Rotation cleaning – What is it and what are its advantages?

Why does a cleanroom need to be cleaned? What is rotational cleaning, and is it really necessary? Why are there so many different chemicals, and how do they work? Which ones should be used? As the Business Development Manager for a cleanroom consumables provider, Rebecca Smith deals daily with such questions.
To answer these questions, she has spoken with experts, reviewed a wealth of information about microorganism resistance and the modes of action of disinfectants, and studied the cleanliness requirements for different cleanroom classes.

Cleaning the cleanroom

The answer to the first question is: Of course, cleanrooms must be cleaned. Although they may look clean, most particles to be removed are not visible to the naked eye. Over time, dirt particles, cell debris, and other contaminants accumulate on the surfaces of the cleanroom and need to be removed. The standard DIN EN ISO 14644-1 defines a cleanroom as follows:
“A room in which the concentration of airborne particles is controlled, designed and used so that the number of particles introduced into, generated in, and deposited in the room is minimized, and in which other purity-related parameters such as temperature, humidity, and pressure are regulated as needed” [10].

Cleaning reduces the deposition of particles in the cleanroom. The best technique for cleaning a surface in a cleanroom is moist cleaning. Typically, cloths soaked with a disinfectant or cleaning solution are used. Mechanical wiping removes a certain amount of particles from the surface. When the cloth and the surface are moist, the adhesion between particles and the surface is further reduced, allowing more particles to be removed.

Killing microorganisms and Good Manufacturing Practice according to EU guidelines

What exactly is rotational cleaning? It refers to microbial load, i.e., the number of microorganisms living on an unsterilized surface. Rotational cleaning aims not only to remove dirt, lint, cell debris, etc., but also to kill any contamination in the form of living organisms. When cleaning a cleanroom, some microbial contamination is undoubtedly removed. However, it is unlikely that all “germs” are eliminated, so those microorganisms that are not removed must be killed.

To reduce the microbial load in the cleanroom, disinfectants are needed—chemicals capable of killing microorganisms. It is likely necessary to alternate between two or more agents to kill all germs. This cycle is called rotation, hence the term rotational cleaning.

The EU guideline for Good Manufacturing Practice (GMP) for human and veterinary medicinal products, (EudraLex Volume 4), Annex 1 on sterile medicinal product manufacturing, states in point 61:
“Decontamination of clean areas is particularly important. They should be thoroughly cleaned according to a written program. When using disinfectants, more than one type should be used. The area should be regularly monitored for the development of resistant strains.” [6]

Why is more than one disinfectant necessary? To prevent resistance. There are two types of resistance—naturally occurring resistance and the development of resistant strains through selection.

Development of resistant strains through selection

In organisms like MRSA, it has been tracked how genetic resistance develops. A bacterium initially killed by methicillin gradually developed genetic resistance, and this antibiotic no longer works against it. It is assumed that the same could happen with the effectiveness of disinfectants against microorganisms in the cleanroom.

Over time, bacteria that were previously sensitive to alcohols could develop genetic resistance to such agents, rendering disinfection ineffective. Therefore, it seems advisable to prevent this effect by using multiple disinfectants. So far, there is no evidence that such resistance actually occurs or is even possible, but precautions should still be taken.

The conditions that favored the development of MRSA differ from those in a cleanroom. To develop resistance, a few bacteria would need to survive the application of a specific effective agent. These survivors can then multiply and pass on the advantage they gained, allowing the resistant strain to grow and thrive.

During subsequent cleaning cycles, only a few of these bacteria survive again, multiply, and pass on their survival advantage, which becomes increasingly stronger. Repeated use of the same agent repeats this process many times. The advantage grows until the strain is completely resistant to the agent. Since cleanrooms are usually cleaned very frequently with strong agents, very few microorganisms survive, making it unlikely that resistance develops through selection. Also, antibiotics have a very specific and targeted mechanism of action, which makes selection more likely. Disinfectants, on the other hand, have a broad spectrum of activity, making resistance less probable.

The development of resistant strains through selection is a gradual process. Resistance is not an inherent property of the organism but develops over time. This distinguishes this type of resistance from naturally occurring resistance.

Naturally occurring resistance

Naturally occurring resistance is based on the fact that different disinfectants work in different ways. Not all are equally effective against all organisms. Some are very effective against bacteria but not fungi, others against viruses but not endospores.

Due to the different modes of action of disinfectants, some microorganisms are inherently better equipped to resist them. This trait is not learned, acquired, or genetically inherited but is inherent in the structure of the microorganism itself. An analogy makes this clear: It’s not learned or trained that tall people have an advantage in basketball; their height is simply a natural trait.

Modes of action of disinfectants

Alcohol-based disinfectants are effective against most microorganisms but not against endospores. They denature proteins within the cell, causing them to clump together and become nonfunctional. This leads to structural loss and collapse of the cell wall.

Quaternary ammonium compounds (QACs or Quats) damage the cell membrane, causing internal components to leak out and decompose. They are effective against bacteria, enveloped viruses, and fungi but are hardly effective against non-enveloped viruses and endospores.

Biguanides alter the permeability of the cell membrane, damaging outer layers and attacking inner layers. This also results in leakage of cell contents. Their effect is similar to that of QACs.

Chlorine is a strong oxidizer. It oxidizes DNA and cellular proteins, destroying their activity. Chlorine-based disinfectants kill nearly everything at higher concentrations, including endospores.

Hydrogen peroxide is highly reactive and acts oxidatively, generating free hydroxyl radicals. These attack essential cellular components. Disinfectants based on hydrogen peroxide kill all microorganisms, including endospores, but can also damage the surfaces being disinfected.

From the mode of action of biguanides on the cell wall and membrane, it follows that such agents are not very effective against microorganisms with very strong cell walls. These microorganisms have a natural resistance to biguanides.

Endospores

Endospores are extremely difficult to kill. They are a dormant stage of bacteria or viruses that occurs under unfavorable external conditions, such as nutrient deficiency, lack of water, temperature or pH changes. They form a very robust shell around the nucleus and essential elements. This stage can survive long periods until conditions become favorable again, at which point the shell becomes permeable, and the cell becomes active again.

Due to this tough shell, endospores are very resistant—they withstand gamma radiation and many disinfectants. Disinfectants based on chlorine or hydrogen peroxide are effective against endospores and are often called sporicidal. Chlorine increases the permeability of the endospore shell, and hydrogen peroxide can break down proteins within it.

Spectrum of activity

Using different disinfectants can broaden the spectrum of activity, i.e., the proportion of microorganisms that can be killed. The spectrum can be visualized similarly to the electromagnetic spectrum. When only visible light is considered, a large part of the spectrum is ignored because X-ray and ultraviolet radiation are also part of the electromagnetic spectrum. Similarly, if only disinfectants effective against bacteria are used, nothing is done against endospores and other microorganisms. Therefore, when selecting disinfectants, those covering the widest possible spectrum should be considered.

If only agents that kill bacteria but not viruses are used, conditions become even more favorable for viruses. In such an environment, even the specially selected disinfectant may no longer be effective against viruses.

Selecting a disinfectant

Which disinfectants should be used? It has been emphasized that using a sporicidal disinfectant is very important, but agents effective against spores are generally so strong that they cannot be used daily. For this reason, it is recommended to alternate sporicidal agents with other effective disinfectants that are more suitable for regular use. Additionally, using an alcohol-based disinfectant is advisable, as it works well against most germs and can also effectively remove residues of other disinfectants.

Summary

Cleanrooms must be cleaned to minimize particle deposition according to the standard DIN EN ISO 14644-1. Rotational cleaning involves alternating different disinfectants to reduce microbial load in the cleanroom.

The EU-GMP guidelines recommend thorough cleaning, a written cleaning program, and, when using disinfectants, the use of more than one agent. The reason for these efforts is to prevent resistance. This includes naturally occurring resistance, where microorganisms are inherently insensitive to a particular disinfectant, and resistant strains that could develop through selection from formerly sensitive microbes.

Different types of disinfectants should be used because their active ingredients differ in their modes of action. They target different types of germs, thus providing a broader spectrum. The choice of disinfectants and their frequency depends on many factors, such as the cleaning process itself, the cleanroom class, the type of contamination, the formulation of the agents, user-friendliness, and environmental considerations. A general guideline is to alternate among three agents: an alcohol-based one, another general disinfectant, and a sporicide.

Sources:
1. Sandle, T. (2012) A guide to cleaning & disinfecting cleanrooms, Surrey: Grosvenor House Publishing.
2. Whyte, W. (2010) Cleanroom Technology, Fundamentals of design, testing and operation, West Sussex: John Wiley & Sons Ltd.
3. Araújo, P. Lemos, M. Mergulhão, Melo, L. Simões, M. (2011) Antimicrobial resistance to disinfectants in bio-films, Science against microbial pathogens: communicating current research and technological advances, p826.
4. Sartain, E. (2005) Disinfectant Rotation, Available: www.cemag.us/print/articles/2005/03/disinfectant-rotation.
5. Martinez, J.E. (2009) The rotation of disinfectants principle: true or false? Available: http://www.pharmtech.com/pharmtech/Article/The-Rotation-of-Disinfectants-Principle-True-or-Fa/ArticleStandard/Article/detail/580032.
6. "Annex 1: Manufacture of Sterile Medicinal Products," Good Manufacturing Practice (GMP) Guidelines (Brussels, Nov, 2008), Available: http://ec.europa.eu/health/files/eudralex/vol-4/2008_11_25_gmp-an1_en.pdf.
7. McDonnell, G. Denver Russell, A. (1999) Antiseptics and disinfectants: activity, action and resistance, Clinical Microbiology Reviews, Jan 1999, vol 12. No 1 147-179.
8. Critical Cleaning Bulletin (2007) contact Weitzel, S. Critical Process Cleaning, CANI, Inc, Available: http://cdn.shopify.com/s/files/1/0186/2832/files/BULLETIN_selection_and_rotation_of_disinfectants.pdf?380.
9. Guideline for disinfectant and sterilization in healthcare facilities, 2008, Centers for Disease Control and Prevention, Available: http://www.cdc.gov/hicpac/disinfection_sterilization/6_0disinfection.html.
10. "Part 1: Classification of air cleanliness" Cleanrooms and associated controlled environments, The European Standard EN ISO 14644-1:1999.

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About the author Rebecca Smith
Her scientific interest led her to study biology, which she completed with a bachelor's degree. Afterwards, she moved into the pharmaceutical sector, working for Merck Sharp and Dohme (MSD). She worked for five years as a pharmaceutical representative for MSD, gaining valuable experience that is also essential for work in the cleanroom industry. For the past two years, she has been responsible for Business Development at Connect 2 Cleanrooms. In this role, she supports cleanroom users in their daily work with their cleanrooms. In her free time, she relaxes by raising her Labrador, Charlie.