Food Safety Magazine

Sanitation | February/March 2016

Biofilm and Pathogen Mitigation: A Real Culture Change

By Siobhan S. Reilly, Ph.D.

Biofilm and Pathogen Mitigation:  A Real Culture Change

Every food processing plant contains a biofilm somewhere in the facility or on equipment. No matter how well the facility cleans and sanitizes, the biofilm may continue to be a source of pathogens and may manifest as disease in animals or humans.

The intent of this article is to offer a solution to prevent, reduce or eliminate this biofilm/pathogen from the food processing plant when there are no other possible corrective actions. Our solution is to inoculate the production environment with probiotic microorganisms proven to be inhibitory to pathogens. This action changes the “culture” or the microbial population inherent to the food processing environment from bad to good. Salmonella and Listeria have been removed from commercial food processing environments by the distribution of dry probiotic formulas. This process is a successful intervention-and-prevention technology.

History and Current Culture of Food Safety
The concept of food safety goes back hundreds if not thousands of years. Food safety practices began accidentally by attempts to keep food in a more favorable state of consumability for longer periods, known as food preservation. Sometimes this did not work (e.g., canning and lead poisoning), but some of these earlier problems did spark the interests of scientists all over the world. In 1673, Antonie van Leeuwenhoek discovered “wee beasties” using his newly developed microscope, but it wasn’t until the 1800s that the association of foodborne illness and microorganisms became a prominent field of research. Parasites and bacteria became a vector for study by researchers James Paget and Richard Owen (Trichinella in 1835), Louis Pasteur (generic bacteria in 1860), Daniel Salmon (Salmonella in 1885), Theodor Escherich (Escherichia coli in 1885), August Gartner (Bacillus in 1888), Emile Pierre-Marie van Ermengem (Clostridium in 1895) and M.A. Barber (Staphylococcus in 1914).[1] Novel recognition of food safety resulted from Upton Sinclair’s book The Jungle (1906). The book outlined horrible conditions in meat processing that prompted the formation of the Pure Food and Drug Act of 1906 and the Meat Inspection Act of 1906. Fast-forward to 1982 with research related to an E. coli O157 outbreak in the United States,[2] and the most pivotal and visible food safety event of our time, the 1996 E. coli O157:H7 outbreak from undercooked beef patties served at a fast-food establishment.[3] This event resulted in a major paradigm shift in that food safety became a focus for millions, and the entire food industry began to pay attention.

In the last 20 years, food safety has become a high priority for the world’s food producers. The meat and poultry industry, governed by the U.S. Department of Agriculture (USDA), charged forward with Hazard Analysis and Critical Control Points (HACCP) systems and aggressive investigations into new processing techniques, detergents, sanitizers, test methods, sampling protocols, etc. The rest of the food industry [mostly regulated by the U.S. Food and Drug Administration (FDA)], eventually came along under the Food Safety Modernization Act (FSMA) of 2011. The combined industries of meat, poultry, juice, seafood, fruits, vegetables, dairy, pet food, nuts, grains, spices, starches, seasonings and even packaging have advanced quickly through the maze of food safety regulations, customer requirements and perceived risk analysis to promote a food safety culture.

The list of ever-growing food safety focus areas includes:[4]

Contamination control: allergens, chemical hazards, cross-contamination, microbiological hazards, physical hazards and reduction methods

Facilities: air/water monitoring, design, Standard Operating Procedures (SOPs), Good Manufacturing Practices, hygienic equipment design and sanitation

Food types: beverages, dairy/eggs, ingredients, low moisture/dry, meat/poultry, natural/organic, produce, ready-to-eat, refrigerated/frozen and seafood

Management: best practices, case studies, food defense, international, recall/crisis, risk assessment and training

Process control: best practices, intervention controls, packaging, process validation and processing technologies

Regulatory: audits/certifications/Global Food Safety Initiative, FDA, FSMA, USDA, guidelines, HACCP, inspection and international standards/harmonization

Sanitation: biofilm control, clean-in-place/out-of-place, cleaners/sanitizers, environmental monitoring, food preparation/handling, personal hygiene/handwashing, pest control and Sanitation SOPs

Supply chain: foodservice/retail, growers/Good Agricultural Practices, imports/exports, regulation, temperature control/cold chain, traceability/recall and transportation

Testing/analysis: allergens, chemical hazards, environmental testing, laboratory management, methods, microbiological hazards, physical hazards and sampling/sample prep

Overwhelming, isn’t it? The food processor has had to evolve, taking most of these programs and combining them with employee training to produce an effective “culture” capable of providing a safe and wholesome food or ingredient. The industry is doing its best to juggle a majority of these programs, but we still have increasing incidents of foodborne illness by some of the same pathogens discovered and vetted decades ago. Why?

The Problem
All that are left after cleaning/sanitizing are the pathogens.

Even with the best cleaning and sanitizing programs, the control of contamination by pathogenic microorganisms continues to be a problem for the food processing industry. Product can be made under optimal conditions, using controlled ingredients, effective lethality steps, intervention strategies and cleaning and sanitizing of equipment by the most careful and fully trained employee. Nevertheless, these steps may be undone by a positive test for a pathogen performed by the company itself, a customer or a governmental agency. The process is then scrutinized, some or all of the equipment/environment is cleaned and sanitized again and samples are analyzed for some suspected defect. The results tend to dictate the next step—either continuing production or starting corrective actions again. But this problem never goes away. A few months later, the same defect is detected, hopefully by the company’s own test-and-release program, corrective actions are again taken, product disposition is determined and the process is repeated over and over again.   

One major contributor to perpetual microbial defects is that some equipment is difficult or impossible to completely clean and sanitize. Thus, pathogens may colonize food product surfaces or other nonfood contact surfaces and aerosolize. They may be detected during routine testing and screening of the food or the food production facility, causing the company or the government to effectively shut down operations and/or recall product from the market. Even worse, if consumers are exposed to the food containing the pathogen, sickness or even death may occur.

The culprit here is biofilm, a bacterial matrix made up of carbohydrates, proteins, nucleic acids and other components that facilitate gene regulation for communication, defense and growth of said pathogen. Some bacteria can produce a biofilm that protects them from their environment and helps them adhere to food equipment surfaces. If a biofilm has developed, which it will, and if the biofilm contains a microbiological defect, which it will, then the probability of that biofilm perpetuating in the equipment and breaking off or aerosolizing into the food is almost certain.[5,6] Interfering with this or preventing it from occurring is crucial to producing a safe food product.

While many products are used to clean (detergent/surfactant) and sanitize (lethal agent), they are often difficult to use, mix and apply. If improperly used, these agents may not work, may be corrosive to equipment and toxic to employees, and may be a dangerous chemical hazard if they contaminate food. At the very least, the detergents and sanitizers are costly and may impart undesirable organoleptic changes in the food. And there are many, many surfaces that can be neither cleaned nor sanitized in a food production facility. These surfaces are, in many instances, harboring pathogens.

A Solution: Probiotics
Probiotics are defined by the World Health Organization as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host.”[7] These probiotics must be alive when they are administered and they must be delivered in an adequate concentration.[8] Two of the most well-known probiotic bacteria include Lactobacillus and Bifidobacterium, which have been researched extensively.[9,10] The key word “alive” is very important during the selection process. These microorganisms, in order to function, must be in the active and aggressive vegetative state. Spore formers (e.g., Bacillus) may not provide consistent or aggressive interactions with other microorganisms in order to be inhibitory since the spore form of these microorganisms is dormant. There are thousands of strains of Lactobacillus and Bifidobacterium, each differing in its ability to grow, metabolize and provide desirable functions as probiotics. With this in mind, careful selection of the strains is the key to making them work. Just because a product is called a probiotic, however, doesn’t mean it functions as intended.

We have screened thousands of Lactobacillus and Bifidobacterium strains to find ones that are effective probiotics. We focus on selection criteria that screen for the most aggressive strain against pathogens (e.g., Salmonella) that has antipathogen biofilm properties. There are several physical and chemical mechanisms in removing and replacing pathogens from a biofilm on a food contact surface, much more than just producing inhibitory substances in a biofilm. In addition to being inhibitory by competing for nutrients or producing some metabolic product that is antimicrobial (e.g., lactic acid), probiotics may act by the following mechanisms:

1) Blanketing: This mechanism simply populates the food contact surface with probiotic biofilm and prevents the pathogen from binding by taking up all the space. Also, the probiotic population may excrete anti-adhesion molecules that change the molecular charge or the hydrophobicity of the surface, preventing pathogen binding. These probiotic-specific molecules will prepare the surface for the new, incoming good bacteria (Figure 1).[11]

2) Biosurfactant production: When a probiotic is able to excrete a biosurfactant, this mechanism may break down a biofilm if it already exists. These “slippery detergents” change surface tension, allowing the surface to become wet and facilitate dispersion of old biofilm or prevent pathogen adherence by letting it slide away.[12] Lactobacillus and Bifidobacterium may compete by developing their own biofilm, but first they will produce a biosurfactant to get rid of any pathogenic biofilm that may be found in a hidden niche on a piece of equipment. These biosurfactants have lower toxicity and higher biodegradability than conventional synthetic surfactants, and they are not easily synthesized by conventional methods (Figure 2).[12]

3) Exopolysaccharide (EPS) production: This mechanism involves a matrix released from the cell that dampens down a pathogen’s ability to remain in the processing environment. Also known as anti-adhesion polysaccharides, EPS may modulate the expression of pathogen genes that produce biofilm or surface adhesins. The generated EPS can then eventually aid in the probiotic’s ability to establish its own biofilm (Figure 3).[13]

Our probiotic compositions are dry powders capable of being dispensed from any suitable aerosol container or even by hand. This allows the transport of the probiotics into and through equipment mounted vertically or high over the factory floor, as well as equipment that has joints with geometries that make it difficult for traditional solutions to reach. The dry aerosol form allows for multidirectional application of the probiotic composition on the desired surface and on surfaces that could easily contain a pathogenic biofilm but never, ever have seen the light of day or a cleaning tool.

Validation Experiments
The objective was to determine whether probiotics influence the removal of pathogenic biofilm from food contact surfaces. The intent was to mimic food passing through a production line that has a pathogenic biofilm and has had the probiotic added to the surface.

Procedures:
•    Food contact surfaces: Two approximately 1.5-L cylindrical canisters were used:

     •    304 stainless steel, 12.4 cm × 15.9 cm

     •    Food-grade plastic, 12.4 cm × 17.8 cm

•    Pathogens: Two pathogens (Salmonella and Listeria) were tested separately. These microbes were isolated from biofilms in commercial food processing facilities following routine cleaning and sanitation. They were grown in tryptic soy broth at 35 °C for 24 hours and subcultured twice more on consecutive days. The cells were prepared by centrifugation at 4,000 rpm for 10 minutes. The pellet was resuspended in 0.1% buffered peptone water (BPW). Serial dilutions (10-fold) were used to prepare the test solutions of Salmonella (~103 CFU/mL) and Listeria (~103 CFU/mL).

•    Biofilm formation: In every trial, the inside of each canister received 1 mL of the Salmonella or Listeria solution applied evenly with sterile pipettes. The 1 mL was distributed over a 2- to 3-hour period with agitation. Once the entire volume was delivered evenly over the surface, canisters were dried overnight at 21 °C in a closed container.

•    Treatments: A separate experiment was conducted with each of two dry probiotic formulas. In control, no-probiotic trials, each canister was dusted on the inside with 3.5 g dry carrier. In treatment, probiotic trials, each canister was dusted on the inside with 3.5 g dry carrier containing probiotics. Formula One contained 3.2 × 1010 CFU/g Lactobacillus plus Bifidobacterium, and Formula Two contained 4.5 × 1010 CFU/g of the same Lactobacillus and Bifidobacterium plus Enterococcus. The applications were left overnight at 21 ° C in a closed container.

•    Processing: In each trial, 454 g dry, sterile, hard food pellets were added to the canisters 15 hours after dry powder application. The canisters were placed on their side on a rotating platform and rotated for 10 minutes at 10 rpm.

•    Sampling and analysis: After 10 minutes, the food was transferred to 4× the amount (1/5 dilution) of BPW and enriched at 37 °C for 22 hours. A secondary enrichment in 5 mL prewarmed brain-heart infusion broth was inoculated with 0.1 mL BPW-enriched sample and incubated for 3 hours at 37 °C. The enriched samples were tested for Salmonella by immunological method AOAC-030301 and DNA-PCR method AOAC-100201. For Listeria, 454 g food were enriched (1/5 dilution) in UVM-modified Listeria enrichment broth for 26 hours at 30 °C. A 0.1-mL portion was then transferred to a secondary enrichment (MOPS-BLEB) for 18–24 hours at 35 °C. Detection of Listeria was by DNA-PCR method AOAC-RI-30502.

•    Time testing: To monitor the removal of the Salmonella and Listeria over time (days) by the probiotic, after sampling as above, another 3.5 g of either the control or probiotic dry powder were added to each container. After 15 hours, another 454 g food were added, and canisters rotated for 10 minutes before sampling. Testing was stopped after three or more consecutive samples were negative for Salmonella or Listeria.

•    Replications: Three replicates for each pathogen and formula were performed as described above on different days.

•    Response variable: Data were recorded as the number of days that samples gave positive tests for pathogens during each trial.

Results and Conclusions:
The presence of dry probiotic formulas decreased pathogenic biofilm persistence during mechanical or abrasive action of dry dog food on both stainless steel and plastic surfaces (Figures 4 and 5). Pathogenic biofilm persistence on the plastic surface was greater in trials with Formula One (Figure 4) than with Formula Two (Figure 5), but this difference is not attributed to the probiotics. Persistence on stainless steel was similar between the trials with Formulas One and Two. When no probiotic was applied, pathogenic biofilms remained for up to 10 days before the abrasion of the food on the biofilm finally removed them. When probiotic was applied, however, Salmonella and Listeria biofilms were removed 2 to 6 days faster.  

The application of the dry probiotic blends was effective and consistent in removing an exceptionally large number (1,000) of pathogen cells (both Salmonella and Listeria) on common food processing surfaces (stainless steel and plastic). This biofilm containing a large number of pathogen cells would not likely ever be seen in practice. With the smaller concentrations of pathogens found in typical plants, we believe that dry probiotics will be even more effective in eliminating and preventing pathogenic microorganisms in any food processing environment.  

Field Application Experience
Currently, these probiotic formulas are being used successfully in several food processing plants across the U.S., and even some involved in past recalls. The dry probiotic application has been effective with a variety of surfaces, equipment and food. These probiotics provide protection in the environment and the food, with benefits to both processor and consumer.

Siobhan S. Reilly, Ph.D., is president and CEO of Log10® LLC in Ponca City, OK. She received a Ph.D. in food science from Oklahoma State University. She can be reached at [email protected]

References
1. Bari, L and DO Ukuku. 2015. Chapter 1, “History and Safety of Food Past, Present and Future,” in Foodborne Pathogens and Food Safety, eds. L Bari and DO Ukuku (Boca Raton, FL: CRC Press).
2. Riley, LW et al. 1983. “Hemorrhagic Colitis Associated with a Rare Escherichia coli Serotype.” N Engl J Med 308(12):681–685.
3. en.wikipedia.org/wiki/1993_Jack_in_the_Box_E._coli_outbreak.
4. www.foodsafetymagazine.com/.
5. Bower, CK and MA Daeschel. 1999. “Resistance Responses of Microorganisms in Food Environments.” Int J Food Microbiol 50(1):33–44.
6. Van Houdt, R and CW Michiels. 2010. “Biofilm Formation and the Food Industry, a Focus on the Bacterial Outer Surface.” J Appl Microbiol 109(4):1117–1131.
7. www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf.
8. Sanders, ME. 2008. “Probiotics: Definition, Sources, Selection, and Uses.” Clin Infect Dis 46(2):S58–S61.
9. Servin, AL. 2004. “Antagonistic Activities of Lactobacilli and Bifidobacteria against Microbial Pathogens.” FEMS Microbiol Rev 28(4):405–440.
10. Gomes, AMP and FX Malcata. 1999. “Bifidobacterium spp. and Lactobacillus acidophilus: Biological, Biochemical, Technological and Therapeutical Properties Relevant for Use as Probiotics.” Trends Food Sci Technol 10(4):139–157.
11. Rendueles, O and J-M Ghigo. 2012. “Multi-Species Biofilms: How to Avoid Unfriendly Neighbors.” FEMS Microbiol Rev 36(5):972–989.
12. Fracchia, L et al. 2010. “A Lactobacillus-Derived Biosurfactant Inhibits Biofilm Formation of Human Pathogenic Candida albicans Biofilm Producers.” Appl Microbiol Biotechnol 2:827–837.
13. Ku, S et al. 2009. “Enhancement of Anti-Tumorigenic Polysaccharide Production, Adhesion, and Branch Formation of Bifidobacterium bifidum BGN4 by Phytic Acid.” Food Sci Biotechnol 18(3):749–754.

Categories: Contamination Control: Microbiological, Reduction Methods; Sanitation: Environmental Monitoring, SSOPs