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Bacterial Attachment to Food and Food Processing Surfaces Can Lead to Biofilm Formation

Summary

Biofilms are a community of microorganisms (such as bacteria) that come together and stick to surfaces. Bacteria “join” a biofilm because it is easier for them to survive environmental threats, such as sanitizers, when they are a part of a biofilm-community. To stop biofilm development, growers, producers, food handlers, and food processors should develop routine sanitation practices of harvest equipment, food-contact and non-food contact surfaces. Without proper sanitation, biofilms have a chance to become “mature.” A mature biofilm is more difficult to remove, and it can be a constant source of microbiological contamination for food and other surfaces. The purpose of this publication is to provide additional information about biofilms and the importance of keeping biofilms from developing on foods and surfaces that come in contact with food.

What are Biofilms?

When most of us think about bacteria, we often imagine them as a single, free-floating cell that lands and grows on a surface such as food or soil. In reality, bacteria naturally come together and collect on many different surfaces, including food, equipment, plants, animals, fresh produce harvest bins/containers/crates/cartons, water lines and water systems, cleaning equipment, and tools. These collections or “communities” of bacteria are called biofilms, and approximately 99% of bacteria worldwide have been a part of a biofilm at some stage of their growth.1, 2 About 40-80% of bacteria exist in biofilms.3 Thus, a biofilm can be defined as surface-attached bacteria that collect to form a “community.” The biofilm community of bacteria are irreversibly attached to a surface and each other. Within the biofilm structure, bacteria are not randomly positioned. Instead, they are positioned in the biofilm at a location that is the most helpful to the bacteria. Biofilms are usually very organized structures and, believe it or not, communicate (using signaling molecules) to build a strong biofilm.

Importance of Biofilms

Biofilms typically consist of different types of bacteria because diversity gives them the advantage of being able to form a thicker and more stable biofilm. In fact, mixed species biofilms show higher resistance to disinfectants.4 A biofilm also contains other particles, including protein, fat, water, RNA/DNA, and ions. As the bacteria build a biofilm, they leave space for water channels that allow the flow of nutrients and oxygen. Sometimes bacteria use the water channels to move throughout the biofilm. There are several advantages for bacteria in a biofilm, but the main benefit is protection from detergents, disinfectants, sanitizers, antimicrobials, and other changes in their environment. One report stated that bacteria in a biofilm were 150 to 3,000 times more resistant to bleach than free-floating bacteria.5 Bacteria in biofilms have a better chance of surviving than free-floating cells when nutrients are scarce or when pH changes (acids or alkali).6 In other words, bacteria in biofilms have a better chance of survival. The bottom line, biofilms make sanitation of food equipment much more difficult and keeping food safe more of a challenge. Foodborne diseases associated with bacterial biofilms on food contact surfaces can occur from shedding of live cells or release of toxins which can contaminate food, causing individual or multiple (in the case of an outbreak) illnesses.

How Biofilms Form

Scientists have studied biofilms for several years. The first biofilm (tooth plaque) was observed by Antonie Van Leeuwenhoek in 1684 on his own teeth using a microscope he made himself. Since that time, scientists figured out that it was important to understand how biofilms form so they could prevent them from developing in areas where food is held, processed, stored, and distributed.

Biofilms form during a five-step process. The figure below (adapted from Vasudevan R.7) shows an image of how the five-step process works.

Step 1: Bacteria or another microorganism (since biofilms are usually mixed populations) attaches to a surface.

Step 2: Other microorganisms attach, forming a layer, and they begin to produce polysaccharides, which is a carbohydrate sometimes called “slime.”

Step 3: Multiple layers of microorganisms attach, forming what is sometimes called a “micro-colony”.

Step 4: The biofilm matures and typically becomes shaped like a “mushroom” from the polysaccharide made by the cells.

Step 5: Some cells leave the biofilm, become free-floating and possibly join a new biofilm on another surface.

The important thing to note is that Step 1 is the reversible stage, and beyond step 1, it is harder (but not impossible) to remove a biofilm. So, if possible, you want to keep biofilms from getting past Step 1.

Figure 1: Five Steps of Biofilm Formation. Biofilms form in a five-stage process.

Figure 1: Five Steps of Biofilm Formation. Biofilms form in a five-stage process.
Step 1: Bacteria attach to a surface.  Step 2: Other microorganisms attach, forming a layer of microorganisms. These microorganisms produce polysaccharides (slime). Step 3: Multiple layers of microorganisms attach to the surface forming a microcolony. Step 4: The biofilm matures, and a mature biofilm is usually shaped like a mushroom. Step 5: Some cells leave the biofilm to become free-floating and possibly join another biofilm. (adapted from Vasudevan R.7).

Biofilms can form more easily in cracked, dented, scratched, “pitted,” corroded or rough surfaces. These areas often contain trapped food particles and are more difficult to clean. It is important to pay close attention to these conditions and fix or replace them when possible. Biofilms also form differently on different types of surfaces, such as stainless steel, plastic, rubber, and food.

Figure 2 shows an image of a biofilm formed on stainless steel. The image was taken by Dr. Claudia Ionita, Post-doctoral Fellow at Clemson University in the Department of Food, Nutrition, and Packaging Sciences. It demonstrates that biofilms can form on just about any surface.

Figure 2: Listeria monocytogenes biofilm on stainless steel.

Figure 2: Listeria monocytogenes biofilm on stainless steel.
Image taken using Atomic Force Microscopy (20X) by Dr. Claudia Ionita, Post-doctoral Fellow at Clemson University in the Department of Food, Nutrition and Packaging Sciences

How are Biofilms Removed

It is important to have a routine cleaning and sanitation procedure for equipment, tools, food-contact and non-food contact surfaces in a facility or operation. After routine cleaning and sanitation, areas should be inspected for cleanliness to make sure nothing was missed (and clean or reclean dirty surfaces as needed). Routine cleaning and sanitation will keep biofilms from reaching the irreversible stage where they are firmly attached. When biofilms are mature, the extra layers prevent the detergent and sanitizer from reaching all the cells. The community provides layers of protection. To remove a mature biofilm, the right formulation and concentration of cleaning and sanitation chemicals must be used (possibly consult with a sanitation chemical company; ALWAYS follow the labeling instructions and ALWAYS use something that can be applied to food-contact surfaces). Cleaning and sanitation chemicals must be applied at the right temperature and for the right amount of time (exposure). Lastly, scrubbing, scraping, and other mechanical actions are needed to remove a mature biofilm from a surface (just like brushing your teeth or having the dental hygienist clean your teeth to remove plaque biofilms). Some scientists have tested a variety of sanitizers against biofilms. One study found that peroxyacetic or peracetic acid worked very well.8 Many establishments choose to do daily sanitation and a harsher cleaning/sanitation procedure once a week with quaternary ammonium chloride-based sanitizers.

Some other advanced methods to control biofilm formation in addition to chemical treatments and physical scrubbing include enzymatic disruption,9 surface coatings, 10, 11 and biosurfactants. 12

Conclusion

Within nature and often within a food processing environment, bacteria prefer to exist in a biofilm rather than a free-floating cell. Therefore, routine and adequate sanitation is necessary to stop biofilms from reaching the ‘mature’ stage on produce harvest and processing equipment, tools, and surfaces.

References:

1Costerton, J. W., Cheng, K. J., Geesey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., and Marrie, M. J. Bacterial biofilms in nature and disease. Annu Rev Microbiol. 1987 41:435-464.

2Dalton, H. M. and March, P. E. Molecular genetics of bacterial attachment and biofouling. Curr Opin Biotechnol 1998 Jun 9(3):252-255.

3Flemming, H. C. and Wuertz, S. Bacteria and archea on earth and their abundance in biofilms. Nat. Rev. Microbiol. 2019 Apr 17(4):247-260.

4Meyer, R. L. Intra- and inter-species interactions within biofilms of important foodborne bacterial pathogens. Front.Microbiol. 2015 Aug 6:841. doi: 10.3389/fmicb.2015.00841.

5 LeChevallier, W.M., C.D. Cawthon, C. D. and Lee, R. G.. Inactivation of biofilm bacteria. Appl. Environ. Micro. 1988 Oct 54(10):2492-2499.

6Jefferson, K. K. What drives bacteria to produce a biofilm? FEMS Microbiology Letters 2004 Jul 236: 163–173.

7Vasudevan R. Biofilms: microbial cities of scientific significance. J Microbiol Exp. 2014 Jun 1(3):84‒98. DOI: 10.15406/jmen.2014.01.00014.

8Humm, B. A research update on the effects of cleaners and sanitizers on food processing biofilms. Food Protection Report 1992 8:2, 5.

9Coughlan, L. M., Cotter, P. D., Hill, C., and Álvarez-Ordóñez, A. New weapons to fight old enemies: novel strategies for the (bio)control of bacterial biofilms in the food industry. Front.Microbiol. 2016 Oct 7:1641. doi: 10.3389/fmicb.2016.01641.

10Rai, M., Ingle, A. P., Gaikwad, S., Gupta, I., and Gade, A. Nanotechnology based anti-infectives to fight microbial intrusions. J. Appl. Microbiol. 2015 Aug 120(2): 527–542. doi: 10.1111/jam.13010.

11Gu, J., Su, Y., Liu, P., Li, P., and Yang, P. An environmentally benign antimicrobial coating based on a protein supramolecular assembly. ACS Appl. Mater. Interfaces 2017 Jan 9(1):198–210. doi:10.1021/acsami.6b13552.

12Zhao, H., Shao, D., Jiang, C., Shi, J., Li, Q., Huang, Q., et al. Biological activity of lipopeptides from Bacillus. Appl. Microbiol. Biotechnol. 2017 101, 5951–5960. doi: 10.1007/s00253-017-8396-0.

If this document didn’t answer your questions, please contact HGIC at hgic@clemson.edu or 1-888-656-9988.

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