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Line of Attack

Cleaning your cleanroom is essential. Although it probably looks clean, most of the particles that need removing are not visible to the naked eye. Over time, particles of dirt, cell debris, residues and so on will build up on the surfaces of the cleanroom and must be removed.

The ISO standard 14644-1 defines a cleanroom as: “A room in which the concentration of airborne particles is controlled, and which is constructed and used in a manner to minimise the introduction, generation and retention of particles inside the room, and in which other relevant parameters, e.g. temperature, humidity and pressure, are controlled as necessary” (1).

When cleaning a cleanroom, the retention of particles inside the room is minimised. It is thought that the best technique to clean a surface is a wet clean, which usually involves an impregnated wipe and a disinfectant or detergent solution. The mechanical act of wiping a surface will remove a number of particles from that surface. If the wipe and surface are wet, this will break more of the bonds that hold particles to the surface and allow you to pick up many more.

Microbial Control

Rotational cleaning refers to the bioburden, which is the number of bacteria living on an unsterilised surface. With this type of cleaning, the aim is not just to remove particles of dirt, fluff and cell debris, but also to kill the living elements of contamination – or microorganisms – that may be present.

By cleaning a cleanroom, a portion of the microbial population will undoubtedly be removed. However, it is unlikely that it will be completely eliminated, which is why companies must take steps to kill any remaining microbes.

To control the bioburden in your cleanroom, you will need to use disinfectants; these are chemicals that have properties that can kill microorganisms. You will probably need to use two or more disinfectants in rotation – hence the name rotational cleaning.

Annex 1: Manufacture of sterile medicinal products, from volume four of the European Union’s (EU’s) Good Manufacturing Practice (GMP) guidelines, point 61, states the following: “The sanitation of clean areas is particularly important. They should be cleaned thoroughly in accordance with a written programme. Where disinfectants are used, more than one type should be employed. Monitoring should be undertaken regularly in order to detect the development of resistant strains” (2).

Why is it necessary to use more than one disinfectant? The simple answer is to stop resistance. There are two main types: naturally occurring resistance and selection for resistant strains.

Selection for Resistant Strains

We have seen how developed genetic resistance has occurred in organisms such as meticillin-resistant Staphylococcus aureus (MRSA). This bacterium, once controlled by meticillin, has developed a genetic resistance over time and can no longer be controlled by that antibiotic. There is a theory that the same thing could potentially happen to microbes in the cleanroom, and that they could become resistant to disinfectants.

The belief is that bacteria currently controlled by alcohols may develop a genetic resistance to certain disinfectants, meaning they would no longer be effective agents. For this reason, it is advisable to use multiple disinfectants to try and prevent this from happening. Although there does not seem to be any evidence to demonstrate this taking place as yet, or even show that this could potentially take place, it is important to take precautions.

There are differences between the environment where MRSA can cultivate and a cleanroom environment. For resistance to develop, a few bacteria must survive a dose of whatever agent has been employed to kill it. These bacteria then have the chance to multiply, and whatever advantage they had over other strains that allowed them to live will be passed on, resulting in a surviving strain which grows and thrives. A number of these new bacteria will then survive another dose of agent, multiply, and pass on the same resistant strain – and the cycle will repeat itself with continued use of that same agent. Eventually, a strain will be totally resistant to that particular disinfectant.

In contrast, because the quantity and frequency of disinfectant use is far greater in the cleanroom environment, very few microbes actually do survive. This means it is unlikely that resistant strains will occur. In addition, antibiotics have a specific and targeted action which makes selection more likely. Disinfectants have a very broad action which means it will be less likely that selection can take place.

The process of selection for resistant strains is developed and gradual – the resistance is not inherent in that organism. This is the main difference between this type of resistance and naturally occurring resistance.

Naturally Occurring Resistance

Naturally occurring resistance is due to the fact that as disinfectants work in different ways, they have different modes of action. This means they are not all equally as effective at killing all varieties of microbes. Some may be very effective against bacteria but not fungi, whereas others may be effective against viruses but not endospores.

Due to the way that disinfectants kill, some micro-organisms will naturally be better able to resist their actions. This is not learned, selected or genetically passed on – it is down to the nature of the micro-organism itself and the properties it already possesses.

Disinfectant Modes of Action

Alcohol-based disinfectants tend to be effective against most microorganisms, but not endospores. Their mode of action is to de-nature proteins in the cell which can cause them to clump together and lose their function. When this happens to the cell wall, it can lose structure and collapse.

Quaternary ammonium compounds (Quats) work by causing disorganisation of the cell membrane, whereby the cell’s insides leak out and degrade. They are effective against bacteria, enveloped viruses and fungi, but have little impact on non-enveloped viruses or endospores.

Biguanides alter the permeability of the cell membrane. They can damage the outer layers and attack the inner layers, which will also cause leakage. They have similar effects to the Quats.

Chlorine is a highly active oxidising agent. It oxidises DNA and cell proteins, destroying their activity. Disinfectants containing chlorine kill most things, including endospores at higher concentrations.

Hydrogen peroxide is highly reactive and acts as an oxidant, producing free hydroxyl radicals which can then attack the essential cell components. Hydrogen peroxidebased disinfectants tend to kill everything including endospores, but this kind of disinfectant is very harsh on the surfaces it cleans.

Therefore, because a biguanide kills by affecting the cell wall and membrane, it may not be particularly effective against a micro-organism with a very strong cell wall. That kind of micro-organism will be naturally resistant to the effects of a biguanide.

Endospores

Endospores are extremely difficult to kill. The endospore is a state that a bacteria or virus can enter into when conditions are unfavourable – lack of food or water, for example, or nutrient, temperature or pH changes. They build an ultra-strong coat around the cell’s nucleus and essential parts to protect it. Endospores can remain in this dormant state until conditions improve, when the coat will break down and the cell returns to normal.

Such a strong coat means that in this state the endospore can be very difficult to kill, as it will resist the effects of gamma irradiation and many disinfectants. Chlorine and hydrogen peroxide are two disinfectants that do have an effect on endospores, and are often referred to as sporicidal. Chlorine can increase the permeability of the endospore coat, while hydrogen peroxide can remove proteins from the coat.

Kill Spectrum

Using different disinfectants will increase the kill spectrum, or the total portion of the microbial population eliminated. It can be helpful to think of the kill spectrum in a similar way to the light spectrum. If we only ever consider visible light, we miss out a huge portion of the light spectrum – what about X-rays and ultraviolet rays? In the same way, if we only ever use disinfectants that kill bacteria, we are not doing anything to combat endospores and other types of micro-organisms. It is important to select disinfectants that, when used in rotation, cover as much of the spectrum as possible, thereby increasing the kill spectrum as far as possible.

We must also consider that if we only use a disinfectant that kills bacteria, but does not harm viruses, we are creating conditions for viruses to thrive. The disinfectant chosen for the cleanroom will therefore be unable to make enough of an impact.

Choosing a Disinfectant

What disinfectants should be used? It is clear that using a sporicide is highly important, but agents that have sporicidal activity tend to be harsh and unacceptable for everyday use. For this reason, it is recommended that a sporicide is used in rotation with another effective disinfectant that is more suitable for regular use. It would also be advisable to use an alcohol, as they have good efficacy against most microbes and can also remove any residues that may build up from using other disinfectants.

Summary

Cleanrooms must be kept clean to minimise the retention of particles inside the space, as stated in the ISO standard 14644-1. The EU’s GMP guidelines recommend that you clean thoroughly, have a written cleaning programme in place and, if using disinfectants, use more than one. This prevents naturally occurring resistance and selection for resistant strains.

Using various types of disinfectant with a range of active chemicals is beneficial because they all have different modes of action. This means that they are effective against numerous types of microbe, and using more than one will maximise your kill spectrum. There are many factors which affect the type and frequency of disinfectant you choose to use, including your process, cleanroom class, residues, what format it is available in, how easy it is to use and the environmental impact. As a guide, it seems sensible to rotate three agents – an alcohol, another general disinfectant and a sporicide – as part of your rotational cleaning system.

References
1. Part 1: Classification of air cleanliness: cleanrooms and associated controlled environments, EN ISO 14644-1:1999
2. Annex 1: Manufacture of sterile medicinal products, EU Guidelines to Good Manufacturing Practice, 2008. Visit: http:// ec.europa.eu/health/files/eudralex/vol- 4/2008_11_25_gmp-an1_en.pdf

Other Sources
1. Sandle T, A Guide to Cleaning and Disinfecting Cleanrooms, Grosvenor House Publishing, 2012
2. Whyte W, Cleanroom Technology, Fundamentals of Design, Testing and Operation, John Wiley & Sons Ltd, 2010
3. Araújo P, Lemos M, Mergulhão, Melo L and Simões M, Antimicrobial resistance to disinfectants in biofilms, Science Against Microbial Pathogens: Communicating Current Research and Technological Advances, Formatex Research Center, p826, 2011
4. Sartain E, Disinfectant rotation, Controlled Environments Magazine, 2005. Visit: www.cemag.us/print/ articles/2005/03/disinfectantrotation
5. Martinez JE, The rotation of disinfectants principle: true or false? Pharmaceutical Technology 33(2): pp58-71, 2009. Visit: www. pharmtech.com/pharmtech/article/ the-rotation-of-disinfectantsprinciple- true-or-false/ articlestandard/article/detail/580032
6. McDonnell G and Denver Russell A, Antiseptics and disinfectants: activity, action and resistance, Clinical Microbiology Reviews, 12(1): pp147-179, 1999
7. Weitzel S, Critical cleaning bulletin, Critical Process Cleaning, CANI, Inc, 2007. Visit: http://cdn.shopify.com/s/files/1/0186/2832/files/BULLETIN_ selection_and_rotation_of_disinfectants. pdf?380
8. Guideline for disinfectant and sterilization in healthcare facilities, Centers for Disease Control and Prevention, 2008. Visit: www.cdc.gov/hicpac/disinfection_ sterilization/6_0disinfection.html


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Rebecca Smith has a BSc in Biological Science. She worked for Merck Sharp and Dohme for over five years as a Medical Sales Representative, developing her expertise and knowledge about the cleanroom industry. For the last two years, Rebecca has been a Business Development Coordinator for Connect 2 Cleanrooms, working with cleanroom users to help them manage the running of their cleanrooms.
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