Occurrence of Staphylococcus aureus in a commercial poultry plant and poultry farm : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Albany, New Zealand

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.