# Quantitative measurements of factors influencing bacterial attachment to meat surfaces

Zulfakar, SS 2013 , 'Quantitative measurements of factors influencing bacterial attachment to meat surfaces', PhD thesis, University of Tasmania.

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## Abstract

Pathogenic microbial contamination of meat can lead to foodborne illness following consumption of contaminated products. In abattoirs, contamination of carcass surfaces normally occurs when microorganisms are transferred from the outer surface of the carcass to sterile underlying tissues during slaughter and subsequent post-slaughter processes. Bacterial attachment to surfaces is a complex phenomenon that is influenced by many factors including the physicochemical properties of the bacterial cell and meat tissues, interacting surface structures and other factors.
The complexity and the heterogeneous nature of meat tissue, as well as physical entrapment within tissue fibres, have made it difficult to describe the specific tissue structures involved in the attachment process. A more systematic approach can be achieved by studying binding interactions between bacteria and specific structures of meat tissue.
Beef carcass prepared for commercial trade mainly consists of muscular and fatty tissue, bones and connective tissues. Approximately 49 to 68% of carcass weight consists of the muscular tissue whereas connective tissues are ubiquitous on the carcass surface, located at the fascia (between the skin and the skeletal tissues) and also within the muscle. The connective tissues are mainly composed of extracellular matrix (ECM) proteins.
This study aimed to determine the mechanisms of bacterial attachment to specific meat surface structures, namely the ECM proteins and muscle cells, by a range of enterohemorrhagic Escherichia coli (EHEC) and Salmonella spp. strains. The influence of physicochemical and environmental factors on attachment to meat structures was also investigated.
Attachment properties of the E. coli and Salmonella strains to four major ECM proteins (collagen I, fibronectin, collagen IV and laminin) were measured by a microtiter plate assay using crystal violet staining, and by epifluorescence microscopy. The effect of temperature (4, 25 and 37°C) and protein concentration were also determined. A wide variation in attachment to ECM proteins among strains was observed. In general, E. coli strains had a higher binding capacity to ECM proteins compared to Salmonella strains. Bacterial attachment was also found to be selective based on the anatomical location of the ECM proteins. Specifically, a higher proportion of strains attached to basement proteins (laminin and collagen IV) than to interstitial proteins (collagen I and fibronectin).
Protein concentration had a minor effect on bacterial attachment to ECM proteins; however attachment was significantly influenced by temperature. Highest attachment levels occurred at 4°C for collagen I and at 25°C for the other three ECM proteins. A strong positive correlation was found between the results of both the crystal violet and epifluorescence methods (r≥0.905, p<0.05) indicating that the former method is useful to study bacterial attachment to ECM proteins, especially in determining the attachment properties of high binding strains.
Based on these results, a subset of strains representing ‘high-‘, ‘intermediate-‘ and ‘low-‘ binding were chosen for further investigation. The influence of pH and salt (NaCl, KCl and CaCl$$_2$$) on attachment to ECM proteins was assessed in vitro. pH, within the range of 5 to 9, had no effect on attachment to ECM proteins, whereas the effect of salt type and concentration (0.1 – 5%) varied depending on strain-ECM protein combination. The effects of three chemical rinses commonly used in commercial abattoirs (2% acetic acid, 2% lactic acid and 10% trisodium phosphate (TSP)) on the attachment were also investigated. Rinses containing TSP were the most effective, producing >95% reduction in attachment to all ECM proteins. Acetic and lactic acids also markedly reduced bacterial attachment to ECM proteins, but at a lower level than TSP.
In addition to ECM proteins, bacterial attachment to animal muscle cells was measured. Specifically, attachment of E. coli and Salmonella strains to primary bovine muscle cells and a cultured muscle cell line, C$$_2$$C$$_{12}$$, at 10 and 37°C was measured by plate-count assay. As shown for ECM proteins, attachment to muscle cells was strain dependant, with temperature being a significant factor. The attachment properties of the two muscle cell types differed significantly, indicating that C$$_2$$C$$_{12}$$ cells are not suitable as surrogates for bovine muscle cells.
Finally, the specificity of interactions between bacterial cells and ECM proteins was studied. In general, addition of certain soluble ECM proteins had significant inhibitory or enhanced effects on binding interactions, depending on the type of ECM protein. For example, while soluble collagen IV inhibited attachment of E. coli M23Sr to laminin, basement membrane proteins increased attachment of E. coli EC614 and H10407 to the interstitial ECM proteins. However, soluble fibronectin did not affect binding interactions. These results could serve as the basis for future studies of potential synthetic analogs to inhibit bacterial attachment to meat surfaces.
Through this study, there is greater understanding of bacterial attachment to specific meat tissues. The knowledge obtained from this study may be beneficial in developing new and more targeted intervention systems for carcass decontamination, potentially reducing carcass contamination, product spoilage and health risk associated with meat.

Item Type: Thesis - PhD Zulfakar, SS attachment, bacteria, extracellular matrix protein, meat surfaces, primary bovine muscle cells Copyright 2013 the author Chapter 2 appears to be the equivalent of a post-print version of an article published as: Zulfakar, S. S., White, J. D., Ross, T., Tamplin, M. L., 2012. Bacterial attachment to immobilized extracellular matrix proteins in vitro, International Journal of food microbiology, 157(2), 210-217Chapter 3 appears to be the equivalent of a post-print version of an article published as: Zulfakar, S. S., White, J. D., Ross, T., Tamplin, M. L., 2013. Food microbiology, 34(2), 369-375Chapter 4 appears to be the equivalent of a post-print version of an article published as: Zulfakar, S. S., White, J. D., Ross, T., Tamplin, M. L., 2013. Cultured C$$_2$$C$$_{12}$$ cell lines as a model for assessment of bacterial attachment to bovine primary muscle cells, Meat science, 94(2), 215-219 View statistics for this item