Biofilm Formation and Food SafetyBY: MARY MADISON

Presentation Overview

Questions?

Microbes of

concern.Control

MeasuresHow to test for

biofilms.

Importance to food

safety.

How are they

formed?

What are “biofilms”

?

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!

What Biofilms do for Microorganisms

*Limits penetration of cleaning and sanitizing agents.

Removal can be difficult!

• 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 Stages of Biofilm DevelopmentStage

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.

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.

Hawaiian Bobtail Squid

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.

• 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

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.

Methods of Detection• Direct• Help determine locations of biofilms• Can require lots of equipment.

1. RODAC2. 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.

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.

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.

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.

Problematic MicrobesL.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.

Closing Summary

THE TAKE HOME POINTS Molds

Prevent Instead of Get Rid Of

Complex

Responsive to

Environment

YeastIndustry Pathoge

ns

Communicate

Dynamic

Bacteria

Everywhere

Questions?

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.