Biofilms: Our Constant Enemies
By John N. Sofos, Ph.D.
Sanitation efforts in food facilities may fail if biofilms have formed in the environment; however, failure or inadequacy of cleaning/sanitation programs leads to biofilm formation in the first place. Obviously, biofilm prevention—or removal if prevention fails—is a major issue in food processing.
Biofilms may be defined as communities of bacterial cells that a) adhere to each other and to surfaces, b) are surrounded, held together and protected by glue-like materials (polysaccharides) that they produce and c) have different gene expression profiles and phenotypes compared with normal cells. Biofilms occur widely in nature and may become major problems in wounds and surgical instruments as well as in foods and processing facilities.
While microbial cells have the ability to attach to surfaces where they can multiply and grow, their attachment to surfaces is facilitated by the presence of a conditioning layer of nutrients or soil on the surface that may be the result of inadequate or infrequent cleaning. Biofilm formation is then enhanced with increased contact time of the cells with the surface and conditions that increase the rate of bacterial growth, such as nutrient level, acidity (or pH) and temperature. Furthermore, cell adherence and biofilm growth are affected by the type of attachment surface and species of bacteria. Attachment of some bacteria to surfaces, such as stainless steel, polypropylene, rubber and glass, may occur within 20 minutes of contact time. Biofilm development may occur within 24 hours and can grow to millimeters in a few days. Biofilm bonds become stronger with time, but cells may also dislodge or slough off and serve as sources of contamination on other surfaces or food. Biofilm-forming bacteria include Listeria, Salmonella, Campylobacter, Escherichia coli, Pseudomonas, lactic acid-producing bacteria and thermotolerant (heat-tolerating) species. They may occur as mixed cultures, but often one species will dominate.
Just Where Are They?
Biofilms may be present on floors, walls, pipes and drains, and surfaces of equipment including stainless steel, aluminum, nylon, Teflon, rubber, plastic, Buna-N and glass. Food-contact surfaces such as conveyor belts, pasteurizers, crevices, gaskets and dead spaces, as well as areas that are hard to clean and sanitize, may harbor biofilms. Also of concern is the potential attachment of microorganisms on food surfaces, which may affect the efficacy of interventions applied to carcasses, meat, produce or other foods to reduce contamination.
Since microorganisms are ubiquitous and unavoidable as environmental contaminants of raw food products, much interest has been focused on research dealing with the formation and control of biofilms in food environments, especially as DNA fingerprinting techniques are used more frequently to compare bacterial isolates from various sources. Such research has demonstrated that pathogens are able to persist in processing environments for long periods of time. For example, L. monocytogenes has been found to persist in food plants for months, and even up to several years.
Bacterial cells in biofilms may be as much as 500 times more resistant to sanitizing chemicals than free-flowing or suspended cells (planktonic) of the same species. Studies have found that sanitizer concentrations and exposure times may have to be increased 10- to 100-fold to be effective against cells in biofilms compared with interventions found to be effective against planktonic cells. Sanitizers and disinfectants are very effective against planktonic cells because they have a larger surface area exposed to the sanitizer, while attachment of cells and formation of biofilms may also lead to the expression of genes that make bacteria more resistant to sanitizers. In addition, the increased resistance of biofilms to antimicrobial compounds may be due to the exopolysaccharide layer that surrounds the biofilm and protects the cells. Obviously, it is crucial to prevent the formation of and to remove and inactivate existing biofilms. Understanding the conditions and mechanisms that allow bacteria to attach to surfaces, including food and food-contact surfaces, is critical in developing new methods to prevent, inactivate or remove attached bacteria from foods and food-contact surfaces.
Among other research activities, the Pathogen Reduction Laboratory at the Center for Meat Safety & Quality of Colorado State University has been extensively involved in studies of biofilm control and biofilm formation by pathogens such as E. coli O157:H7 and L. monocytogenes on food-contact surfaces and food products. Some selected findings include the following:
E. coli Findings:
• E. coli O157:H7 remained detectable on stainless steel for up to four days in mixed organic acid and water carcass decontamination runoff fluids (washings).
• It may attach to stainless steel and high-density polyethylene food-contact surfaces not only at abusive temperatures (60 °F) but also during cold storage (40 °F), demonstrating the need for development of effective sanitation programs for various plant environments.
• Contaminated beef fat was better than ground beef in the transfer of E. coli O157:H7 to beef fabrication contact surfaces.
• Drying of beef residues on surfaces resulted not only in cell attachment, but also in cell entrapment on surfaces.
• If introduced into the processing environment, E. coli O157:H7 could be considered a source of product contamination since the pathogen can attach and grow under limited nutrient availability if temperature permits.
• Plastic beef-contact surfaces may allow more biofilm formation by E. coli O157:H7 than stainless steel, and inoculated E. coli O157:H7 cells allowed to dry on stainless steel before exposure to reduced nutrient but moist conditions had a stronger strength of attachment but a slower growth rate than cells that remained hydrated.
• If allowed to dry on surfaces, E. coli O157:H7 may have an increased strength of attachment, making its removal more difficult, and thus demonstrating the importance of proper cleaning and sanitizing of equipment surfaces after each use.
• Beef residues may facilitate attachment, while spoilage bacteria may outgrow E. coli O157:H7 in biofilms.
• Application of decontamination interventions on beef carcass sides immediately before fabrication should be useful in pathogen control because such processes decreased attachment and, subsequently, pathogen levels on fabrication equipment surfaces.
• Cell attachment to beef fabrication surfaces varied among nine strains of E. coli O157:H7, making strain selection important in biofilm studies.
• No differences were observed in biofilm formation between quorum-sensing positive and negative strains of E. coli O157:H7.
• Surface material did not influence the fate of biofilm cells exposed to sanitizers.
• When biofilms are present, sanitizers should be applied at the highest allowable concentrations for extended dwell times.
• Adequate cleaning before sanitation is essential.
• L. monocytogenes cells have the ability to adhere to various food-contact surfaces used in food processing, food service and at home, including polyethylene, polypropylene and laminates; if not properly cleaned, they form biofilms that are resistant to sanitizers.
• Multi-species biofilms containing high levels of L. monocytogenes developed and survived for up to 14 days on high-density polyethylene and polypropylene surfaces at room temperature.
• L. monocytogenes survived and was recovered by wiping from kitchen countertop (laminate) surfaces at room temperature (77 °F; 50% and 90% relative humidity) in the presence of food residues (ham homogenate) for at least 96 hours. Populations recovered at 90% relative humidity were higher than those recovered at 50% relative humidity.
• Biofilm survival was greater on rough than on smooth high-density polyethylene surfaces.
• Sanitizer efficacies were higher against older biofilms on smooth surfaces versus those on rough surfaces.
• There were no statistical differences in the efficacy of various wiping materials in removing pathogen cells from laminate surfaces, but numbers of cells recovered ranged from 3.6 to 1.9 logs per square centimeter at 50% relative humidity.
• Although sanitizers were effective against attached L. monocytogenes cells, efficacy decreased as the biofilm developed.
• Sanitizers (acetic or lactic acid-, sodium hypochlorite-, quaternary ammonium-, or hydrogen peroxide-based) were effective in reducing L. monocytogenes, especially in younger biofilms.
• A lactic acid-based sanitizer (pH 3.03) was the most effective, while quaternary ammonium-based sanitizers of higher pH (10.5–11.5) were more effective than those of lower pH (6.2–8.7).
• Of products commonly found in households, effectiveness against three pathogens (Salmonella, E. coli O157:H7 and L. monocytogenes) increased in the order: household bleach (0.0314%) > hydrogen peroxide (3%) > undiluted vinegar > baking soda (50% sodium bicarbonate), while pathogen sensitivity followed the order Salmonella > E. coli O157:H7 > L. monocytogenes.
• Sanitizer activity increased at warm temperatures (130 °F) and longer exposure times (10 minutes).
• Sanitation of cutting boards should be performed with selected sanitizers after each use, or at least daily, in order to achieve maximum efficacy.
Prevention of biofilm formation may be accomplished by avoiding conditions that lead to cell attachment and selecting conditions that make the environment unfavorable for microbial growth; however, this is not often possible. Proper cleaning and sanitation work best for biofilm prevention while its removal is only necessary if prevention fails. Biofilm removal and inactivation is achieved by combining proper cleaning and sanitizing agents, adequate exposure time, proper temperature and mechanical action. This combination dissolves the biofilm and the organic material to which it adheres, allowing the sanitizer to inactivate the released, sensitive cells. Extensive scrubbing with proper chemicals is important in biofilm removal. Incomplete biofilm removal may promote growth of remaining cells, and appearance of sporadic bacterial colonies on agar plates from sanitized equipment swabs may indicate presence of biofilms.
The results presented are derived from studies supported in part by the National Integrated Food Safety Initiative of the USDA Cooperative State Research, Education and Extension Service, the National Cattlemen’s Beef Association, the American Meat Institute Foundation, the National Pork Board, and the Colorado Agricultural Experiment Station.
John N. Sofos, Ph.D., is a University Distinguished Professor, Center for Meat Safety & Quality and Food Safety Cluster of Infectious Diseases Supercluster, Colorado State University, Department of Animal Sciences, Fort Collins, Colorado. He can be reached at telephone: 970.491.7703; Fax: 970.491.5326; e-mail: [email protected].
The author would like to thank the following postdoctoral fellows and graduate students for their contribution to the research findings summarized above: J. Adler, D. Dourou, G. Geornaras, S. Gupta, S. Parikh, C. Simpson, P. Skandamis, J. Stopforth, H. Yang and Y. Yoon.