Need help assisting with my microbiology paper on staphylococcus

Sean Rivera

Professor Ryan Manow

Micorbiology 233

11/27/19

Staphylococcus aureus Lab Report

Introduction:

In the late 1800s, while trying to figure out why infections and sepsis occur in wounds, Alexander Ogston used a microscope to look at a sample of pus (taken from an infected person) and found a Gram-positive bacterium (Myles, I. and Datta, S., 2012). Ogston named that bacteria
Staphylococcus aureus, since it was spherical and grew in ‘grape-like bunches’ and (when cultured) it was gold in color (Myles, I. and Datta, S., 2012). In the late 1800s,
S. aureus was a problematic bacterium and it is still problematic today (Myles, I. and Datta, S., 2012). It is estimated that at
S. aureus has colonized anywhere from 30% to 70% of the world’s population (Liesenborghs L, Verhamme P, Vanassche T., 2018). Using the more conservative estimation (30%) suggests that
S. aureus has colonized at least 2.325 billion people (of the almost 7.75 billion people on Earth) (“Current World Population” 2019). It is estimated that millions of infections in the soft tissue and the skin occur per year in the United States alone (Myles, I. and Datta, S., 2012). Some diseases that can occur due to chronic
S. aureus infections can include type 2 diabetes, pneumonia, and renal failure (Myles, I. and Datta, S., 2012).
S. aureus is more likely to colonize those undergoing dialysis, surgery, and/or other situations where there is broken skin or mucus-barriers, which allows the bacterium to make its way into the blood (Kim et al., 2019). If
S. aureus gets into the bloodstream it can cause bacteremia, which can lead to infective-endocarditis, or colonization of the heart’s innermost tissue layer, which can then lead to heart failure (Odutola, A., et al., 2019). The bacterium can also cause sepsis (body wide inflammation), pneumonia (fluid in the lungs), and osteomyelitis (a bone infection) (Odutola, A., et al., 2019). The large number of infected individuals and the severe types of diseases it can cause highlight just how dangerous this bacterium can be for humans.

From the late 1800s to the mid 1900s, treatment for S. aureus infected individuals was limited to topical phenol applications, but in the mid 20th century penicillin was introduced as a viable treatment option (Myles, I. and Datta, S., 2012). That treatment, however, had its limitations; within a few years of being introduced to penicillin,
S. aureus had mutated and could now produce penicllinase (which gives it penicillin-resistance) and shortly after (in 1961) methicillin-resistant
S. aureus (MRSA) had been discovered after alternative antibiotics were used to treat the disease (Myles and Datta, 2012). The MRSA strain is notably different that the methicillin-susceptible
S. aureus strain in that it has the
mecA gene, which codes for a different penicillin-binding-proteins (PBP2a) allowing the bacterium to continue synthesizing its peptidoglycan layer, which is necessary for the cells to survive and also the target of many antibiotics (Stapleton and Taylor, 2002). Since
S. aureus is associated with so many diseases, it is important to note that many of the initial infections actually begin in the hospital (Kim, Greenfield, Snyder, Steinmaus, and Riley, 2018).
S. aureus infections are a huge concern for people in the hospital as patients are more likely to have compromised immune systems, which is more likely to lead to a more severe staph-infection (“Healthcare-associated Infections:
Staphylococcus aureus in Healthcare Settings” 2019). It is such an issue that in some Hong Kong hospitals, they started testing people when they were admitted to see if they carried the MRSA strain or not (so they could try to reduce the spread of the bacterium) (Leung, Lee, and Lai 2013).

Materials and methods:

To test whether or not the people in our class were carriers of
S. aureus, we cultured our bacteria on mannitol salt agar (MSA) plates. The plates consisted of the selective agent sodium-chloride (not all bacteria can survive in high-salinity environments,
S. aureus is one that can) and the differential agent mannitol (which is used as a source of nutrients for the bacteria on the plate) (Leboffe and Pierce 2010). To see the effect of the differential agent, phenol red is also in the agar plate; it changes color depending on the pH (the more acidic the environment, the more yellow the plate will become [it starts off as a reddish color, and will stay red if the pH remains neutral or becomes basic]) (Leboffe and Pierce 2010). Since the majority of staphylococci genus can endure the high salinity of the MSA plate, they are able to grow on the plate; however, few are able to use the mannitol for fermentation (Leboffe and Pierce, 2010).
S. aureus is one of the staphylococci species that can ferment mannitol, which produces acid and lowers the pH of the plate where the bacterium is growing; which is evident by the phenol red producing a color shift to yellow (Leboffe and Pierce, 2010). The inside of the nose, the ear, and the tonsils were the environments that were swabbed to test to see if
S. aureus had colonized there (as those are environments where the bacterium is commonly found). To test whether or not we had those respective areas colonized by
S. aureus, we used sterile swabs pick up the bacteria growing on the inside of our nose (the lowermost portion) and our ears (mostly on the outside of the ear, but also just inside of the ear canal). After dividing an MSA plate in half (with the use of a marker), the bacteria from the nose was streaked on one side and the bacteria from the ears was streaked on the other. Similarly, each tonsil was swabbed with its own sterile swab and streaked on a second MSA plate (also divided in half). The cultures were then incubated at roughly 37°C for about two days, after which, the plates were examined to check for growth and color changes.

Results:

This is a picture of the MSA plate containing the bacteria from my ears (the left side of the plate) and my nose (the right side of the plate).

This is the picture of the MSA plate containing the bacteria from my tonsils (each of them on their respective sides).

As indicated by the colonies growing on the MSA plates, it was clear that I was definitely a carrier of a staphylococci bacterium (or two); as it is one of the few genera that can handle the high salinity of the skin (and the MSA plate) (Leboffe and Pierce, 2010). Looking at the bacteria cultured from my nose, it was relatively clear that
S. aureus was not present there; there was not even a hint of yellow on that half of the MSA plate.
Staphylococcus epidermidis is most likely to be the bacterium present on this plate, though it is possible that it could be a different staphylococci species (as there seem to be quite a few that are part of the human’s normal microbiota), but with estimations that 100% of the human population are carriers of
S. epidermidis, it should be safe to assume that that is the bacterium growing on the plate if there was no change in pH (Lawrence and Lawrence, 2011)
. Conversely, the bacteria cultured from my ear were able to produce enough acid to decrease the pH enough to change the color of the phenol red to a bright yellow; indicating that
S. aureus had colonized my ears. Looking at the MSA plate containing the bacteria from my tonsils, it is apparent that
S. aureus has not colonized there yet; as there was no change in color on the plate. However, it is likely that
S. epidermidis was present there as well due to the fact that there was plenty of growth on the plate. Everyone in the class had reported that they were carriers of
S. aureus; 22% of the class was noted as nose-carriers, 74% as ear-carriers, and 85% as tonsil-carriers.

This is a graph representation of how many students were carriers of
S. aureus at different sites on the body.

Discussion:

As a class, our results are fairly interesting. It has been estimated that 30-70% of the population are carriers of
S. aureus (Liesenborghs L, Verhamme P, Vanassche T., 2018). These estimations could easily be skewed towards either end of the spectrum; it really depends on what sites were tested for the presence of
S. aureus; overall, 100% of our class proved to be carriers of
S. aureus, but the numbers varied widely between different sample sites.
S. aureus can appear on the throat, nose, groin, and other places; with the nose being the most likely site for the bacterium to colonize (Kadariya et al., 2019). Based on that, if we were to only pick one site to test for the presence of the bacterium, the nose (which is thought to be the most common) seems like it would be a fairly good site to test. However, based on our classes results, that is not always the case. Had we only tested the nose, we would have believed that only 22% of the class were carriers of the bacterium, despite the fact that 100% of our class are carriers of the bacterium.

According to the CDC, the bacterium is spread when a person comes into direct contact with the bacterium (either through skin to skin contact or by coming into contact with something that an infected person had touched), so our results could be biased as it is entirely possible that a handful of people had spread it to other people (“Methicillin-resistant
Staphylococcus aureus (MRSA) General Information” 2019). A big problem associated with
S. aureus carriage is that it is very likely that there are little-to-no symptoms (as it can be part of our microbiota), so it can easily be spread from person-to-person (Kadariya et al., 2019). With all of us being in close proximity to each other and using a lot of the same lab equipment, it would be easy for the bacterium to spread throughout the class (especially if something was not cleaned after use [I do not believe I have ever washed, or seen anyone wash, the handles on the inoculating loops]). I know that I do not really have many of the symptoms associated with
S. aureus colonization, so I easily could have been spreading it to the rest of my class if I was rubbing my ear and then using some of the shared lab equipment or I could have picked it up when using that lab equipment and then touching my ear.

Since it seems like
S. aureus is roughly as problematic as it was when it was first discovered as it is now, it should be safe to say that most people will not want to be colonized by it (as it would make it that much easier to develop some of the more severe symptoms associated with the bacterium in the future). Some of the ways to get rid of the organism are to use a diluted bleach bath, bleach is a free-radical generator that can destroy parts of the bacterial cell needed to stay alive, so the mecA gene should have no effect against the harm that bleach will cause the organism (Huang, Abrams, Tlougan, Rademaker, Paller, 2009). Another way to get rid of the bacteria is by using mupirocin, though it only works on bacterial colonies in the nose (“Mupirocin (Nasal Route)” 2019). These two ways of getting rid of the bacterium seem to be very effective at reducing the amount of the bacterium present on individuals.

References:

Diluted Bleach Bath Helps Reduce Atopic Dermatitis, Staphylococcus aureus. (2009, August 28). Retrieved November 27, 2019, from https://www.medscape.com/viewarticle/707974.

Kadariya, J., Thapaliya, D., Bhatta, S., Mahatara, R. L., Bempah, S., Dhakal, N., & Smith, T. C. (2019). Multidrug-resistant Staphylococcus aureus Colonization in Healthy Adults Is more Common in Bhutanese Refugees in Nepal than Those Resettled in Ohio.
BioMed Research International,
2019, 1–11. doi: 10.1155/2019/5739247

Kim, M. W., Greenfield, B. K., Snyder, R. E., Steinmaus, C. M., & Riley, L. W. (2018). The association between community-associated Staphylococcus aureus colonization and disease: a meta-analysis.
BMC Infectious Diseases,
18(1). doi: 10.1186/s12879-018-2990-3

Lawrence, R. A., & Lawrence, R. M. (2011).
Breastfeeding: a guide for the medical profession. Maryland Heights, MO: Mosby/Elsevier.

Leboffe, M. J., & Pierce, B. E. (2010).
Microbiology Laboratory Theory and Application (3rd ed.). Colorado: Morton Publishing.

Leung, E. C. M., Lee, M. K. P., & Lai, R. W. M. (2013, September 19). Admission Screening of Methicillin-Resistant Staphylococcus aureus with Rapid Molecular Detection in Intensive Care Unit: A Three-Year Single-Centre Experience in Hong Kong. Retrieved November 27, 2019, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3793295/.

Liesenborghs, L., Verhamme, P., & Vanassche, T. (2018). Staphylococcus aureus, master manipulator of the human hemostatic system.
Journal of Thrombosis and Haemostasis,
16(3), 441–454. doi: 10.1111/jth.13928

Mupirocin (Nasal Route) Proper Use. (2019, February 1). Retrieved November 27, 2019, from https://www.mayoclinic.org/drugs-supplements/mupirocin-nasal-route/proper-use/drg-20064917.

Myles, I. A., & Datta, S. K. (2012). Staphylococcus aureus: an introduction.
Seminars in Immunopathology,
34(2), 181–184. doi: 10.1007/s00281-011-0301-9

Odutola, A., Bottomley, C., Zaman, S. A., Lindsay, J., Shah, M., Hossain, I., … Mackenzie, G. A. (2019). Staphylococcus aureus Bacteremia in Children of Rural Areas of The Gambia, 2008–2015.
Emerging Infectious Diseases,
25(4), 701–709. doi: 10.3201/eid2504.180935

Staphylococcus aureus in Healthcare Settings. (2011, January 17). Retrieved November 27, 2019, from
https://www.cdc.gov/hai/organisms/staph.html.

Stapleton, P. D., & Taylor, P. W. (2002). Methicillin resistance in Staphylococcus aureus: mechanisms and modulation. Retrieved November 27, 2019, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2065735/.

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