Effect of temperature on bacterial attachment to muscle extracellular matrix proteins

Meat surfaces become contaminated with bacteria during slaughter and processing. Understanding bacterial attachment process to specific structures of meat could lead to interventions that reduce health risk and product spoilage. Seventeen bacterial strains consisting of ten Escherichia coli and seven Salmonella strains were tested on its attachment properties to muscle extracellular matrix (ECM) proteins (collagen I, fibronectin, collagen IV and laminin), measured by using a crystal violet stain assay. The effect of temperature on attachment was also reported. Only five of 17 strains showed significant attachment to ECM proteins. Highest attachment levels occurred at 4°C for collagen I and at 25°C for the remaining three proteins. Higher number of bacterial strains attached to laminin and collagen IV as compared to the other proteins. Our study demonstrated that attachment properties to ECM displayed a distinct variation among strains and that temperature had a major effect on this process. Introduction Meat is among the top food vehicles that causes foodborne illnesses. Microbial contamination of meat carcass surface is certain to occur during slaughter and post-slaughter processing (Jackson et. al, 2001). Attachment to the targeted surface is the essential initial step for any contamination events to occur (Selgas et. al, 1993). Due to the complexity and the heterogenous nature of meat tissue, studies of bacterial attachment to specific structures within meat would be beneficial to understand the specific mechanisms of the attachment process. Extracellular matrix proteins (ECM) are the major components of meat connective tissues which are located at the fascia and within the muscular tissues itself (Taylor, 2004). In this study, we investigated the bacterial attachment properties to ECM and the effect of temperatures relevant to meat production on attachment process. Materials and Methods Ten E. coli and seven Salmonella strains were used in this study (Table 1). Bacterial attachment to ECM proteins was measured using a modified crystal violet staining method (Štryiak et. al, 1999). Attachment assay were done on ECM (collagen I, fibronectin, collagen IV and laminin) coated 96-well plates. Attached cells were quantified by measuring absorbance values using a microplate reader (Bio-rad® Benchmark microplate reader, USA) at 595nm. The absorbance values obtained were corrected by subtractions of the average absorbance values from un-inoculated control wells which served as blanks. Experiments were performed in triplicate and each experiment was repeated twice. The averages of three absorbance values were calculated. Results for all experiments were presented as mean ± standard error of the mean (sem). Data were analyzed using ANOVA followed by Tukey’s test. All statistical tests were performed using the IBM SPSS Statistics 19. Results were considered significant when p < 0.05, unless otherwise stated. Table 1: List of bacterial strains Strain ID Bacterial species Serotype EC01 Escherichia coli M23Sr (with streptomycin resistance) EC02 Escherichia coli O157:H7 sakai EC03 Escherichia coli O157:H7 EC04 Escherichia coli O157:H- EC05 Escherichia coli O157:H12 EC06 Escherichia coli O26:H11 EC07 Escherichia coli O111:H- EC08 Escherichia coli O127:H6 EC09 Escherichia coli O78:K80:H11 EC10 Escherichia coli M23 (parent strain) ST135 Salmonella enterica Typhimurium 135 ST9 Salmonella enterica Typhimurium 9 SI Salmonella enterica Infantis SA Salmonella enterica Anatum SC Salmonella enterica Chester SM Salmonella enterica Mississippi SS Salmonella enterica Sofia Results and Discussion Results from this study showed wide variation in attachment to ECM between the strains tested (Figure 1). Overall observations indicated that only five strains attached significantly to ECM at all temperatures. In general, Salmonella strains attached in lower numbers than E. coli strains. Attachment to ECM was noticeably selective based on the anatomical location of the proteins. More strains showed relatively high attachment to basement membrane proteins (laminin and collagen IV) than interstitial proteins (collagen I and fibronectin). This indicates that attachment to ECM is a specific interaction that involves specific receptors which are not present on all strains (Ofek et al., 2003). We also demonstrated that attachment to ECM is highly influenced by the temperature. Highest attachment levels of cells were observed at 25°C for all proteins except at 4°C for collagen I. We relate this behavior with the presence of bacterial adhesins such as exopolysaccharides and curli that might mediate the attachment process, in which temperature is known to affect their expressions (Olsén et. al, 1993). These particular findings on the effect of temperature are especially important as it is one of the important control parameter in an abattoir. It would also provide the information on the likeliness of bacterial attachment process to occur throughout the various post-slaughter processes. From this study, we believed that by understanding the mechanisms of bacterial attachment to ECM proteins could lead to the development of new interventions targeted at specific structures of the meat surface to help in reducing carcass contamination, product spoilage and health risk associated with meat.