Why clean my cleanroom when it is already clean? What is rotational cleaning? Do I really need to go that far? Which chemicals should I use? As a Business Development Coordinator for a cleanroom consumables provider, I get asked questions like this every day. To answer these questions, it is helpful to talk to the experts, consult the wealth of information there is available on micro-organism resistance and disinfectant mode of action, and read about the standards of cleanliness required to keep a cleanroom up to specification. The following gives a simple guide to the reasoning behind rotational cleaning.
To answer the first question, yes cleanrooms do need to be cleaned. While it may look clean, most of the particles that need removing are not visible to the naked eye. Over time particles of dirt, cell debris, residues etc. 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, i.e. temperature, humidity and pressure, are controlled as necessary.’
By cleaning the cleanroom, the retention of particles inside the room is minimised. It is widely thought that the best technique to clean a surface in a cleanroom 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 more particles to be picked up.
Microbial control and EU GGMP grade cleanrooms
What is rotational cleaning? Rotational cleaning makes reference to bioburden, which is the number of bacteria living on an unsterilised surface. With this type of cleaning, operators are not just trying to remove particles of dirt, fluff, cell debris and such, they are also trying to remove and kill the living elements of contamination, which are the micro-organisms that may be present. In cleaning the cleanroom, a portion of the microbial population will undoubtedly be dislodged. However, it is unlikely that all of it will be removed, which is why steps must be taken to kill any microbes that have not been eliminated by cleaning.
Disinfectants are used to control the bioburden in the cleanroom as they have properties that can kill micro-organisms. But probably two or more disinfectants will need to be used in rotation, and this is why the process of killing micro-organisms in the cleanroom is called rotational cleaning. Annex 1 – Manufacture of sterile medicinal products – Volume 4 EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary use, 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.’
Why use more than one disinfectant? The simple answer is to stop resistance. There are two main types of resistance: naturally occurring and that derived through selection for resistant strains.
Selection for resistant strains
The development of genetic resistance has been reported in organisms such as Methicillin-resistant Staphylococcus aureus (MRSA). Just as this bacterium, once controlled by methicillin, has developed a genetic resistance over time and can no longer be controlled by that antibiotic, there is also the potential for this to happen to microbes in the cleanroom with disinfectants.
Over time, bacteria that were once controlled by alcohols, for example, would develop a genetic resistance to that disinfectant, making it no longer an effective agent. For this reason it is advisable to use different disinfectants. Although there is scant evidence in the literature to show that this has happened or even the potential for this to happen, precautions still need to be taken.
There are differences between the environment where MRSA developed and that of a cleanroom. In simple terms, for resistance to develop, a few bacteria must just about survive a dose of whatever agent has been employed to kill that type of bacteria. These bacteria then have the chance to multiply, and the genetic advantage they had over other strains that allowed them to survive will be passed on, leaving behind a surviving strain that grows and thrives.
Once again, if a few of these bacteria survive a dose of agent and then multiply, the advantage that allowed them to survive is passed on yet again and gets stronger. This happens over and over with continued use of that same agent. The advantage grows until what is left is a strain that is totally resistant to that agent.
In the cleanroom environment, because the policy is to overspecify the quantity and frequency of disinfectant used, very few microbes actually do survive. This means that it is unlikely that selection for resistant strains will occur. Also, antibiotics have a very specific and targeted action, which makes selection more likely. Disinfectants, on the other hand, have a very broad action, making it less likely that selection can take place. The process of selection for resistant strains is one that develops gradually. The resistance is not inherent in that organism and this is the main difference between this type of resistance and the naturally occurring resistance.
Naturally occurring resistance
Naturally occurring resistance occurs due to the fact that disinfectants have different modes of action. This means that not all disinfectants are equally effective at killing all microbes. Some may be very effective against bacteria but not fungi, whereas some may be effective against viruses but not endospores.
Depending on the method by which that particular disinfectant kills, some micro-organisms will naturally be better able to resist its actions. This is not learned, selected for or genetically passed on – it is down to the nature of the micro-organism itself and the properties it already has. In the same way, tall humans may naturally be better at basketball. It is not that they have learned to be good, or have a better understanding of the game that was passed on to them by their parents – they just happen to be closer to the hoop.
Disinfectant mode of action
Alcohol-based disinfectants tend to be effective against most micro-organisms, but not endospores. Their mode of action is to denature proteins in the cell that 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 (QACs or Quats) work by causing disorganisation of the cell membrane and the cell’s insides to leak out and degrade. They are effective against bacteria, enveloped viruses and fungi, but have little activity 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 and this will also cause leakage. They have similar effects to 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. These free radicals can then attack the essential cell components. Hydrogen peroxide-based disinfectants tend to kill everything including endospores, but this kind of disinfectant is very harsh on the surfaces it cleans.
So because a biguanide kills by affecting the cell wall and cell membrane, it may not be very effective against a micro-organism with a very strong cell wall. That type of micro-organism would 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, for example, lack of food, lack of water or nutrients, temperature or pH changes. They build an ultra-strong coat around the cell’s nucleus and essential parts to protect it. They can remain in this dormant state until conditions improve, when the coat will break down and the cell returns to normal. This 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. Chlorines and hydrogen peroxide are two disinfectants that do have an effect on endospores, and are often referred to as sporicidal. Chlorines can increase the permeability of the endospore coat and hydrogen peroxide can remove proteins from the coat.
Kill spectrum
Using different disinfectants will increase the kill spectrum – the total portion of the microbial population that can be killed. It can be helpful to think of the kill spectrum in a similar way to the light spectrum. If one considers only visible light, a huge portion of the light spectrum is being ignored, what about X-rays and UV rays? In the same way, if disinfectants that kill only bacteria are used, nothing is being done to combat endospores and other types of micro-organisms. Companies need to select disinfectants that, when used in rotation, cover as much of the spectrum as possible, therefore increasing the kill spectrum to the maximum.
It must also be taken into consideration that using a disinfectant that kills bacteria but not viruses creates the conditions for viruses to thrive. It will create an environment where viruses will flourish, and the disinfectant chosen to try and prevent this from happening will be unable to make an impact.
Choosing a disinfectant
What disinfectants should you use? 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 well, as they have good efficacy against most microbes and can also remove any residues that may build up from the use of other disinfectants.
In conclusion, a cleanroom needs to be kept clean to minimise the retention of particles inside the room, as stated in the ISO standard 14644-1. Rotational cleaning is the use of more than one disinfectant in rotation to control the bioburden in the cleanroom.
EU-GMP guidelines recommend that cleanrooms are cleaned thoroughly, that there is a written cleaning programme and, if using disinfectants, more than one is used to prevent resistance. This applies both to naturally occurring resistance, where microbes are just not affected by a particular disinfectant, and to selection for resistant strains, where microbes that were once controlled by a disinfectant have developed resistant strains that are no longer controlled by the same disinfectant.
Use different types of disinfectant with different active chemicals because they have different modes of action. This means that they are effective against different types of microbe, and using more than one maximises the kill spectrum.
There are many factors that will affect the choice and frequency of use of disinfectant, including the process and cleanroom class, residues, what format the disinfectant is available in, how easy it is to use and the environmental impact, among others. So, as a guide it seems sensible to rotate three agents – an alcohol, another general disinfectant and a sporicide.
Bibliography
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