Cheek Swab Reveals Presence of Stapholococcus aureus in Oral Cavity of some Individuals
Uaf undergraduate and BIOL 342 student
From childhood we learn about the different environments around the world ranging from the tundra of the arctic to the arid sands of the desserts; with further research we discover the animals that are able to thrive in these environments through adaptations to the conditions. It’s easy to focus on a narrow range of environments to ones we are able to encounter through exploration, and to limit our knowledge of organisms to animals we may come across in those areas we can reach. But what about environments that we don’t think about as being environments? Environments aren’t always large tracts of land or a lake system. What if the area a creature inhabits is unlike any you will ever come across and yet at the same time you are more connected to than any place that you could find on a travel? What if the environment… is you?
With over 700 bacterial species known, the human oral cavity is a perfect home for many bacteria. Bacteria are able to grow on several sites in the mouth including the tongue dorsum, lateral sides of tongue, buccal epithelium, hard palate, soft palate, supragingival plaque of tooth surfaces, subgingival plaque, maxillary anterior vestibule, and tonsils (Aas et al. 2005). There are numerous benign as well as opportunistic pathogens that inhabit the oral cavity as well including Staphylococcus aureus, Streptococcus mitis, and Steptococcus pyogenes as well as species of the yeast Candida (Lab 10). The objective of this study to isolate a bacteria and perform both physiological and genetic tests to determine its identity as well as how and where it survives. With a simple swabbing of the inside my cheek, I am making the attempt to find an organism that calls the human oral cavity home.
A sterile swab was used to rub several times against the inside of my cheek, avoiding other areas of the mouth. The sample was quickly inoculated onto two microbial growth media mediums: a tryptic soy agar (TSA) plate, as well as a Sabouraud’s agar (SA) plate, where it was allowed to grow at around 23 ºC for several days.
In lab the original TSA sample was kept and four different quadrant streaks were carried out throughout the next couple weeks in order to purify the isolate. After these attempts to purify the culture, a series of physiological and genetic tests were used in order to obtain an identity of the bacteria isolated from the cheek swab. The physical tests included a Gram stain, fluid thioglycollate test, oxidase test, catalase test, and an API-20E test, while DNA purification followed by a BaseSpace analysis was used as a genetic test of the isolate.
Three weeks into the project, the most recent TSA plate was used in order to perform a Gram stain (Lab 4 protocol), which would identify if the cell wall of the bacteria had a thick (positive Gram stain) or thin (negative Gram stain) peptidoglycan layer. A positive Gram stain dyes the cells a dark purple due to crystal violet being encased in the cell wall due to dehydration of the peptidoglycan layer followed by addition of Safranin, whereas crystal violet is removed by ethanol from a Gram negative cell and the cell turns a light pinkish red after the introduction of Safranin.
Five weeks from original sampling, three physiological tests were performed on a fresh culture sample. The fluid thioglycollate test was carried out (Lab 6 protocol) in order to determine the oxygen class of the isolate. The solidified agar is a barrier to oxygen, so the bottom of the test tube is anoxic while the top has oxygen available. This test reveals if the bacteria is able to grow only in the presence of oxygen (strict aerobe), grow regardless but better with oxygen (facultative aerobe), grow just below the surface (microaerophile), only grow without oxygen’s presence (strict anaerobe), or equally throughout (aerotolerant anaerobe). The oxidase test was used (Lab 6 protocol) to determine the presence or lack of cytochrome oxidase, which differentiates bacteria into pseudomonad species (oxidase positive) and enteric species (oxidase negative). The catalase test reveals the presence of the catalase enzyme, which protects cells that have it from reactive oxygen species including hydrogen peroxide. By neutralizing these oxygen species, the cell is protected from oxidative damage. The API-20E test is comprised of twenty physiological tests which characterize the Gram negative bacteria. The characteristics tested are an indication of what the bacteria is able to ferment as well as production of certain enzymes and ability to oxidize reactants.
A DNA extraction was performed (Lab 5 protocol) in order to obtain the genomic sequence of the bacterial isolate. The extraction is carried out in three main steps: cell lysis, removal of inhibitors and proteins, and obtaining a pure solution of DNA. Cell lysis is accomplished by physical means of using beads to batter the sample, chemical means to dissolve the cell wall, and by enzymes that break down chunks of cell wall, which allow the cell’s contents to be exposed. Proteins and inhibitors are used by introducing chemical substances that extract these from the solution followed by centrifugation. During the centrifugation, the DNA is bound to a solid matrix while the inhibitors and proteins are part of the supernatant. In the last step, DNA is removed from the solid matrix, centrifuged again, and appears in the resulting supernatant.
The last test on the isolate performed was to determine its antibiotic susceptibility (Lab 9 protocol). Seven antibiotic discs in total were placed among the isolated culture to determine if zones of inhibition were produced by the antibiotics that prevented its bacterial growth. The antibiotics tested included tobramycin, cefazolin, vancomycin, cefoperazone, trimethoprim, gentamicin, and amikacin. The results were compared to known susceptibility thresholds of each antibiotics in order to determine efficacy of the antibiotic.
After the first 24-hour period after inoculation, no colonies were evident on either plate. After 72 hours, numerous colonies of varying sizes were forming on the TSA plate compared to one large colony on the SA plate. After five days, the number of colonies on the TSA plate had reached double digits while the SA plate still had one major colony with a couple smaller colonies on the side. After one week’s time, the TSA plate had around 30 colonies; there was one large colony, four colonies larger than 1mm, and the amount of pencil-tip sized colonies was in the double digits. On the SA plate, however, there was no noticeable growth than observed on day 5.
Figure 1. A comparison between colonies formed on SA plate (left) and TSA plate (right).
The results of the physiological tests will be discussed first. Under the microscope, there was Gram negative bacteria along with Gram positive bacteria. The sample was continuously purified thereafter until the resulting bacteria was completely Gram positive. The fluid thioglycollate test revealed that growth occurred more on the surface than the underlying agar. The oxidative test on the bacterial isolate was a yellow/buff color, which occurs when the test is negative. The catalase test showed that the isolated bacterial bubbled when exposed to hydrogen peroxide. This is a positive result to the catalase test. The original API-20E test revealed that the bacterial contaminant did not have gelatinase and thus couldn’t break down gel, could oxidize nitrate to nitrite, had a positive citrate test, and was able to ferment glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, meliblose, amygdalin, and arabinose. The bacteria had large zones of inhibition on all antibiotics tested.
Figure 2. Picture of contaminated Gram stain. Figure 3. Example of zone of inhibition.
Figure 4. Picture of thioglycollate test. Figure 5. Picture of contaminant API-20E test.
The genetic results showed that the number of contigs in the sample tested was in the hundreds, the total length was in the millions, and the largest contig was over 100,000 bp. The result of the BaseSpace analysis was that 97% of all contigs were able to be read; of those contigs, there was a 99.94% match with known samples of Staphylococcus aureus.
Figure 6. Pie diagram and bar graph results of BaseSpace test revealing Staphylococcus aureus as isolate.
TSA favors bacterial growth while SA provides a better medium for fungal growth to thrive. The reason more colonies grew on the TSA plate was because more colonies were bacterial and made more use of the medium that favored them. The reason colonies existed on the SA plate was because either there was a fungus within the cheek, or there was bacteria that was able to survive with the resources available in the SA plate.
The original Gram stain result of both Gram positive and Gram negative bacteria proves the bacterial sample was not pure. A purified sample of bacteria would be one or the other, unless the bacteria is Gram variable, which is very rare and did not match the known scientific literature with the results of the genomic sequencing. The gram positive result on the culture after further purification did agree with scientific literature. Under the microscope, the bactieria formed clusters of round cells.
The fluid thioglycollate test shows the bacteria is a facultative anaerobe as it grows more when oxygen is available, but is able to survive when oxygen is not present. The negative result on the oxidase test reveals the bacterial isolate is an enteric species, while the positive catalase test reveals that the culture has a resistance to reactive oxygen species and thus protects the cell from oxidative damage The API-20E test results showed that there was a Gram negative bacteria present in the culture that was not present in later Gram stains. Unfortunately, an API-Staph was not carried out since the writer of this paper missed the part about API-20E working on Gram negative bacteria only when he had decided the isolate was Gram positive. The antibiotic disc test revealed the isolate is highly susceptible to all seven antibiotics tested when compared to their susceptibility thresholds.
The number of contigs, total length, and the size of the largest contig were all statistically meaningful (Lab 7 protocol). The high amount of readable contigs as well as the nearly identical comparisons between laboratory results and scientific literature suggest to the utmost that the bacteria isolated from the cheek swab is Staphylococcus aureus.
Using the API-20E test, the Gram negative result, and the observations of the bacterial contaminant under a microscope, it would be possible for future research to determine the identity of this contaminant. It is interesting that the contaminant yielded a catalase positive, oxidative negative, and citrus positive result, all of which are shared by the Gram positive Staphylococcus aureus that was isolated. I attribute this to the similarity of the environment from which these bacteria were isolated.
The remainder of this paper will discuss the bacterial isolate. Staphylococcus aureus is found in clusters of round cells in the nasal passages, oral cavity, lower reproductive tract, and skin of humans as well as other animals such as the dogs of healthcare workers (Masalha et al. 2001). It’s estimated that 20-30% of people are long term carriers (Tong et al. 2015). Usually a commensal bacteria, it becomes an opportunistic pathogen when circumstances allow and cause ailments ranging from minor skin infects and food poisoning to life-threatening diseases such as pneumonia, endocarditis, toxic shock, and sepsis (MedlinePlus 2017). Once it’s inside the bloodstream, Staphylococcus aureus has enzymes to clot blood and destroy proteins, can secrete toxins, and has super-antigens that cause organ failure by causing the immune system to become hyper-reactive. Antibiotic-resistant strains such as methicillin-resistant Staphylococcus aureus (MSRA) are becoming increasingly abundant and are a major concern in the medical field due to transmissions in hospitals. Over half a million people in US hospitals contract Staph infections, and most are caused by Staphylococcus aureus (Bowersox 1999). Staphylococcus aureus is here to stay; it would benefit us to further research this bacteria to better understand it to aid in limiting its role as an opportunistic pathogen that has taken a countless amount of lives.
Aas J; et al. (2005). “Defining the Normal Caterial Flora of the Oral Cavity’. J Clin Microbiol. November 2005. 43(11) 5721-5732.
Bowersox, John (27 May 1999). “Experimental Staph Vaccine Broadly Protective in Animal Studies”. NIH. Archived from the original on 5 May 2007. Retrieved 28 July 2007.
Masalha M; et al. (2001). “Analysis of Transcription of the Staphylococcus Aureus Aerobic Class Ib and Anaerobic Class III Ribonucleotide Reductase Genes in Response to Oxygen”. Journal of Bacteriology. 183 (24): 7260—7272.
MedlinePlus [Internet]. “Staphylococcal Infections”. Bethesda, MD: National Library of Medicine, USA. Skin infections are the most common. They can look like pimples or boils.
Tong SY; Davis JS; Eichenberger E; Holland TL; Fowler VG (July 2015). “Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management”
UAF Lab handouts provided to me by Mary Beth Leigh.