Do we pose the biggest risk? Our skin and cleanroom contamination


10th June 2024


Where there are people there is a risk of contamination. Part of developing a successful contamination control assessment is identifying this risk, whether that is through inadvertent touching and transferring contamination or simply the shedding of skin detritus.


Here Dr Tim Sandle explains why understanding our own microbiomes are key to this process.
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Human microbiome of the skin


Insights into the microbiome of the skin aids in assessing the risk. Research outcomes have shown that there is a high population1  and a considerable diversity of microbial species on the outer layer of the skin (around 1000 species upon human skin from 19 phyla 2,3 ). This diversity is not, however, evenly distributed. 


The distribution of microorganisms varies by topography, with different types of microorganism resident in different locations. There are three main ecological areas of the skin - sebaceous, moist, and dry. Microbial divergence includes Propionibacteria and Staphylococci (the main species found in sebaceous areas), Gram-positive cocci, primarily the Micrococcaceae, found on the arms and legs, with Gram-positive rods found in high numbers on the torso (dry areas). Staphylococci are found together with some Gram-negative bacteria 4 on moist areas. Ecologically, sebaceous areas have a greater species richness compared with the moist and dry regions.


Why is there ecological diversity?


The reasons for the topographical variations relate to the physicochemical properties of the skin 5. The pH of the skin, for example, varies between neutral and alkali. Another important variation is with skin temperature, which ranges between 25-37oC. Another factor is the level of humidity, with the dampest places being the groin region, the armpits and between the toe webs.


In these relatively moist areas, proportionately higher numbers of Gram-negative rods are found, with Acinetobacter being the dominant genus 6 . Oxygen levels provide another variation, with the lower oxygen regions across the forehead and within hair follicles providing environments for anaerobic bacteria to colonise.   


Variation is not only across different body locations – it also varies between individuals, with men carrying more microorganisms than women 7. A further complication to understanding the skin microbiota is that the skin microbiome may not be stable and may change over time as a person ages 8.



Environmental monitoring and cleanrooms


The information about the human microbiome, in terms of the rich depth of variety of microorganisms on the skin, introduces several implications for the environmental monitoring of aseptic processing environments. The foremost implication arises from comparing and contrasting the human microbiome research with the findings from cleanroom environmental monitoring using standard methods. Moreover, there is a difference between the diversity of microorganisms found from microbiome research compared with what is ordinarily recovered from the culture methods.


Published studies of cleanroom microflora have largely been based on cultural methods for environmental monitoring 9,10,11. These studies have shown that the most common types of microorganisms recovered are Gram-positive bacteria with a close phylogenetic affiliation. The most commonly isolated genus are Micrococcus spp., Staphylococcus spp., with occasional isolates of Corynebacterium spp., Bacillus spp., and infrequent fungi. In an aseptic filling area, Gram-negative bacteria are rarely isolated.


However, the human microbiome research would suggest that the predominance of Gram-positive cocci should not be as great as the literature on cleanroom flora suggests. Conversely, the levels of Gram-positive rods (such as Corynebacterium spp.), Gram-negative rods (such as Acinteonacter spp.) and anaerobic bacteria (such as Propionibacterium spp.) should be - based on even shedding of skin detritus - higher in number. 


The differences between what is recovered from the cultural based methods, and the data pertaining to the microbial ecology of the skin, raises a concern with the efficacy of culture-based methods for environmental monitoring. These methods are conventionally active air sampling, settle plates, surface monitoring using swabs and contact plates, and direct personnel monitoring using gown and finger plates. 



The limitations of cultivation


Cultural based methods are limited for a number of reasons, with one key factor being that there is no universal agar or set of incubation conditions (time and temperature) that will recover all culturable microorganisms. The most widely used agar for environmental monitoring is soybean casein digest medium. However, this medium cannot grow all types of bacteria associated with the human body. For example, although not directly related to skin flora, the high presence of Streptococcal bacteria in the oral cavity and its absence from most microbiological monitoring surveys tallies with reports that Streptococcus does not grow well on unmodified forms of this medium 12.


A second reason relates to 'culturability' itself. Many bacteria, although maintaining metabolic activity, are non-culturable due to their physiology, fastidiousness, or mechanisms for adaptation to the environment 13. Some authors argue that only 10% of the bacteria found in cleanrooms are culturable 14




Should we be worried about our environmental monitoring data?


It is unsurprising that not all of the microorganisms resident and transient to human skin can be detected using conventional monitoring methods. Whilst this difference is no doubt of academic interest, does it necessarily mean that environmental monitoring regimes conducted in pharmaceutical environments are inefficient to the degree that  a product could be subject to a higher risk than previously thought?


In answering, I contend that this is not necessarily the case. Given the limitations of the culture methods used for environmental monitoring (as set out above), the methods remain a reliable measure of operator-derived microbial contamination in clean rooms in terms of risk mitigation and for detecting adverse trends. The objective of environmental monitoring is to act as a ‘spot check’ for indicators of cleanroom contamination. What is important is how levels compare with historical trends. Thus, it stands that if levels of bacteria are high, compared with previously seen levels, some facet of the cleanroom or the behavior of the operators is at fault and needs addressing. If the incidents remain very low, the cleanroom is functioning as designed and operator behavior is of an acceptable standard.




Anaerobic organisms?


One area where environmental monitoring may need to be adjusted is in regard to anaerobic microorganisms, especially given the relatively high levels of Propionibacterium spp. that are associated with hair follicles. In most cases, anaerobes will not pose a significant risk due to their inability to reproduce in cleanrooms (although the ability of some anaerobes to survive should not be underestimated 15). However, where nitrogen gas or compressed air lines are used as part of the filling process then the microbiologist may wish to give consideration for the monitoring of anaerobes.





An implication is with operator gowning for entry into cleanrooms. This relates to the question of whether gowning practices are adequate to exclude all microorganisms from the richest areas of the skin microbiome. This is a pertinent point given that most bacteria free-floating in cleanroom air current are not free-living but are instead the result of direct particle shedding of desquamated skin cells and subsequent re-suspension of skin detritus in the air stream 16.


This should lead to a consideration of the types of cleanroom undergarments used and an examination as to whether these provide an effective barrier, especially for the moister parts of the body. Other considerations related to gowning are the importance of the outer gown covering all parts of the body (including the forehead), the level of training required for operators in relation to gowning, the way that gowning qualification is conducted, how long a cleanroom suit should be worn for (in relation to material integrity against operator perspiration) and the environment in which gowns are donned, where higher air-change rates might prove effective.




The information relating to human microbiological data deviations can help support investigations in the event of an environmental monitoring result exceeding an action level. 


Where hitherto unexpected microorganisms are recovered within cleanrooms, the deeper understanding of the human microbiome might suggest that these rarer microorganisms are of human skin origin rather than from an environmental failure. This has implications for the way in which the investigation is conducted and the resultant corrective and preventative actions.



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2.    Gao, Z., Tseng, C.H., Pei, Z., and Blaser, M.J. (2007). Molecular analysis of human forearm superficial skin bacterial biota. Proc. Natl. Acad. Sci. 104: 2927–2932

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7.    Kong, H.H. and Segre, J.A. (2012). Skin Microbiome: Looking Back to Move Forward, Journal of Investigative Dermatology, 132: 933–939

8.    Costello, E.K., Lauber, C. L., Hamady, M., Fierer, N., Gordon, J.I., Knight,R. (2009).  Bacterial community variation in human body habitats across space and time. Science ,326: 1694–1697

9.    Favero, M., Puleo, J., Marshall,J., Oxborrow, G. (1966). Comparative levels and types of microbial contamination detected in industrial cleanrooms. Appl.Microbiol. 14 (4): 539–55

10.    Wu, G. F.; Liu, X. H. (2007). Characterization of predominant bacteria isolates from clean rooms in a pharmaceutical production unit. J. Zhejiang Univ.Sci.B, ,8 (9): 666–672

11.    Sandle, T. (2011): ‘A Review of Cleanroom Microflora: Types, Trends, and Patterns’, PDA Journal of Pharmaceutical Science and Technology, 65 (4): 392-403


12.    Bandettini R, Melioli G. (2012). Laboratory diagnosis of Streptococcus pneumoniae infections: past and future, J Prev Med Hyg; 53(2):85-8


13.    Nagarkar, P. P., Ravetkar, S. D., Watve, M. G. (2001). Oligophilic bacteria as tools to monitor aseptic pharmaceutical production units. Appl. Environ.Microbiol. 67 (3): 1371–1374


14.    La Duc, M. T., Dekas, A.; Osman, S., Moissl, M., Newcombe D., Venkateswaran, K. (2007). Isolation and characterization of bacteria capable of tolerating the extreme conditions of cleanroom environments. Appl. Environ.Microbiol. 73 (8): 2600–2611


15.    Hambraeus, A. and Benediktsdotter, E. (1980). Airborne non-sporeforming anaerobic bacteria, J. Hyg, 84: 181-189


16.    Hospodsky, D., Qian, J., Nazaroff, W.W., Yamamoto, N., Bibby, K., Rismani-Yazdi, H., and Peccia J. (2012). Human occupancy as a source of indoor airborne bacteria, PLoS One. 2012;7(4):e34867 (on-line publication)