We live in a microbial soup. We are a microbial soup; the human body contains an estimated 10 times more bacterial cells than mammalian cells.1
Those of us outside of the biotechnology or pharmaceutical arena may not focus on the potential for living organisms to impact surfaces of controlled environments or even on surfaces of the product itself. However, microbes can grow exponentially if there is warmth, a food source, and moisture. These three conditions are readily found in controlled environments and as part of manufacturing processes, including critical cleaning.2
While 150 years have been devoted to the study of single species of microbes growing in culture media, most microbes live in multi-species, organ-like, societal structures termed biofilms.3 Biofilms are a potential problem in manufacturing facilities, including in controlled environments. Trying to manage contamination without understanding biofilms would be akin to making decisions about human physiology based on in vitro studies alone.
Evolution
Biofilms have an evolutionary advantage over discrete colonies; they are designed to survive. In forming biofilms, microbes make their own protective armor — extracellular polymeric substances (EPS). EPS contains polysaccharides and proteins as
well as other biobased materials. EPS provides protection from the environmental challenges such as UV radiation, pH changes, and osmotic shock. EPS can absorb and retain moisture, preventing desiccation. Channels within the biofilm
increase the availability of water and nutrients. When biofilms contain multiple organisms there can be what ecologists call “symbiosis” or mutually beneficial societal behavior. This can mean a greater quantity and diversity of food production; if fermentation happens, the biofilm can have its own on-site gastropub. EPS can contain extracellular DNA (eDNA)4. eDNA is considered to have a role in biofilm formation and development — a recent study demonstrated that eDNA has the ability to bind several classes of antibiotics.5 The main point to consider is that the biofilm has multiple components, polysaccharides, proteins, eDNA, and lipids. Each of the components can play different roles in the structure and function of the biofilm.
Persistent problem, holistic approach
Overall, the effect of biofilms is undoubtedly positive, or life on earth would be problematic or at least quite different. However, biofilms are persistent, and some are harmful. Dental plaque is a biofilm. Some implant-based infections are associated with biofilms. Because the living portion of biofilms are protected by the EPS armor, disinfection become more of a challenge. Biofilms have been reported in systems for drinking water. Biofilms have been found on produce and in the grout in tiles floors (e.g. kitchens, bathrooms, and industrial facilities). In the manufacture of critical products, the implication is that biofilms have the potential to compromise the controlled environment, the process, and even the product.
Battin calls for “a holistic approach that integrates biology, physics, and chemistry” to understand systems with a “biodiversity equal or superior to the biodiversity of plants in a tropical rainforest or fish in the ocean.”6 By analogy, cleaning and sanitation processes have to be coordinated in controlled environments and in critical manufacturing applications, and the processes have to be rigorously demonstrated and defined. Since cleaning of controlled environments is typically aqueous-based, cleaning has to combine the cleaning agent with an appropriate level of physical cleaning action. Particularly in dealing with biofilms, if the cleaning is incomplete, disinfection will also be incomplete.7
Designing to deter biofilms is also important.8 Hygienic design is a common practice in the food and beverage (F&B) industry where food processing equipment and rooms are designed not to have the nooks and crannies where bacteria could hide, grow, and create a protective biofilm. The machines have smooth surfaces that are easy to clean. Grinstead points out that there is a high degree of similarity between the conventional “soils” in the F&B industry and structural components of biofilms (i.e. proteins, lipids, and other organic polymers). Alkaline cleaners are good for removing most carbohydrates, and chlorinated alkaline cleaners are used in protein removal.9 These are the kind of lessons learned in the F&B world that could readily be applied to industrial and biomedical cleanrooms.
A rose by any other name
It should also be pointed out that there are a number of definitions of biofilms. Definitions have evolved and become more refined along with our own understanding of biofilms. We occasionally hear the term used to describe microbes that are trapped in “stuff” (e.g. machining oil, cleaning agent residue) where the “stuff” is not actually produced by the microbe itself. Even if microbes trapped in “stuff” would not be considered a biofilm by most experts, people involved with controlled environments still need to be concerned about appropriate cleaning and disinfection. An understanding of biofilm and biofilm management is likely to help, even with mixed soils that are not true biofilms. The purpose of this article is to introduce the community to the complex world of microbial biofilms. In an upcoming article, we plan to describe the current state of regulations, test methods for growing biofilms, and validation of “biofilm kill.”
References
1. J. Ackerman. “How bacteria in our bodies protect our health,” Scientific American 306 (6): 36, 2012. http://bit.ly/1EDS8tj
2. Kanegsberg, B., J. Unmack, and E. Kaengsberg. “Live Dirt Part I — Bacteria and Fungi, Controlled Environments, May 2003. http://bit.ly/1EwlBEj
3. Davey, M.E. and G. A. O’Toole. “Microbial Biofilms: from Ecology to Molecular Genetics,” Microbiology and Molecular Biology Reviews, p. 847-867, Dec. 2000.
4. Flemming, H-C., T. Neu, and D. J. Wozniak. “The EPS Matrix: The ‘House of Biofilm Cells,” Journal of Bacteriology, Nov. 2007, p. 7945-7947.
5. Aspe, M., L. Jensen, J. Melegrito, and M. Sun. “The Role of Alginate and Extracellular DNA in Biofilm-Meditated Pseudomonas aeruginosa Gentamicin Resistance,” Journal of Experimental Microbiology and Immunology (JEMI), Vol. 16: 42-48, April 2012.
6. Battin, T.J. Perspective: “Chartering new waters of biofilm ecology and evolution,” npj Biofilms and Microbiomes, March 2015.
7. Sandle, T. “Bacterial Adhesion: An Introduction,” Institute of Validation Technology Network, June 2013. http://bit.ly/1zvacWo
8. Carpentier, B. “Biofilms and Microorganisms on Surfaces After Cleaning and Disinfection,” Food Safety Magazine, April/May 2011. http://bit.ly/1DND0Wl
9. Grinstead, D. “Biofilms in the food and beverage industries,” in Cleaning and Sanitation in Food Processing Environments, P. Framtamico, B. Annous, and J. Guenther eds., Woodhead Publishing, 2009.
Barbara Kanegsberg and Ed Kanegsberg (the Cleaning Lady and the Rocket Scientist) are experienced consultants and educators in critical and precision cleaning, surface preparation, and contamination control. Their diverse projects include medical device manufacturing, microelectronics, optics, and aerospace. info@bfksolutions.com
Stephen Lyon is a senior scientist with Sealed Air Corp. and focuses on the microbiology of the built environment. With more than three decades of experience in the water/wastewater/environmental fields, he uses his training in several disciplines to better understand emerging issues in the building care and food and beverage industries. stephen.lyon@sealedair.com
This article appeared in the May/June 2015 issue of Controlled Environments.
