1. Biofilm Formation and
Food Safety
BY: MARY MADISON

2. Presentation Overview
Questions?
Microbes of
concern.
Control
Measures
How to test
for
biofilms.
Importance
to food
safety.
How are
they
formed?
What are
“biofilms”?

3. What are “biofilms”?
• Biofilms are microbial cells that have aggregated together on a
surface.
• Biofilms can be made up of one type of cell or a multitude of cell
genera.
• Sites for biofilm formation include natural materials, metals,
plastics, and more.
• All that is needed for a biofilm to form is moisture, nutrients, and a
surface.
• Everyone in the world has had some instance of biofilm formation
in their body!

4. What Biofilms do for Microorganisms
• Biofilms are held together by
sugary molecular strands
known as EPS or “extracellular
polymeric substances”.
• The cells that secreted the EPS
are protected by the formation
of the resulting biofilm.

5. 5 Stages of Biofilm Development
Stage 1.
• Initial reversible attachment of planktonic
microbes to a surface. Mostly physical.
Stage 2
• Permanent chemical attachment.
• Single layer slime development. (EPS)
Stage 3
• Early vertical development.
• Start of 3D structure formation.
Stage 4
• Multiple towers are formed with channels
linking them. (nutrient movement)
Stage 5
• Mature biofilm formed.
• Dispersal of more planktonic microbes.

6. Microbial Communication in the
Formation of Biofilms
• Cell-to-cell signaling , known as quorum sensing, is a system of
stimulus and response correlated to population density.
• Many species of bacteria use quorum sensing to coordinate gene
expression according to the density of their local population.
• Quorum sensing allows for bacteria to display a unified response-
advantageous to the population by facilitating tolerance to stress and
providing access to nutrients and more favorable environmental niches.
• The type of mechanisms and the participants of the mechanism differ
from organism to organism.

7. Model of quorum sensing
and gene expression in
Vibrio fischeri. (Gram -)
• LuxI produces AHL which
makes it possible for LuxR
to bind the DNA ‘Lux Box’.
• However, high concentration
of AHL is required for LuxR-
Lux Box binding.
• LuxR-Lux Box binding
activates luciferase genes,
profucing bioluminescent
molecules and even more
AHL.

8. • A major concern in the food industry is the continuous recontamination of foods
due to persistent bacteria in processing equipment.
• Removal of persistent bacteria and biofilms requires costly efforts leading to lost
productivity and environmental issues.
• Estimations of food poisoning cost in the United States vary widely, but a report
by the FDA in 1997 suggested that there were between 6.5 and 33 million cases
per year, resulting in 9000 deaths and costing $6.5–34.9 billion.
• Products that are a contaminated with microbes may be subject to recall,
resulting in expensive losses for companies.
Impact on the Food Industry

9. Impact on Food Safety
• Product contamination occurs from sloughing bacteria that are shed
periodically by the film and can reattach on equipment down stream or
in the food product itself.
• If the cells released are pathogenic, the product may be considered
adulterated, and cause a foodborne illness outbreak.
• 21 U.S. Code § 342 - Adulterated food
• Example: From April to July 2008, more than 1,329 people across 43
states were infected with Salmonella poisoning. They were linked to
fresh tomatoes from Mexico and Florida. Washing water was reported
to be contaminated.
• Restaurants would not serve tomatoes on any of their food products.

10. Methods of Detection
• Direct
• Help determine locations of biofilms
• Can require lots of equipment.
1. RODAC
2. Bioluminescence
• Indirect
• Great for determining if sanitation and cleaning procedures are
being effective on the whole.
• Does not give location!
1. Flushing with sterile buffer and enumerating the collected micro-flora.
2. Residual protein and glucose detection swabs.

11. Direct Testing for biofilms
• RODAC plate: ‘Replicate Organism Direct Agar Contact’
• A direct method of testing the surface of objects to
determine viable organisms.
• Also helps determine if method of sanitation is working
and microbial load.
• Limited area of detection
• ATP Testing
• ATP can be detected by bioluminescence.
• Luciferin + Luciferase Light
• ATP detected cannot be distinguished between residue left
over from cleaning or microbial organisms.
• Just indicates a surface’s cleanliness.

12. Control Measures
Improved Cleaning
• Turbulent two-phase flow.
• reduce biofilm levels by 6 log cycles.
• air +detergent
• Ice Pigging
• A semi-solid that is pumped like a liquid and flows through changes in diameter, bends passes
through fittings without blockage.
• Ideal for pipes
Temperature Cycling
• Programmed temperature spikes could control the growth of thermo-
resistant microbes.
• Good use in the milk industry
• Can help extend run time of pasteurization between cleanings.

13. Control Measures
Molecular Brush
• Blocking of attachment
• Coat surface with an inert material that physically blocks microbial attachment.
• Polyethylene glycol (PEG)
• Microbial adsorption depends on density of the polymer.
• Emerging research on its effectiveness between microbes.
• Pseuedomonas sp. versus L. monocytogenes on stainless steel, (Wei et al., 2003)
Developing Options
• Bacteriophages as a control agent.
• Bacteriophage: virus that infects bacteria.
• Lytic bacteriophages acting on EPS-synthesising bacteria produce polysaccharases, degrading
capsular and other EPS and permitting access to the cell surface.
• Host-specific, there should be no risk to other parts of the industry.

14. Problematic Microbes
L.monocytogenes
• Commonly found in sites such as floor drains, conveyor belts,
stainless steel surface equipment, and cold rooms.
• Associated foods: unpasteurized milk, soft cheeses, raw fruit and
vegetables, and ready to eat meats (delis).
• Can grow at refrigerated temperatures.
Salmonella spp.
• Associated foods: ray meat, poultry, eggs, milk and dairy products,
nuts.
• Extremely heat sensitive.
• Can survive in lower water activity conditions than most bacteria.
(Peanut Butter)
Escherichia coli O157:H7
• Found in the tracts of infected animals, and attach to grooved surfaces (cutting
board).
• Associated foods: ground beef, unpasteurized milk, unpasteurized apple cider,
and leafy greens.
• Unique in being able to survive in acidic foods such as apple cider and
mayonnaise.

15. Closing Summary
THE TAKE HOME POINTS
Molds
Prevent
Instead of
Get Rid
Of
Complex
Responsive
to
Environment
Yeast
Industry
Pathogens
Communicate
Dynamic
Bacteria EverywhereQuestions?

16. References
• Annous, Bassam A., Pina M. Fratamico, and James L. Smith. "Quorum
Sensing in Biofilms: Why Bacteria Behave the Way They Do." Journal of
Food Science 74 (2009): R24-37. IFT.org. Web. 11 Apr. 2014.
• Blaschek, Hans, Hua Wang, and Meredith Agle. "Ch. 6." Biofilms in the
Food Environment. Ames, IA: Blackwell Pub., 2007. 95-110. Print.
• Brooks, John D., and Steve H. Flint. "Biofilms in the Food Industry:
Problems and Potential Solutions." International Journal of Food Science
& Technology 43.12 (2008): 2163-176. Print.
• Cramer, Michael. "Biofilms: Impact on the Food Industry." Food Safety
Magazine. Food Safety Connect, June-July 2012. Web. 10 Apr. 2014.
• Tarver, Toni. "Biofilms: A Threat to Food Safety." 1 Feb. 2009`: 46-52. IFT
Publications. Web. 10 Apr. 2014.
• Wei, J., Ravn, D.B., Gram, L. & Kingshott, P. (2003). Stainless steel
modified with poly(ethylene glycol) can prevent protein adsorption but
not bacterial adhesion. Colloids and Surfaces B: Biointerfaces, 32, 275–
291.