Team:Paris Bettencourt/Contribution
EpiGrow, Optimization of S. epidermidis Growth
Introduction
The composition and distribution of the skin microbiome can be influenced by a variety of factors. The skin is always exposed to the external environment, intrinsic host factors along with extrinsic factors such as climate and habitat have a great influence on the microbial flora that colonize the skin.[4] To effectively implement our goal of developing Staphylococcus epidermidis as a chassis for synthetic biology of the skin microbiome, it was essential to explore the growth of our target organism.
S. epidermidis represents approximatively 90% of the aerobic skin flora, and about 5% of the overall skin microbiome species. It t behaves mainly in a mutualistic way. It is a non-motile gram-positive coccus,and a facultative anaerobe. This means that it thrives on growing using respiration but that it can also use fermentation if required.
Our team chose to probe the growth kinetics of S. epidermidis with respect to change in 3 parameters that we could expect to fluctuate on the skin in response to different environmental/ physiological cues.[4] We reasoned that to gain such insight will be relevant to formulating a synthetic probiotic. All of our experimental work was done using Staphylococcus epidermidis strain ATCC12228, which was kindly gifted to us by the L'Oréal Paris microbiology lab.
Additionally, we were intrigued by the question of if there is a change in the growth kinetics in 2D vs 3D environments. To explore further we decided to build an artificial skin model. This is an ongoing part of this project.
Temperature
Normal human skin temperature on the trunk of the body varies between 33.5 and 36.9 °C.[4] The surface of the skin exhibits significant regional temperature variations, it is lower over protruding parts, like the nose and higher over muscles and active organs.[4] Also, fluctuations can be seen within one region and from person to person, due to a variety of reasons (same as homeostasis of body temperature) like health condition, time of the day, sex, age, state of consciousness, etc.[4]
Since S. epidermidis is one of the most frequently isolated species from the skin and is also considered to be ubiquitous in healthy individuals, we decided to probe its growth rate with respect to varying temperatures.
We chose to test the temperature range from 32°C-42°C. The temperature of the skin can be lower than the body temperature. In fact, it may reach temperatures as low as 33°C especially in the extremities such as hands, feet and nose.[4] And high skin temperature can be caused due to fever, which in severe conditions may rise above 40°C.
To study the effect of temperature on the growth of S. epidermidis, we grew the microbe in TSB (Tryptic Soy Broth) media at different temperatures between 32°C and 42°C for 15 hours. The optical density of the growing solution (ƛ=600nm) was measured every hour and the growth rate was calculated.
pH
The most important role of the skin is that it acts as a barrier between our bodies and the outside world, and the skin’s pH is one of its key protective mechanisms. The pH of our skin is constantly challenged by external aggressors such as pollution, temperature changes, and harsh chemicals.
The acidic quality of the skin plays an important role as a protective barrier. The acid mantle is key as it neutralizes alkaline-based aggressors (such as harsh surfactants) and maintains the optimal acid environment in which skin’s natural flora can thrive. The skin’s pH generally fluctuates between 5.6 and 6.4 (armpit, genital area) depending on the region of the body it is covering.[4] The skin’s acidity results from lactic acid from its own keratinocytic byproducts, amino acids from sweat, fatty acids from sebum, as well as the metabolites of the microbes colonizing its various surfaces.[4]
To study the effect of pH on the growth of S. epidermidis, we grew the microbe in TSB media at different pH values ranging from pH 3 to pH 10 for 15 hours. The optical density of the growing solution (ƛ=600nm) was measured every hour and the growth rate was calculated.
Salinity
With respect to the skin, there can be variations in salinity caused mainly by sweat and sebaceous glands. Sweat is produced by the eccrine glands and apocrine glands located largely around the armpits, foreheads, soles of the feet, palms of hands, etc. Secretions of sweat from sweat glands to the skin are high in salt concentration and as the amount of sweating increases, the amount of salt reaching the skin’s surface increases too. The evaporation of water from the release of heat enables the salts to remain present on the skin.[4]
To study the effect of varying NaCl concentration on the growth of S. epidermidis, we grew the microbe in TSB media at different NaCl concentration values for 15 hours. The optical density of the growing solution (ƛ=600nm) was measured every hour and the growth rate was calculated.
Conclusion
The aim of these experiments was to understand if we can boost the growth of S. epidermidis in the laboratory by changing a few parameters.
With respect to the temperature range we tested, the results show a trend which is on par with our expectations. The growth of S. epidermidis is stunted at lower temperatures, and the microbe grows best at 37°C which is the normal human body temperature.
Surprisingly our results of the pH test were different from what we expected. The pH of the human skin generally varies from 5.6 - 6.4. However, the calculated growth rates indicate that S. epidermidis thrive better in the neutral and basic pH conditions.
It has been previously reported that S. epidermidis can survive in high saline conditions. From the salinity tests we conclude that S. epidermidis is indeed halo tolerant and it grows best at NaCl concentration of about 0.5 percent.
References
- 1. Bierman, William (1936-04-04). "The Temperature of the Skin Surface". Journal of the American Medical Association. 106 (14): 1158. doi:10.1001/jama.1936.02770140020007. ISSN 0002-9955
- 2. Kanitakis, Jean (2002-07-02). "Anatomy, histology and immunohistochemistry of normal human skin". European Journal of Dermatology. 12 (4): 390–9, quiz 400–1. ISSN 1167-1122. PMID 12095893
- 3. Benedict, FG; Miles, WR; Johnson, A (June 1919). "The Temperature of the Human Skin". Proceedings of the National Academy of Sciences of the United States of America. 5 (6): 218–22. Bibcode:1919PNAS....5..218B. doi:10.1073/pnas.5.6.218. PMC 1091574. PMID 16576376
- 4. Edmonds-Wilson S,Nurinova N, Zapka C et al. “Review of human hand microbiome research”.Journal of Dermatological Science (2015), 3-12,80(1). doi.org/10.1016/j.jdermsci.2015.07.006
- 5. Marples, M. J. The Ecology of the Human Skin. Charles C Thomas Publisher. Springfield, Ill. (1965) pgs103-154
- 6. Aly, Raza. Clinical Skin Microbiology. Springfield, IL: Thomas Books, 1987. 11-35.
- 7. Elias, Peter M., and Kenneth R. Feingold, eds. Skin Barrier. Danbury: Marcel Dekker Incorporated, 2006