Opinion Review Article | Open Access | Published 13th October 2023
Tougher than all the rest? Why Micrococcus luteus can survive for a long time in your cleanrooms.
Tim Sandle, BPL | EJPPS | 283 (2023) | Click to download pdf
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Micrococci are obligate aerobic Gram-positive cocci with the ability to survive in diverse and stress conditions. One of the most abundant organisms on the outer layer of human skin is Micrococcus luteus (something established prior to the advances in understanding (1), arising from the metagenomics sequencing studies of the Human Microbiome Project) (2) and this bacterium, along with Kocuria rhizophila  (a strain that was once included as M. luteus), is a common contaminant recovered from cleanrooms. This bacterium is ubiquitous in nature and can be easily found in natural environments.
M. luteus is easily spotted on a general nutrient medium agar plate, characterized by the production of yellow water-insoluble pigments. The bacterium dates back at least 120 million years.  
 
Surviving in the cleanroom
While these are well-established facts, the ability of M. luteus to survive for prolonged periods within the cleanroom environment is less commonly understood. Excluding endospores, for the common vegetative bacteria, M. luteus displays a level of robustness for prolonged survival within the cleanroom if it is not directly eliminated using a disinfectant. The survival state the bacterium enters into differs from actively growing states and is a form of dormancy.
Those microorganisms that survive for prolonged periods in cleanrooms are those that can tolerate, or undergo adaptive changes to, extreme desiccation and nutrient scarcity. M. luteus is capable of switching its cellular process to tolerate oligotrophic conditions. An oligotroph is a descriptor for an organism that can live in an environment that offers very low levels of nutrients (3).
M. luteus also demonstrates low sensitivity to elevated hydrostatic pressure where a minimal pressure between 100 and 350 MPa is required before cellular damage occurs. Such organisms can undergo biochemical and physiological changes, essentially experiencing a reduced metabolic state which correlates with their ability to tolerate severe external stresses including starvation.
Dormancy is comprised of different states, including persistent and viable but nonculturable (VBNC) states (4). Here cell division is suspended.
The bacterium also displays some tolerance to UV light (5) and, in a dormancy state, to higher temperatures than normal due to structural changes to its DNA (6).
 
Dormancy
The ability of M. luteus to survive starvation for long periods is an important factor in the maintenance of its viability. In terms of dormancy, this is where the cells are metabolically active, respiration competent, and capable of incorporating amino acids into proteins, but not actively growing (7).
Not all cells of M. luteus will survive and enter dormancy. There will be an initial rapid fall in cell numbers; after this, the small remaining population of M. luteus appears to be able to adapt to the harsh realities of the cleanroom environment.  The degree to which a high cell density at the onset of starvation is necessary for sufficient numbers to endure prolonged nutrient limitation is unknown. Phenotypically, the activation of a stress protein is thought to enhance its survival.
 
Starvation and stress protein
Upon starvation, a static population of starved cells develops, and these enter a minimal metabolic state. This has been evidenced by sample collection and survival assays, through launching and recovery, and exposure to simulated conditions in the laboratory. For example, one study found how a significant number of M. luteus cells starved in a prolonged stationary phase (up to 2 months) and then held on the bench at room temperature without agitation for periods of up to a further 2–7 months, could be resuscitated (8).
Here a common mechanism for reactivation is shared within the actinobacteria (the phylum of Gram-positive bacteria to which M. luteus belongs). This is Universal Stress Protein A WP_010079616.1 (commonly referred to as UspA616). Researchers have shown using mass spectrometry this is a statistically significant protein upregulated in latent cells compared to exponentially growing cells (9). Studies also show that the loss of this protein either blocks cell proliferation (enforced latency) or causes cell death under stress.
 
Recovery and resuscitation
 Dormant M. luteus has a mechanism of reversing this state when conditions arise conducive to growth and proliferation. This is both within the cleanroom or if captured through an environmental monitoring method. The gene for this revival factor, Rpf (regulation of pathogenicity factors gene cluster) is found widely among the Gram-positive, high G + C bacteria like the Micrococci and the Staphylococci.  
On agar, even with this protein expressed, recovery can however take longer. Stressed M. luteus shows a temporary lengthening of the time required for colonial growth and pigment formation when grown on laboratory media. It is for this reason that due consideration needs to be applied to the selection of the incubation regime (time and temperature) for the processing of environmental monitoring plates (factors that should not rest upon testing media with full-adapted laboratory strains alone).
As well as tolerance to low-nutrient conditions, it is also notable that the bacteria widely distributed in cleanrooms are mainly Gram-positive strains (10) that show a high resistance to selected disinfectants (11).
 
Summary
What can we learn from this information? The survival characteristics of M. luteus add to our understanding of the VNBC state, in that organisms may be present but not necessarily recoverable using conventional environmental motoring methods. Or where organisms can be recovered, the time required for resuscitation might be longer.
Does this matter? Environmental monitoring can never recover all organisms, there are no universal conditions and environmental monitoring best functions in terms of spot checks with a picture of facility control formed from trend analysis. However, the ability to survive places an important emphasis on appropriate gowning and behaviours supported by a thorough cleaning and disinfection programme.
 
References
 Kloos WE, Musselwhite MS. Distribution and persistence of Staphylococcus and Micrococcus species and other aerobic bacteria on human skin. Appl Microbiol. 1975 Sep;30(3):381-5. doi: 10.1128/am.30.3.381-395.1975. PMID: 810086; PMCID: PMC187193
Grice EA, Kong HH, Renaud G, Young AC. NISC comparative sequencing program; Bouffard, G. G.; Blakesley, R. W.; Wolfsberg, T. G.; Turner, M. L.; Segre, J. A. A diversity profile of the human skin microbiota. Genome Res. 2008;18(7):1043–50
 Davey, HM, Kaprelyants, AS, Kell, DB (1993) Flow cytometric analysis, using rhodamine 123, of Micrococcus luteus at low growth rate in chemostat culture. In: Lloyd, D (Ed.) Flow Cytometry in Microbiology, Springer-Verlag, London, pp 83–93
 Ayrapetyan M, Williams TC, and Oliver JD. 2015. Bridging the gap between viable but non-culturable and antibiotic persistent bacteria. Trends Microbio, l23:7–13.
 Bamji, MS, Krinsky, NI (1966) The carotenoid pigments of a radiation-resistant Micrococcus species. Biochim Biophys Acta 115: 276–284
Greenblatt, CL, Davis, A, Clement, BG, Kitts, CL, Cox, T, Cano, RJ (1999) Diversity of microorganisms isolated from amber. Microb Ecol 38: 58–68
Kaprelyants, AS, Kell, DB (1993) Dormancy in stationary-phase cultures of Micrococcus luteus: flow cytometric analysis of starvation and resuscitation. Appl Environ Microbiol 59: 3187–3196
Mukamolova, G.V., Yanopolskaya, N.D., Kell, D.B. et al. On resuscitation from the dormant state of Micrococcus luteus. Antonie Van Leeuwenhoek 73, 237–243 (1998)
 Mali S, Mitchell M, Havis S et al. . A proteomic signature of dormancy in an actinobacterium: Micrococcus luteus. J Bacteriol. 2017, DOI: 10.1128/JB.00206-17
Wu, Gf., Liu, Xh. Characterization of predominant bacteria isolates from clean rooms in a pharmaceutical production unit. J. Zhejiang Univ. - Sci. B 8, 666–672 (2007)
Chapman, J.S., 2003. Disinfectant resistance mechanisms, cross-resistance and co-resistance. Int. Biodeter. Biodegr., 51(4):271–276
Author Information
Corresponding Author: Tim Sandle, Head of Microbiology Bio Products Laboratory , UK Operations, England Email: timsandle@btinternet.com