 51.5°C to 56°C and 138s to 300s, respectively. The four plants investigated by Notermans et al. (1982) had different temperature and time at scalding stage as well. Generally, there are two kinds of scalding: soft scalding (50- 53°C) and hard scalding (58-60°C) (Corry & Atabay, 2001). The viable counts of Salmonella on Chickens were less when using 52°C or 56°C scalding temperature while Campylobacter isolated from chickens showed a lower level when treated with 60°C scalding temperature (Slavik et al., 1995). The purpose of scalding is to loose chicken feathers, making the feathers easily removable in the defeathering stage (Davies & Board, 1998). Traditional scalding of poultry processing only uses one single tank while modern industries usually scald chickens through a multi-stage process which consists of three or four scalder machines (Veerkamp, 1995; Veerkamp & Hofmans, 1973). This invention has been proved to reduce the loads of microorganisms on chicken carcasses after scalding, as scalding is a critical hazard point (Davies & Board, 1998; Veerkamp & Heemskerk, 1991). It is likely because of the warm temperature accelerates the growth of some bacteria and the cross-contamination from other carcasses through water (Tsola et al., 2008). The following figure (Figure 2.2.1) shows different effects of traditional scalding and multi-stage scalding methods on controlling of viable cells of microorganisms (Veerkamp, 1995). Dodd et al. (1988) examined the viable counts of Cheng QIAN LITERATURE REVIEW 9 S. aureus during scalding and defeathering. The results showed that after scalding, the viable counts of S. aureus were reduced significantly while the defeathering stage showed the opposite situation. Figure 2.2.1 Different effects of traditional scalding and multi-stage scalding methods on controlling of viable cells of microorganisms (Davies & Board, 1998; Veerkamp, 1995). 2.2.3 Defeathering During the defeathering stage, the feathers of chickens are removed in a plucker where rotation with rubber fingers and water spraying are also applied (Corry & Atabay, 2001). It is reported that chicken carcasses are contaminated with S. aureus at a high rate during defeathering stage when compared to other processing stages (Mead & Dodd, 1990; Notermans et al., 1982). For example, Mead et al. (1988) found that the viable counts of S. aureus after defeathering (107 CFU/swab) were 1000-fold Cheng QIAN LITERATURE REVIEW 10 higher than that of S. aureus before defeathering (104 CFU/swab). They explained that the reason probably was because of the conditions during plucking, such as high temperature, humidity and available nutrients. Also, staphylococci can attach to the rubber fingers in a plucker at environmental temperature of 28°C for 6 hours (Mead et al., 1995). The introduction of spray washing with water can reduce the viable counts of microorganisms (Mulder et al., 1978). Chlorinated water has been reported to prevent cross-contamination effectively during defeathering (Mead & Scott, 1994). A washing system was invented to lower the costs of water and improve the overall hygienic quality during defeathering. After spraying water onto the carcasses, the water is pasteurised and reused in scalding and next plucker ((Veerkamp, C. H & Pieterse, C cited by (Davies & Board, 1998)). 2.2.4 Evisceration Evisceration of chicken carcasses is to remove the head, intestines, heart, liver and wings etc.. At the end of evisceration, chicken portions are sorted for edible parts and washed again (Davies & Board, 1998; Tsola et al., 2008). The entrails and other parts of chicken carcasses are detached and removed by using a vacuum scrubber if the evisceration stage is conducted automatically (Scheier & Haynes, 1974). Stals (1992) pointed out that the hygienic quality of carcasses were improved with automated transfer from slaughtering line to evisceration line due to less chances of cross- contamination by workers. Evisceration stage is considered as a critical control point in HACCP (Hazard Analysis and Critical Control Points) as a result of contamination from intestinal contents (physical) or microorganisms (microbiological) (Tsola et al., Cheng QIAN LITERATURE REVIEW 11 2008). Davies and Board (1998) described the process of opening chicken carcasses in details which is illustrated in Figure 2.2.2. Cutting out the cloaca Cutting open the body cavity Taking out the intestines by eviscerator Inspection Figure 2.2.2 Generalised process of opening chicken carcasses (Davies & Board, 1998). 2.2.5 Chilling Immersion chilling, air chilling and spin chilling are the three common chilling methods for poultry products. The chilling method is chosen depending on the types of final products. Immersion chilling with water is normally applied to freeze carcasses while water chilling and air chilling are both suitable for fresh poultry meat or non-frozen poultry meat (Davies & Board, 1998; James et al., 2006). The original aim of chilling process of carcasses is to inhibit the propagation of various microorganism (James et al., 2006). However, it was reported that the viable Cheng QIAN LITERATURE REVIEW 12 counts of microorganisms on broiler carcasses were not significantly different between water chilling and air chilling (Mulder & Veerkamp, 1974). In contrast, Blood and Jarvis (1974) found that water immersion chilling method could reduce contamination of carcasses more effectively than air chilling. The usage of water volume and the chlorine concentration had an impact on the final viable counts of coliform and other bacteria. The combination of more water usage and high concentration of chlorine in water, the less loads of microorganisms are on the finished products (Blood & Jarvis, 1974). On the other hand, the taste, texture and appearance of final poultry products, to some extent, are influenced by the rate of chilling. Tough texture of poultry meat will be obtained if the meat is chilling rapidly while pale soft exudative (PSE) meat will be produced when chilling process is very slow (James et al., 2006). 2.2.6 Packaging There are many packaging methods available commercially, such as vacuum packaging, traditional wrapping packaging, carbon dioxide flushing packaging and modified atmosphere packaging (MAP) (Sebranek et al., 1996; Thomas et al., 1984). The materials and methods used to pack poultry meat depend on the type of products and the slaughter process. For example, fresh or non-frozen poultry carcasses that are processed under high temperature of scalding and immersion chilling are normally packed with ice, avoiding the occurrence of discoloration of the carcasses (Thomas et al., 1984). Thomas et al. (1984) compared the microbiological quality and flavour of dry broilers that were packed using carbon dioxide flushing, vacuum packaging and stretch wrapping. They found that among these three packaging methods, vacuum packaging performed well with regards to the shelf-life. Narasimha Rao and Cheng QIAN LITERATURE REVIEW 13 Sachindra (2002) pointed out that the colour of poultry meat that was packed by MAP remained longer than that of poultry meat under vacuum packaging. The growth of pathogens and spoilage microorganisms is the main concern during packaging. Only MAP can inhibit the growth of Pseudomonads on chicken breasts with skin. However, Lactobacilli, Enterobacteriaceae and Brochothrix thermosphacta can even propagate after chicken meat is MAP packed (Jiménez et al., 1997). Thus, it is important to keep the residence times short and keep the temperature low during both packaging and distribution stages (Davies & Board, 1998). 2.3 Contamination of raw chicken and chicken products 2.3.1 Microbiological contamination Raw poultry meat can be contaminated with various pathogens and spoilage microorganisms. Microbiological contamination of poultry meat is more common than physical and chemical contaminations, as most Crital Control Points (CCPs) are decided according to the potential contamination with bacteria (Tsola et al., 2008). Figure 2.3 shows the occurrence of microbial counts in retail chicken parts and processed chicken products in Spain (Álvarez-Astorga et al., 2002). Cheng QIAN LITERATURE REVIEW 14 Figure 2.3 Occurrence of microbial counts in retail chicken parts and processed chicken products in Spain. In different countries, some common pathogens have been isolated from poultry, including Aeromonas spp., Campylobacter spp., Clostridium perfringens, Listeria spp., Salmonella spp., Shigella and Streptococcus spp., Staphylococcus aureus, etc (Waldroup, 1996). Testing the occurrence of foodborne pathogens and spoilage microorganisms at poultry processing stages in a plant is a basic way to investigate the contamination of poultry products (Gibbs, Patterson, & Thompson, 1978; Mead et al., 1993; Notermans et al., 1982). For example, Lindblad et al. (2006) examined the prevalence and concentrations of microorganisms on 636 chilled broilers in Sweden. The results showed that, about 29%, 18%, 9% and 97% of all samples were Cheng QIAN LITERATURE REVIEW 15 contaminated with Listeria monocytogenes, Clostridium perfringens, Yersinia enterocolitica and Enterococcus, respectively. 2.3.2 Contamination sources Humans, live chickens and processing equipment are the three dominant sources that induce contamination or cross-contamination of foodborne microorganisms or pathogens on chicken meat (Chaffey et al., 1988; Jacxsens et al., 2009; Mead et al., 1988). Cross-contamination always occurs among live chickens during raising and transportation where chickens stay closely together with each other (Davies & Board, 1998). Gibbs, Patterson, and Thompson (1978) analysed the origin of S. aureus in a poultry processing plant and they found that the contamination source of this microorganism is live chickens rather than humans or processing equipment. Hygienic working practices, such as wearing protective clothing and washing hands frequently, are usually developed to ensure that carcasses are free from being contaminated by workers (Aarnisalo et al., 2006). Chicken carcasses can be contaminated with micrococci, staphylococci, propionic bacteria through worker’s skin and E. coli and Salmonella can contaminate products by workers’ hands (Jacxsens et al., 2009). Good hygiene practices are also required to prevent cross- contamination during the cooking of raw chicken meat (Haysom & Sharp, 2004). Through poultry processing, carcasses need to go through a series of equipment. The contact between carcasses and the surface of equipment is a high risk of cross- contamination, as some bacteria can attach to the surface (Kusumaningrum et al., 2003). Therefore, in order to avoid cross-contamination from equipment, it is Cheng QIAN LITERATURE REVIEW 16 important to design and use a series of good hygienic equipment to produce poultry products (Aarnisalo et al., 2006). The level of contamination varies from different poultry products in a production line. For example, the chicken wings had a much higher viable counts of Campylobacter than other chicken portions (Habib et al., 2008). To investigate the microorganisms contamination of poultry products throughout processing, sampling locations should be identified (Mead et al., 1988; Notermans et al., 1982). Microbial Assessment Scheme (MAS) is a systematic procedure to investigate the distribution of bacteria during processing in a plant, which includes the identification of critical sampling locations, the selection of microbiological parameters and sampling method of analysis, the assessment of sampling frequency, final data processing and interpretation (Jacxsens et al., 2009, 2011). 2.3.3 Decontamination of poultry products Food Safety Management System (FSMS) is a comprehensive system to prevent microbiological contamination, to inhibit the growth of foodborne bacteria or pathogens in a food product and also to decontaminate food products (Jacxsens et al., 2011). There are many decontamination methods which are normally classified as chemical decontamination and physical decontamination. However, physical decontamination is more favourable than chemical decontamination, as the potential residues may be found in final products (Corry et al., 2007). Table 2.2 listed some examples of chemical and physical decontamination methods for poultry meat (Bolder, 1997). In European Union countries, it is not permitted to use chemical decontaminants (Corry et al., 2007; Whyte et al., 2003). Cheng QIAN LITERATURE REVIEW 17 The effect of immersion in hot water of chicken carcasses was studied by Corry et al. (2007). The result showed that decreases in viable counts of E. coli K12 and Campylobacter jejuni AR6 were observed using 80°C for 20s immersion and 75°C for 30s immersion. However, on a smaller scale, hypochlorite was reported to reduce the contamination by Salmonella and Campylobacter spp. more significantly and effectively than detergent or hot water (Cogan et al., 1999). It was also proved that gamma radiation doses of 1.50 KGy was able to kill all S. aureus in deboned chicken meat samples before and after storage (Thayer & Boyd, 1992). Table 2.2 Examples of chemical and physical decontamination methods for poultry meat. Method Chemical: Chlorine (hypochlorite, ClO2) Organic acids (lactic acid, acetic acid, buffered lactic acid, gluconic acid, etc.) Inorganic phosphates (trisodium phosphate, polyphosphates) Organic preservatives (benzoates, propionates) Bacteriocins (nisin, magainin) Oxidizer (hydrogen peroxide, ozone) Physical: Water (rinse, spray, steam) Ultrahigh pressure Irradiation Pulsed-field electricity Ultrasonic energy UV light Source: (Bolder, 1997) 2.4 Characterisation of Staphylococcus aureus 2.4.1 Staphylococcus aureus Staphylococcus aureus is one of the micrococcaceae families which have spherical cells with grape like clusters (Bhatia & Zahoor, 2007; Fratamico et al., 2011). Figure 2.4.1 shows an electron micrograph of S. aureus (Montville & Matthews, 2008). S. aureus are Gram-positive coccus and facultative anaerobic bacteria (Bhatia & Zahoor, 2007). They are not motile and cannotform spores (Silva, 2012). During their Cheng QIAN LITERATURE REVIEW 18 metabolism, mannitol is fermented to acid and, protein A, lipase, coagulase, thermonuclease and hemolysin are produced (Ray, 2001; Silva, 2012). As mentioned in section 1, they can live in a wide range of temperatures from 7 to 48°C, low water activity (0.86), pH ranging from 4.2 to 9.3 and high sodium chloride concentrations of 15%, therefore, food matrix is a favourable environment for them to grow (Bhatia & Zahoor, 2007; Kérouanton et al., 2007; Ray, 2001). It is important to notice that S. aureus is not the causative agent of food poisoning because the cells can be killed at 66°C in 12 min or at 72°C in 15s (pasteurisation treatment), but instead, it is the enterotoxin produced by the bacteria which is heat-resistant that causes food poisoning (Fratamico et al., 2011). Figure 2.4.1 Electron micrograph of S. aureus (Montville & Matthews, 2008) People are one of the carriers of S. aureus most of which is present in nose, skin and hair. Food contamination of S. aureus usually occurs during processing and preparation. People contaminate food and food products by poor hygiene hands or Cheng QIAN LITERATURE REVIEW 19 dirty utensils (Montville & Matthews, 2008; Ray, 2001). Another source of S. aureus contamination is from animals such as cows, dogs and birds. For instance, cow mastitis is caused by S. aureus which influences the quality of the milk (Harvey et al., 1982; Montville & Matthews, 2008). Harvey et al. (1982) pointed out that strains of S. aureus which contaminate food from human were more common than that from animals. 2.4.1.1 Methicillin -res is tant Staphylococcus aureus (MRSA) Methicillin-resistant Staphylococcus aureus (MRSA) is a typical type of S. aureus that is resistant to penicillinase-resistant penicillin (Lee, 2006). It was first discovered in the 1960s and after that hospitals and communities had shown the presence of MRSA (Gosbell, 2004). MRSA is one of the reasons for hospital-related infection as well as the cause of community diseases among people (Khanna et al., 2008). In Canada, MRSA causes nosocomial infections in humans and the infections have been increased since 1990s with MRSA possibly being a potential cause for foodborne illness (Crago et al., 2012). It was reported that MRSA was first found in pigs as an animal carrier with a high prevalence (Crago et al., 2012; De Neeling et al., 2007). Recently, other animals, such as cows, dogs, cats, horses and chickens, have been reported to carry MRSA especially animals with scratches or wounds, causing a potential threat to meat products (De Boer et al., 2009; Kitai, Shimizu, Kawano, Sato, Nakano, Uji, et al., 2005). MRSA isolates have been found in not only meat products, but also other different kinds of food products, such as milk and cheese (Normanno et al., 2007; Pereira et al., 2009; Pu et al., 2009). Meat products are most likely to be contaminated by MRSA if carcasses are contaminated from MRSA-positive animals and the Cheng QIAN LITERATURE REVIEW 20 environment during slaughtering (De Boer et al., 2009). Also, food workers infected with MRSA are another source of contamination during processing (Lozano et al., 2009). The unique methicillin resistance characteristic of this organism is encoded by mecA gene which can be detected by PCR (De Boer et al., 2009; Khanna et al., 2008; Lee, 2006). MRSA Chromogenic agar, MRSA latex agglutination test and DNase assay are other alternative methods that have been used in relevant researches (Lozano et al., 2009). 2.4.2 Outbreaks of Staphylococcus aureus Microbiological food poisoning is considered as the most serious food poisoning. Two reasons count for this: bacterial infection and food intoxication (Bhatia & Zahoor, 2007). S. aureus has the ability to produce enterotoxins in food leading to food intoxication. Outbreaks caused by enterotoxins of S. aureus have been widely reported all over the world, especially in the early 1900s (Ray, 2001). For example, 25%-35% of all microbial outbreaks in Japan were contributed to staphylococcus toxins before 1984 while in America, the percentage outbreaks of staphylococcus toxins was 14% (Bhatia & Zahoor, 2007). Raw poultry and poultry products are more vulnerable to be contaminated with S. aureus which has been proved by a study in UK, investigating the incidence among raw poultry and poultry products (75%), fish or shellfish (7%) and milk products (8%) (Waldroup, 1996). In NZ, from 1989 to 1999, 24 staphylococcal food poisoning (SFP) cases were reported. It was also reported that food products, such as yogurt, hot ham sandwiches, chicken salad, hot turkey sandwiches etc. were the SEs carriers which were responsible for totally 1762 cases in NZ (Ministry of Primary Industry, 2001). Cheng QIAN LITERATURE REVIEW 21 2.4.3 Disease and symptoms The symptoms of food poisoning produced by S. aureus includes nausea, vomiting diarrhoea, salivation, headache and sweating (Fratamico et al., 2011; Lee, 2006; Ray, 2001). The type of symptom depends on the amount of toxins and the type of that toxin (Bhatia & Zahoor, 2007). The disease caused by staphylococcal toxins not only occurs on humans but also on poultry, such as arthritic lesion of joints, foot abscesses, skin dermatitis (Mead & Dodd, 1990). It is reported that food with 100 to 200 ng toxins that are produced by S. aureus can infect a healthy man after consuming the food in 30 mins to 8 hours (Fratamico et al., 2011; Ray, 2001). However, there might be a little influence on human as our immune system will start to recover in 24 to 48 h with low intake of toxins (Montville & Matthews, 2008). With regard to the prevention of infections of staphylococcal toxin diseases, inspection of raw ingredients, sanitation of plant environment and good personal hygiene are essential (Ray, 2001). 2.4.4 Staphylococcal enterotoxins (SEs) Some strains of S. aureus are able to produce one or more staphylococcal enterotoxins (SEs) which are the causative agents of staphylococcal food poisoning (SFP) (Lee, 2006; Matyi et al., 2013; Notermans et al., 1982). Food intoxication of S. aureus is caused by consuming food that has already been contaminated with S. aureus and most importantly the strains have produced enterotoxins into the food matrix (Bhatia & Zahoor, 2007; Ray, 2001). SEs are single chain proteins with a low molecular weight (Bergdoll, 1999). The 2D structures of some SEs were illustrated by Hennekinne et al. (2012) having a cysteine loop in the centre of each one (Figure 2.4.2) (Bergdoll, 1999). Cheng QIAN LITERATURE REVIEW 22 Figure 2.4.2 Structures of some staphylococcus enterotoxins (Hennekinne et al., 2012). Raw poultry and poultry products are vulnerable to be contaminated with S. aureus because the pH, water activity of carcasses and temperature in some stages during manufacturing are suitable for S. aureus to grow and even synthesise SEs (Kérouanton et al., 2007; Pepe et al., 2006). SEs are named alphabetically according to the time that they have been discovered (Montville & Matthews, 2008; Roberts & Greenwood, 2002; Silva, 2012) Ten SEs (A to J) have been discovered before 2003 (Roberts & Greenwood, 2002). Until 2007, fourteen different SEs have been discovered from SEA to SEO without SEF while one year after Silva (2012) pointed that SER, SES, SET (new SEs) and enterotoxin-like proteins (SEls): SElU, SElV and SElW have been recognised. Poultry meat has been reported to carry enterotoxinic S. aureus isolates. Kitai, Shimizu, Kawano, Sato, Nakano, Kitagawa, et al. (2005) detected SEB, SEA, SEC, SED, SEA+SEB and SEA+SEC from 360 S. aureus Cheng QIAN LITERATURE REVIEW 23 isolates of 444 raw chicken meat samples in Japan. Strains that originate from birds usually produce SEC and SED while Pepe et al. (2006) found that 62% of S. aureus isolates from birds were SEA producer. 2.4.4.1 Inactivation of s taphylococcal enterotoxins There are three ways to inactivate SEs: irradiation inactivation, chemical inactivation, biological inactivation and thermal inactivation (Bhatia & Zahoor, 2007). SEs in food matrices are not suitable to be inactivated by irradiation as a relatively high dose is required to achieve the reduction of SEs level (Read & Bradshaw, 1967). It is reported that 16%-26% SEA could not be inactivated even under a dose of 23.7 KGy (Modi et al., 1990). Some researchers have used several chemical components to inactivate SEs. For example, Stelma Jr and Bergdoll (1982) used bromoacetic acid at pH 7.0 to induce the carboxmethylation of histidine residues in SEA which can inhibit activity of SEA’s antibody. Suzuki et al. (2002) inactivated SEA successfully with electrolysed anodic NaCl solution [EW(+)]. For food with more organic compounds, high level of chlorine is also a solution with the combination of phosphate-buffered saline and hypochlorite (HOCI) (Suzuki et al., 2002; Warren et al., 1974). Biological inactivation needs to be further studied as SEs are tolerant to proteolytic enzymes. Lactic acid bacteria can slightly reduce the concentration of enterotoxin, but it is still unclear about the specific reason (Bhatia & Zahoor, 2007). Thermal inactivation is the main study area to inactivate SEs, although SEs are stable to heat treatment. Z-values of SEs are 25-33°C, D-value at 121°C ranging from 8.3-34 min and F-value at 120°C is almost 30 mins (Bhatia & Zahoor, 2007). The time and temperature required to inactivated SEB were investigated by Read and Bradshaw (1966) using the double-gel-diffusion technique. The combination of thermal Cheng QIAN LITERATURE REVIEW 24 inactivation and chemical inactivation probably is a more effective approach. Satterlee and Kraft (1969) pointed out that a heat treatment at 80°C of SEB of meat proteins in phosphate-saline buffer showed a more effective inactivation as 67.5% of the activity was lost at the beginning of 15 min and the SEB in ground-beef slurry showed a relatively faster loss of activity. Moreover, many factors play a role in inactivation of SEs, such as pH, initial concentration of SEs, ionic strength etc. (Bartlett et al., 1971; Denny et al., 1971; Schwabe et al., 1990). 2.5 Isolation of Staphylococcus aureus There are several agars that can be used to isolate S. aureus such as colony counting with Baird-Parker agar, colony counting with rabbit plasma fibrinogen agar (RPFA), enrichment culture and Petrifilm (Roberts & Greenwood, 2002). The selection of isolation methods depends on the type of products being analysed (Roberts & Greenwood, 2002; Silva, 2012). For example, for dried food products, MPN technique is a proper one due to potential low numbers of coagulase-positive colonies and the injuries of bacteria cells (Roberts & Greenwood, 2002). Apart from selective agar (Baird-Parker agar and RPFA), several agars are also available, such as Mannitol Salt agar (MSA), Egg Yolk Azide agar, Vogel-Johnson agar, Bovine Fibrinogen agar (BFG agar), Milk-Salt agar. Notermans et al. (1982) used BFG agar to investigate the contamination of chicken carcasses with S. aureus during processing in a plant. Tryptic Soy agar was used to study the effect on reduction of S. aureus on mechanically deboned chicken meat (MDCM) by different doses of gamma radiation (Thayer & Boyd, 1992). However, Silva (2012) pointed out that Baird-Parker agar is the most common and widely used agar to enumerate S. Cheng QIAN LITERATURE REVIEW 25 aureus which have been proved by many researchers in their studies. The time and temperature required to incubate S. aureus are 24 to 48 h at 35 or 37°C (Gibbs, Patterson, & Thompson, 1978; Gundogan et al., 2005; Harvey et al., 1982; Lee, 2003, 2006; Mead et al., 1988; Tsola et al., 2008; Waters et al., 2011). Petrifilms are convenient and flexible to use and it offers a good repeatability and reproducibility (Aarnisalo et al., 2006). It contains a cold-water-soluble gelling agent with modified Baird-Parker agar inside which is a selective and differential agar to identify S. aureus colonies. Basically it involves inoculation of sample onto the Petrifilm and then incubate the Petriflim for 24±2 hours (3M™, 2010b). 3M Petrifilm Rapid Staph Express Count (PSE) system was used to identify S. aureus isolated from chicken products (Pepe et al., 2006). The red-violet colonies on the plate are regarded as S. aureus (3M™, 2010b). 2.5.1 Confirmation tests of suspect S. aureus isolates To confirm presumptive S. aureus colonies, Gram-staining, catalase test, coagulase test, carbohydrate fermentation, DNase, phosphatase tests etc. can be tested. The systematic confirmation biochemical tests of S. aureus have been outlined in Bergey’s Manual (Bergey et al., 2001). Lancette and Bennett (2001) summarised the enumeration and confirmation methods including plate count method and Most Probable Number (MPN) technique of S. aureus (Figure 2.5.1 & Figure 2.5.2). Cheng QIAN LITERATURE REVIEW 26 Figure 2.5.1 Schematic procedures of enumeration and confirmation (coagulase test) of S. aureus using plate count method APHA 2001(Lancette & Bennett, 2001; Silva, 2012). Cheng QIAN LITERATURE REVIEW 27 Figure 2.5.2 Schematic procedures of enumeration and confirmation (coagulase test) of S. aureus using plate count method APHA 2001 (Lancette & Bennett, 2001; Silva, 2012) 2.6 Detection of staphylococcal enterotoxins (SEs) To identify SEs, many approaches, such as molecular biological methods, Reverse phase latex agglutination (RPLA), the enzyme-linked immuno-sorbent assay (ELISA), biosensors and polymerase chain reaction (PCR), are available nowadays. ELISA and Cheng QIAN LITERATURE REVIEW 28 RPLA can detect SEs more sensitively and efficiently than other common microbiological methods (Rose et al., 1989). Hennekinne et al. (2012) classified them into bioassays, molecular biology (PCR) and immunological methods (ELISA). 2.6.1 Immunological methods Immunological methods involve using monoclonal antibodies to detect SEs which are more difficult to conduct as pure toxins are required (Harvey et al., 1982; Hennekinne et al., 2012; Kitai, Shimizu, Kawano, Sato, Nakano, Kitagawa, et al., 2005; Šimkovičová & Gilbert, 1971). ELISA is able to detect SEA to SEE at a very low level (1 ng/g food), but only SEA to SEE, SEG, SHE and SEIQ can be detected using this method (Bhatia & Zahoor, 2007; Chiang et al., 2008; Morandi et al., 2007; Schlievert & Case, 2007). Wieneke (1991) compared four immunological methods that were used to detect SEs: SET-EIA (staphylococcal enterotoxin ELISA), SET- RPLA, ELISA-M (ELISA-membrane) and ELISA-T (ELISA-tube) kits in terms of reagents used, time required, extraction procedure and cost (Table 2.6) Table 2.6 Comparison of ELISA-B, SET-RPLA, ELISA-M and ELISA-T kits for the detection of staphylococcal enterotoxins. Kits ELISA-B SET-RPLA ELISA-M ELISA-T Enterotoxins can be detected SEA-SED SEA-SED SEA-SEE SEA-SEE Detection of individual enterotoxins Yes Yes Yes No Cost (Pound/ number of tests per kit) £66/10 £114/20-40 £230/25 £165/20 Time (h) 24 16 4 1.5 Sensitivity (ng/mL) 0.1-1 0.5 0.5 0.2 Extracts used (mL) 20 0.2 20 0.5 Source: (Wieneke, 1991) Cheng QIAN LITERATURE REVIEW 29 2.6.2 Molecular biology methods Molecular biology methods for detecting enterotoxins that are produced by S. aureus strains from contaminated food products are based on genes encoding. Polymerase Chain Reaction (PCR) has been widely used by researchers to detect SEs in chicken meat (Bhatia & Zahoor, 2007; Hwang et al., 2007; Kérouanton et al., 2007). Pepe et al. (2006) examined SEs from S. aureus stains of breaded chicken samples using PCR to detect SEs genes. Kérouanton et al. (2007) summarise the nucleotide sequences of primers which can be obtained from pervious relevant literatures for each staphylococcal enterotoxin. Specificity and rapidity are usually used to describe the advantages of PCR (Maurer, 2006). PCR can detect the presence of SEs according to the specific gene sequences of each enterotoxin even from heat-treated food products (Bhatia & Zahoor, 2007). PCR is based on amplifications of the target genes in a PCR thermocycling machine which provides an optimum temperature for DNA to denature, anneal and synthesise (Maurer, 2006). The temperature of a standard PCR reaction first increase to 96°C separating the template DNA strands and then decrease to 55°C (annealing temperature) allowing primers to anneal to each DNA strand. Finally, the temperature increases to 72°C (extension temperature) which is the optimum temperature environment for Taq polymerase (Innis et al., 1999). Therefore, extracted DNA template, designed primers, deoxynucleotide triphosphates (dNTP) mixture, Taq polymerase, buffer and water are the essential reagents to run a PCR reaction. Experimental design and optimisation of a PCR protocol for an experiment of interest are usually required to amplify targeting genes successfully without non-specific amplifications, as PCR is so sensitive that reaction components and working conditions must provide an optimal environment for amplifications of a DNA Cheng QIAN LITERATURE REVIEW 30 template. Such parameters as primer design, concentrations of each reagent and cycling conditions are required to optimise (Weissensteiner et al., 2010). A single PCR uses only one pair of primers (forward and reverse) in each reaction while multiplex PCR application involves multiple primer pairs which can amplify several target genes in one single reaction (van Pelt-Verkuil et al., 2008). Compared to single PCR, multiplex PCR is more efficient if many DNA samples or target genes in a sample need to be tested (Innis et al., 1990). On the other hand, non-specific PCR products are a common problem of Multiplex PCR because the primer pairs in each reaction may anneal to each other if the annealing temperature or the design of each primer are not optimal (van Pelt-Verkuil et al., 2008). Food samples need to be prepared before starting a PCR reaction. The preparation of food samples varies with the type of food which has been explained and summarised by Maurer (2006), including collection of from food sample, food sampling process, concentration or amplification of pathogens, template extraction and concentration (Figure 2.6). Cheng QIAN LITERATURE REVIEW 31 Process food sample (Homogenisation, Washing) Concentrate pathogens (Enrichment, Immunocapture, Buoyant density centrifugation) Template extraction (Heat, Detergent, Chemical Solvent) Concentrate template (Alcohol precipitation, Binding matrices) PCR Figure 2.6 Preparation of food samples for PCR analysis (Maurer, 2006). 2.7 Identification the strain of S. aureus Identifying the specific strains of S. aureus has been applied to investigate the source of contamination or an outbreak which can provide the relatedness within a range of isolates (Bannerman et al., 1995; McCullagh et al., 1998). To date, several methods are available to subtype S. aureus isolates, such as Phage Typing, Biotyping, Plasmid Typing, Pulsed-Field Gel Electrophoresis (PFGE), Amplied Fragment Length Polymorphism (AFLP) and Multilocus Sequence Typing (MLST) (Chaffey et al., 1988; Enright et al., 2000; Harbottle et al., 2006; McDougal et al., 2003; Murchan et al., 2003). Biotyping and Phase Typing were widely used around 1900s to distinguish the strains of S. aureus (Devriese, 1980; Gibbs, Patterson, & Thompson, 1978; Cheng QIAN LITERATURE REVIEW 32 Harvey et al., 1982; Notermans et al., 1982). The occurrence of S. aureus and its contamination source in a poultry processing plant were investigated using Phase Typing, Plasmid Typing, PFGE and MLST (Chaffey et al., 1988; Gibbs, Patterson, & Thompson, 1978; Notermans et al., 1982). However, Phase Typing has several drawbacks such as poor reproducibility, low discriminatory power, high labour work and time-consuming (Bannerman et al., 1995; Harbottle et al., 2006). Plasmid typing is considered a better method for typing strains of S. aureus, but disadvantages such as average discriminatory and intense work still exist (Bannerman et al., 1995; Chaffey et al., 1988). PFGE and MLST both are molecular typing method with MLST possessing the highest reproducibility and discriminatory (Harbottle et al., 2006; Lv et al., 2014). MLST is a more rapid method which only needs 1-2 days to identify one strain while it takes at least 3 days using PFGE method (Bannerman et al., 1995). MLST method also makes it possible to transform and share the data between laboratories all over the world via Internet (Robinson & Enright, 2004). The MLST method is based on sequencing the DNA fragment (~500bp) of seven house-keeping genes on both strands (Maiden et al., 1998). An allele number can be obtained from the MLST database which is available from the MLST website after submitting trimmed sequences (http://www.mlst.net/). The unique seven allele numbers of a specific strain are assigned a sequence type (ST) which is also called allelic profile (Urwin & Maiden, 2003). Figure 2.7 shows the overall steps on how to perform the MLST method (Spratt, 1999). The strains of pathogens, such as Cryptococcus neoformans, Cryptococcus gattii, Pseudomonas aeruginosa, Escherichia coli and S. aureus, have been successfully identified using MLST in some studies (Johnson et al., 2007; Lacher et al., 2007; Lv et al., 2014; Meyer et al., 2009). Moreover, Waters et al. (2011) used MLST to investigate the multidrug- resistant of S. aureus isolates in poultry meat in the United States. Cheng QIAN LITERATURE REVIEW 33 Figure 2.7 Overview of procedures for MLST method (Spratt, 1999). The contamination sources in food products can be traced based on the sequence types (STs) of microorganisms isolates collected from processing line. The MLST data have been widely used to investigate the relatedness within some S. aureus isolates by Based Upon Repeat Sequence Types (eBURST) programme (Mellmann et al., 2008). Compared to using data of Pulsed-Field Gel Electrophoresis (PFGE), Randomly Amplified Polymorphic DNA Analysis (RAPD) and Phage Typing, MLST data can group isolates into a most unambiguous clonal complex (Grundmann et al., 2002). eBURST analyses the genetic relationships between each sequence type (ST) according to the similarity of the seven allele (de Sousa & De Lencastre, 2003; Feil et al., 2003; Grundmann et al., 2002; Schulte et al., 2013). Normally, STs that have identical allele at least five of the seven analysed genes are grouped into a clonal complex (CC). The stringent group definition can be modified if the research requires (MLST database, 2015). Single-locus variants (SLVs) were described as ST that differs from other STs in the group at only one allele number. Therefore, a clonal Cheng QIAN LITERATURE REVIEW 34 ancestor in a group of STs is identifiable as the one with the largest number of SLVs (Grundmann et al., 2002; MLST database, 2015). 2.8 Critical sampling locations in poultry processing plants 2.8.1 Industrial environment In section 2.2, the whole poultry processing procedures have been discussed, however, in order to understand the source of contamination of S. aureus during processing and also to eventually prevent the contamination of S. aureus, critical sampling locations should be identified. Many previous researchers have reported some critical sampling locations in terms of S. aureus contamination in poultry plants. For example, Notermans et al. (1982) collected their chicken samples before and after scalding, after defeathering, evisceration, washing and chilling. Mead et al. (1988) identified the critical sampling locations of broiler chickens as after bleeding, scalding, defeathering and chilling. Mead et al. (1993) regarded seven stages during poultry processing as sampling locations and they were after bleeding, scalding, defeathering, evisceration, washing, chilling and packaging. Similarly, Chaffey et al. (1988) examined chicken neck skin after bleeding, scalding, plucking and chilling to find the source of contamination of S. aureus. According to the distribution of S. aureus in a poultry plant that was reported by Gibbs, Patterson, and Thompson (1978), the viable counts of S. aureus were relatively high before scalding, defeathering, before and after evisceration. Cheng QIAN LITERATURE REVIEW 35 2.8.2 Farm environment Live poultry is considered as another potential contamination source of S. aureus causing skin lesion of live birds or poultry products contamination (Ministry of Primary Industry, 2001; Thompson et al., 1980). The symptom of infected chicken is dermatitis and depression will then occur (Kuramasu et al., 1967). Kibenge et al. (1982) collected their samples from chickens in an Australian poultry farm and the sampling sites were chickens with lesions, skin, nostril of normal chickens, air samples in the poultry sheds and inanimate hatcheries. Poultry skin is widely known as the source of S. aureus contamination especially in bruised skin. It is probably because that S. aureus grow better in bruised skin than normal skin (Kuramasu et al., 1967; Ministry of Primary Industry, 2001). Nostrils of live chicken are another potential sampling sites with high level of S. aureus as necrosis of skin was believed to originate from the inside surface of chicken wings after preening (Kuramasu et al., 1967). 2.9 Sampling methods of poultry meat In poultry industries, it is essential to select a uniform sampling method to examine and control the microbiological quality of final products in order to simplify and standardise sampling procedures (Gill et al., 2005). Excision, swabbing, contact methods and rinse are the four available methods to sample poultry meat before microbiological analysis. Each method has advantages and disadvantages. For example, excision and swabbing are the two popular sampling methods due to their convenience of conducting and Cheng QIAN LITERATURE REVIEW 36 good reproducibility (Capita et al., 2004). However, excision needs more preparation steps of samples before plating than swabbing, such preparation steps are homogenisation and filtration (Pepperell et al., 2005). Also, excision method involves destruction of poultry samples normally by cutting or scrapping (Capita et al., 2004; Pepperell et al., 2005). Probably these factors can explain why swab technique is widely used in poultry plants (Capita et al., 2004). Cogan et al. (1999) and Gibbs, Patterson, and Thompson (1978) used swabbing method in their studies to investigate microbial contamination of chicken carcases in a poultry plant and in kitchen respectively. Only excision method is preferred by EU to a standardised level of analysing microbiological performance of poultry carcasses (Pepperell et al., 2005). It is reported that swabbing method recovers less microorganisms than excision and rinse methods (Capita et al., 2004; Gill et al., 2005; Korsak et al., 1998; Pepperell et al., 2005). In general, using swabbing, the viable counts of bacteria of poultry carcasses were >0.5 log unit less than using excision and rinse, while there was no significant difference between excision and rinse (Gill et al., 2005). There are many factors that affect the variations of swabbing, such as the type of bacteria, the time of swabbing, the storage time before swabbing, swabbing area size, pressure used when swabbing etc. (Capita et al., 2004). Contact methods requires few materials such as agar syringes and membrane filter blots, but this method can only be used when the counts of microorganisms is less than 100 CFU/cm2 (Capita et al., 2004). In addition, there is no significant different on the counts of bacteria when samples are treated with contact method or swab technique (Salo et al., 2000). Cheng QIAN MATERIALS AND METHODS 37 3. Materials and Methods Phase I 3.1 Identification of key processing steps in the plant 3.1.1 Industral level Collection of samples was back-tracked from the final products at further processing to primary processing. The samples were collected as follows: (1) further processing plant: Final Products, Frozen Mechanically Separated Meat (MSM), Frozen Skin, Frozen Skin-On Breast; (2) secondary plant: Fresh MSM, MSM carcass, inside Mechanically Deboning Machine (MDM), MDM conveyor, Fresh Skin, Skinner conveyor and Fresh Skin-On Breast; 3) primary plant: Rubber Fingers in Plucker. Figure 3.1.1 shows the hierarchical relationships between the sampling sites. Cheng QIAN MATERIALS AND METHODS 38 Further Processing Secondary Processing Primary Processing Final Produd cts Final Products Frozen MSM Frozen Skin- on Breast Frozen Skin resh MSMFresh MSM Inside MDMMDM Conveyor MSM Carcass Fresh Breast Skin-on Fresh Breast Skin-on Fresh SkinFresh Skin Skinner Machine Conveyor Skinner Machine Conveyor RuRR bu ber Fingers in Pluckers Rubber Fingers in Pluckers Figure 3.1.1 Hierarchical relationships of sampling sites in the poultry plant. Arrows indicate the route followed to collect Final Products. MSM=Mechanically Separated Meat; MDM=Mechanically Deboning Machine. The results of the three frozen ingredients in the further processing plant discussed in section 5.1.2 showed high contamination by S. aureus. More samples were examined in the secondary processing plant focusing on Fresh MSM, Fresh Skin and Fresh Skin-on Breast (SO BF) to trace back the potential contamination sources. Eighteen samples (n=18) were collected in three batches (6 samples per batch) from fresh meat. Also, samples were collected during 6-h processing for three iterations (three batches) to investigate whether there was potential accumulation of S. aureus on the meat samples or surfaces of equipment. Cheng QIAN MATERIALS AND METHODS 39 3.1.2 Farm level As mentioned in section 2.8.2, nostrils and skins of live chickens were regarded as the most likely sources of S. aureus (Ministry of Primary Industry, 2001; Thompson et al., 1980). Therefore, swabs from skins and nostrils of live chickens were randomly collected at a selected local poultry farm. 3.2 Collection of swab samples 3.2.1 Processing environment Samples were collected from a commercial poultry processing plant located in Auckland, New Zealand for seven consecutive months from May (early winter) through to November (beginning of summer) 2014. From reception of live birds to final products, industrial poultry processing mainly uses mechanical equipment using standardised technology including one or more cooking steps. The entire factory consists of primary processing, secondary processing and