Project Summary

Formation, Destruction and Removal of Escherichia coli O157:H7 Biofilms on Materials that Comprise Meat-contact Surfaces in Beef Fabrication and Grinding Processes

Principle Investigator(s):
John Sofos,Yohan Yoon, Catherine A. Simpson, Dimitra Dourou, Jeremy M. Adler, Avik Mukherjee, Ifigenia Geornaras, Lawrence Goodridge, John A. Scanga, Keith E. Belk, and Gary C. Smith
Colorado State University
Completion Date:
May 2008



Biofilms are composed of matrix-encapsulated bacteria adherent to each other and/or to surfaces (Marshall, 1992; Hood and Zottola, 1995). They form clusters of cells or microcolonies enclosed within an exopolysaccharide (EPS) matrix and surrounded by pores and channels that link individual clusters (Lappin-Scott and Bass, 2001). Biofilms can be found in nearly all environments (Hamilton, 1987; Marshall, 1992; Costerton et al., 1994, 1995). Thus, a major concern in food processing facilities is cross-contamination of foods with cells from biofilms. Biofilms are notoriously difficult to remove, and can act as repeated sources of contamination as cells in biofilm exhibit an increased resistance to many sanitizers (Bower and Daeschel, 1999; Chmielewski and Frank, 2003; Brightwell et al., 2006). Correspondingly, contaminated processing equipment is a leading cause of domestic outbreaks of foodborne Escherichia coli O157:H7 infections (Rangel et al, 2005). 

Substantial efforts to control pathogens at the harvest-floor level can be nullified by contaminated food-contact surfaces encountered during fabrication processes. There is evidence suggesting that equipment surfaces associated with beef slaughter, as well as the processing environment, may be potential sources of cross-contamination of carcasses with E. coli O157:H7. Barkocy-Gallagher et al. (2001) recovered E. coli O157:H7 and E. coli O157 isolates from feces, hides and carcasses at four different beef processing plants. However, only 65.3% (post-evisceration) and 66.7% (in the cooler) of individual isolates from carcasses matched those recovered pre-evisceration. Tutenel et al. (2003) also showed that molecular types of E. coli O157:H7 in the plant environment were different than those on the hides of the slaughtered animals. A study by Gun et al. (2003) demonstrated the presence of E. coli O157:H7 on equipment surfaces and in the environment (abattoir floor, benches including conveyors, knives, aprons, saw, hooks, hands) of a beef abattoir. Eight strains of E. coli O157:H7 were isolated from carcasses and six strains from environmental samples such as knives (two strains), hands (two strains), aprons (one strain) and the floor (one strain). E. coli O157:H7 has also been recovered from fabrication-floor conveyor belt food-contact surfaces during preoperational and mid-shift inspections (Chmielewski and Frank, 2003; Rivera-Betancourt et al., 2004). Aslam et al. (2004) reported that the genetic fingerprints of generic E. coli isolates from animal hides, carcasses and conveyors were genetically related to isolates from ground beef. Inadequate cleaning of equipment surfaces can, thus, result in contamination of beef products. One study (Farrell et al., 1998) examined the potential for attachment of E. coli O157:H7 to a meat grinder surface during the grinding of artificially contaminated beef, and the inactivation of attached cells during cleaning and sanitizing. Results showed that the pathogen was transferred to the meat grinder within 5 min of contact, and that detergent washing and sanitizing of the grinder with chorine (200 ppm) or peroxyacetic acid (0.2%) resulted in low recoveries of viable bacteria; however, enrichment procedures showed a higher survival rate indicating that cells, potentially injured, remained on the surface. These studies support the concern that biofilm formation in beef packing plants may have serious consequences due to the potential for contamination of unadulterated food products through biofilms. Consequently, biofilms have become one of the major concerns in the food industry related to food safety (Stopforth et al., 2002). As directly quoted from Chmielewski and Frank (2003), “the significance of biofilms in food processing is not well understood because of the lack of direct observation of biofilms in this environment and a lack of research using model systems that closely simulate the food system environment”. For these reasons, the development of E. coli O157:H7 biofilms on meat-contact surface materials commonly encountered during beef carcass fabrication and a multitude of factors (i.e., type of surface material, fluid flow, fluid level, temperature, substrate, natural flora) which may inhibit or promote such development, were investigated. Furthermore, the efficacy of sanitizers in inactivating E. coli O157:H7 cells attached to meat-contact surfaces was evaluated, as well as the survival/growth of the pathogen in ground beef cross-contaminated with biofilm- and planktonic-derived cells surviving exposure to sublethal sanitation.

Cell density-dependent signaling systems, known as quorum sensing, occur in many bacteria (Smith et al., 2004). Quorum sensing controls a variety of microbial physiological functions including motility, conjugation, competence, sporulation, virulence, and biofilm formation (Hammer and Bassler, 2003). Autoinducer-2 (AI-2) is a type of quorum sensing molecule found in both Gram-positive and -negative bacteria, including E. coli, Salmonella, Vibrio cholerae, enterococci, Mycobacterium tuberculosis, and Helicobacter pylori, among others (Surette and Bassler, 1998; 1999). The novel interest has been raised in designing systems to disturb or control cell-to -cell signaling processes in bacteria. In our efforts to control pathogens and enhance food safety, studies were also conducted to investigate quorum sensing-regulated biofilm formation, and specifically, the potential involvement of quorum sensing in pathogen growth and biofilm formation. 

The stated objectives for this work were: To evaluate the potential for Escherichia coli O157:H7 biofilm formation and the efficacy of sanitizers to control biofilm formation on food-contact surfaces, and to investigate the potential for pathogen cells, surviving in biofilms after sanitation, to survive in cross-contaminated beef products, as well as factors affecting biofilm formation and the potential contribution of cell-to -cell communication in biofilm formation.

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