Determination of Staphylococcus epidermidis On the Door Handle

Determination of Staphylococcus epidermidis On the Door Handle

Abstract

The purpose of the experiment was to find out the bacteria present on the door handle. The unknown bacterium was swabbed from the door handle, and multiple tastes done to identify it. These tastes included Gram Stain, Mannitol Salt Agar; MSA, Catalase, and Blood Agar Plates; BAP. After the experiment, the bacteria turned out to be Gram-positive cocci, MSA negative since it remained pink, catalase-positive, and BAP test turned green, implying it was alpha-hemolytic. Therefore, the unknown bacterium was Staphylococcus epidermidis because it has all these characteristics. Despite Staphylococcus epidermidis being a normal human flora on the skin, it is pathogenic among individuals with compromised immunity; hence infected individuals need treatment or removal of implants.

Introduction

Bacteria are small and single-celled organisms. They exist everywhere within the ecosystem. Bacteria have varying adaptations that enable them to live under extreme pressure and temperature conditions (Choi et al., 2018, p. 2). The human body has many bacteria when compared to human cells. However, most of these bacteria are harmless, and most of the time, they are helpful to individuals. For instance, commensal bacteria act on the host’s immune system, inducing a protective response that helps prevent invasion and colonization by pathogens. Also, other useful bacteria help in various body functions like digestion for the body to function normally. Just a small number of these bacteria will cause diseases in human beings. Disease-causing bacteria are called pathogenic. Besides, these bacteria have varying shapes, which may be spiral-shaped, rod-shaped, or even spherical (Choi et al., 2018, p. 3). Therefore, the main objective of this experiment was to determine the bacteria present on the door handle through a series of tests like Gram stain, Mannitol Salt Agar; MSA, Catalase, and Blood Agar Plates; BAP.

The gram stain test is mainly used in determining the chemical composition of the cell wall of a bacterium. The bacterial cell wall may stain negative or positive. Bacteria with a thick peptidoglycan layer will always stain positive, which will retain the crystal violate stain (Tripathi & Amit, 2020, p. 2). However, those bacteria with a thin peptidoglycan layer and having a lipopolysaccharide membrane will always stain gram-negative.  because such will allow the washing away of crystal violate by adding ethanol. These bacteria will stain red or pick when safranin; a counterstain is added, though, for gram-positive, they will stain violate when a counterstain is added. Therefore, the color is used in distinguishing gram-negative and gram-positive bacteria. Understanding the chemical make-up of a bacterial cell wall helps in manipulating the bacteria for various purposes (Tripathi & Amit, 2020, p.3). For instance, in medical research, Gram stain may help in checking the site for the bacteria like the skin wounds, genitals, lungs, or throats. Also, Gram stain may help in checking bacteria present in certain fluids of the body like urine or blood.

Mannitol Salt Agar; MSA was the second test. It is a differential and selective growth medium. MSA inhibits the growth of some bacteria and, at the same time, encourages the growth of others (Ahmadunissah et al., 2021, p. 69). The medium has high sodium chloride concentration of about 7.5 to 10%, which inhibits the growth of many of the bacteria. Therefore, this inhibitory aspect of MSA renders it selective against certain Gram-positive bacteria and most Gram-negative bacteria. Some bacteria likely to tolerate this high salt concentration include those of the genus Micrococcus, Enterococcus, and Staphylococcus (Ahmadunissah et al., 2021, p. 70). Similarly, it is a differential medium for Staphylococcus mannitol-fermenting that contains carbohydrate indicator phenol red which is a pH indicator that detects acids that are produced through staphylococci mannitol-fermenting. Yellow zones and yellow colonies will be produced by Staphylococcus aureus, whereas red or pink colonies that have no change to the medium color medium will be produced by other coagulase-negative staphylococci. An organism with the potential of fermenting mannitol will form a byproduct of an acid which turns the phenol red in the agar yellow (Ahmadunissah et al., 2021, p. 73). Therefore, MSA helps in determining those bacteria that can metabolize mannitol.

Catalase was the third test done during the experiment. Catalase is an enzyme found in living organisms exposed to oxygen, and it catalyzes the process that decomposes hydrogen peroxide into oxygen and water (El-Sawaf et al., 2022, p. 3). The enzyme has four irons that contain heme groups allowing it to react with hydrogen peroxide. Catalase test is important when determining if a Gram-positive coccus is streptococci or staphylococci. The appearance of bubbles signifies that the bacteria is catalase-positive, while failure for any bubbles to appear means that the bacteria are catalase-negative. Micrococcus spp. and Staphylococcus spp. are catalase-positive, while Enterococcus spp. and Streptococcus spp. are catalase-negative (El-Sawaf et al., 2022, p. 3). In case a Gram-positive coccus turns out to be catalase-positive and then presumed to be a Staphylococci, a coagulate test follows, which assays coagulase presence hence differentiating between Staphylococcus epidermidis, which is coagulase-negative and Staphylococcus aureus which is coagulase positive. Hydrogen peroxide produced is toxic to organisms and may cause cell lysis if it is not broken down; therefore, the presence of catalase enzyme helps in the hydrolysis of hydrogen peroxide to produce oxygen and water protecting the organism from the lethal effect of hydrogen peroxide (El-Sawaf et al., 2022, p. 3).

Blood Agar Plates; BAP was the final experimental test done to identify the bacteria on the door handle. BAP has mammalian blood, mostly for a horse or sheep, at a typical concentration between 5-10% (Choi et al., 2018, p. 3). BAP is differential and selective media. It is well-enriched for isolating fastidious organisms and detecting hemolytic activity. β-Hemolytic will always display lysis and total digestion of the content of red blood cells that surrounds the colony. Red blood cells will undergo partial lysis when α-Hemolysis is used. The cell membrane remains intact and appears brown or green due to the conversion of methemoglobin from hemoglobin (Choi et al., 2018, p. 4). Gamma hemolysis results when there is no occurrence of any hemolysis.

Methods and Materials

All the following protocols are cited by Williams (2022).

Gram Stain

1. Prepared a wet mount by dripping one water drop onto a clean glass from the spray bottle.
2. Flamed the loop and selected a bacteria colony.
3. Transferred the bacteria onto a water drop on the slide and slightly spread it to disperse the cells.
4. Held the slide using a clothespin and moved the slide back and forth over the Bunsen burner flame. Avoided holding the slide in the flame to prevent it from breaking.
5. Continued until all the water evaporated from the slide. Confirmed when it looked cloudy.
6. Placed the slide over gram stain stray and started staining.
7. Added a few crystal violet drops onto the specimen enough to cover the specimen and kept it for 20 seconds.
8. Gently rinsed off the stain using a squirt bottle, ensuring that the squirt was above the specimen on the slide and allowed the water to run over the specimen while avoiding direct squirting.
9. Added Gram’s iodine and kept it for 1 minute, then rinsed with water.
10. Decolorized with alcohol by adding alcohol drops until the purple color stopped coming off the specimen, though some collations were visible on the bacteria.
11. Rinsed with water.
12. Added safranin and kept it for 20 seconds, then rinsed with water.
13. Allowed the slide to dry and used a microscope (up to 100x) to view the cells.

Mannitol Salt Agar; MSA

1. Poured the cooled Mannitol Salt Agar into a sterile petri dish and allowed it to cool to room temperature.
2. Inoculated the plate using the colony from the isolation steak.
3. Incubated at 37⁰C for 24 hours.

Catalase

1. Used plastic applicator to obtain unknown bacteria.
2. Spread the bacterial colonies on a clean glass slide in a small area.

• Added hydrogen peroxide drops to the colonies on the slide.

1. Observed for bubbling.

Blood Agar Plates; BAP

1. Used bacterial culture to inoculate BAP.
2. Incubated at 37⁰C for 24 hours.

Results

Table 1: Gram Stain Results  

Description	Results
Purpose	To differentiate bacterial cell wall, whether it is Gram + or Gram –
Resultant color of the Stained Morphology	Violet
Gram Status	Positive
Shape	Cocci
Possible Bacteria Present	Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumonia, Streptococcus pyogenes, Staphylococcus hominis, Micrococcus roseus Viridans streptococci, Staphylococcus intermedius, Staphylococcus argenteus

 

Table 2: Mannitol Salt Agar; MSA

Description	Results
Purpose	To assess if the bacterium can metabolize mannitol
Resultant color of the colonies	Pink
MSA status	Negative
Possible Bacteria Present	Staphylococcus epidermidis, Micrococcus roseus

 

Table 3: Catalase

Description	Results
Purpose	To determine the presence of catalase enzyme
Observation	Bubbles produced
Catalase status	Positive
Possible Bacteria Present	Staphylococcus aureus, Escherichia coli, Pseudomonas aeuroginosa, Micrococcus luteus, Staphylococcus hominis, Staphylococcus epidermidis, Pseudomonas aeuroginosa, Micrococcus roseus, Staphylococcus intermedius, Staphylococcus argenteus

 

Table 4: Blood Agar Plates; BAP

Description	Results
Purpose	To assess the presence or absence of hemolytic enzyme
Observation	The green color of the cell membrane was visible
BAP Status	α hemolytic
Possible Bacteria Present	Streptococcus pneumoniae, Streptococcus viridans, Streptococcus mitis, Pseudomonas aeuroginosa, Viridans streptococci, Staphylococcus argenteus, Staphylococcus intermedius, Staphylococcus epidermidis

 

Discussion

The swabbed bacterium from the door handle was finally discovered to be Staphylococcus epidermidis. From the first test conducted: Gram Stain, Staphylococcus epidermidis was found to meet all the specified characteristics of a Gram-positive cocci bacteria (Tripathi & Amit, 2020, p.4). When observed under a microscope, it was found that the bacteria stained violet when the counterstain was added. The implication created was that its cell wall was made up of a thick peptidoglycan layer which retained the crystal violet stain color. Therefore, the violet color retained categorized the bacteria to be Gram-positive. In addition to that, the bacteria were arranged in grape-like clusters, and their shape looked like an ovoid hence concluded to be cocci. Hence, Gram-positive cocci bacterium (Tripathi & Amit, 2020, p.4). However, there were other bacteria that met the same characteristics of Gram-positive cocci, such as Staphylococcus argenteus, Staphylococcus hominis, Staphylococcus aureus, and Streptococcus pneumonia, although they did not meet other tests hence disqualified.

For the Mannitol Salt Agar test, the observation made displayed a pink color. The pink color implied that the bacteria under observation could not ferment mannitol which could have resulted in changing the pink color of the red phenol indicator into yellow (Ahmadunissah et al., 2021, p.73). The red phenol indicator is the pH indicator for MSA. Therefore, of the pink color, the MSA status was concluded to be negative. The bacteria suspected to be present in this case include Staphylococcus epidermidis and Micrococcus roseus. During this test, some of the bacteria like Staphylococcus aureus were disqualified because they can grow on MSA and metabolize mannitol hence changing the pink color of the red phenol indicator into yellow (Ahmadunissah et al., 2021, p. 73). Therefore, the suspected bacteria, in this case, were Staphylococcus epidermidis and Micrococcus roseus because they could not metabolize mannitol.

During the catalase test, there were bubbles emanating from the bacteria after adding hydrogen peroxide. The bubbles indicated that the bacteria under study had a catalase enzyme that hydrolyzed hydrogen peroxide into oxygen and water (El-Sawaf et al., 2022, p. 3). Therefore, the catalase status was concluded to be positive. Some of the suspected bacteria, in this case, included Staphylococcus aureus, Escherichia coli, Pseudomonas, Staphylococcus hominis, Staphylococcus epidermidis, and Staphylococcus argenteus.

The last test involved Blood Agar Plates. The green color was observed on the cell membrane. The BAP status was concluded to be alpha-hemolytic. Some suspected bacteria included Streptococcus pneumoniae, Streptococcus viridans, and Staphylococcus epidermidis. Some of these bacteria were disqualified and remained with Staphylococcus epidermidis as the only bacteria observed under the other tests. Mostly, Staphylococcus epidermidis display Beta hemolysis and sometimes Gama hemolysis. Although, Pinheiro et al. (2015) found that “there has been a description of α-hemolysis of Staphylococcus epidermidis in almost 57% of coagulase-negative staphylococci and that of β-toxin amounting to 75%” (p. 3693). Similarly, the study by Moraveji et al. (2014) stated that “Staphylococcus epidermidis display all the three types of hemolysis, namely: Alpha, Beta, and Gama” (p. 6). For instance, according to Moraveji et al. (2014), “when a horse or rabbit blood are used to assess BAP, they will display Alpha hemolysis. Bata hemolysis will only appear when using sheep blood” (p. 6). Therefore, from the two arguments by both Pinheiro et al. (2015, p. 3693) and Moraveji et al. (2014, p. 6), it was worth concluding that the BAP test turned green due to the presence of Staphylococcus epidermidis indicating presence of alpha hemolysis.

Generally, the bacterium under study was concluded to be Staphylococcus epidermidis because its characteristics appeared on all the tests done. The genus Staphylococcus has more than 40 species, and one of them is Staphylococcus epidermidis. S. aureus is a normal human flora, and it exists on the human skin (Lee & Fatima 2021, p. 4). Also, the bacteria exist in marine sponges and is a facultative anaerobe. Mostly, the bacteria are not pathogenic, although patients who have compromised immune systems may develop an infection. Most of these infections are acquired within the hospital. The bacterium is a concern for individuals with surgical implants or catheters since it forms biofilms that grow on these devices (Choi et al., 2018, p. 5). In addition, considering that Staphylococcus epidermidis is a normal skin flora, it also contaminates laboratory specimens.

The symptoms of Staphylococcus epidermidis infection in a human host include wound infection where the bacterial germs grow within the wound section. Boils infection is another symptom of this bacterium, and it occurs by the development of a pus pocket on the oil gland or hair follicle, making the skin around the infected area swell or become red (Lee & Fatima 2021, p.4). Sinus infection is another symptom, and it causes inflammation of the cavities surrounding the nasal passages. Endocarditis is another symptom of S. epidermidis; despite being a rare infection, it is fatal in case it occurs, and it infects the endocardium, which is the inner lining of the heart (Choi et al., 2018, p. 5).

Treatment of Staphylococcus epidermidis may require the removal of indwelling devices or catheters where the bacterium may be infecting. Though, the removal of some of these devices may require surgery (Lee & Fatima 2021, p. 2). However, if S. epidermidis causes wound drainage or boil infection, then it is advisable for the doctor to make an incision that will drain the fluid collected in that region. Under severe conditions of Staphylococcus epidermidis infection, parenteral therapy is advisable. The chances are that the bacteria may resist methicillin. Therefore, empiric therapy is advisable by the use of IV vancomycin. However, if the pathogen is susceptible to methicillin, then beta-lactam antibiotic treatment is recommended, such as the use of oxacillin and nafcillin (Lee & Fatima 2021, p.5). Therapy duration depends on the clinical presentation.

Statistics show that Staphylococcus epidermidis causes infection from medical devices implanted into human beings. Lee and Fatima (2021) stated that “almost 20% of the patients having cardiac devices become infected by this bacterium” (p. 2). Also, S. epidermidis causes high infections of endocarditis in both native and prosthetic valves. Prosthetic valve endocarditis amount to almost 40% of infections from this bacterium (Lee & Fatima 2021, p. 2). Neonates having low birth weight experienced high infection resulting from the sepsis of S. epidermidis, which was about 4.8% and 9.4%. Catheter-related infections from this bacterium cause mortality of almost 20 to 30% (Lee & Fatima 2021, p. 3).

Works Cited

Ahmadunissah, Asmirah, Shafiq Aazmi, and Aziyah Abdul-Aziz. “Antibiotic resistance properties of Staphylococcus epidermidis isolated from hospitals in Selangor.” Science Letters 15.1 (2021): 69-81. https://doi.org/10.24191/sl.v15i1.11795

Choi, Qute, et al. “Manual versus automated streaking system in clinical microbiology laboratory: Performance evaluation of Previ Isola for blood culture and body fluid samples.” Journal of clinical laboratory analysis 32.5 (2018): e22373. https://dx.doi.org/10.1002%2Fjcla.22373

El-Sawaf, G. A., et al. “Identifying biofilm-forming strains of Staphylococcus epidermidis isolated from intravascular-catheterized patients by icaA and icaD genes.” CMU J. Nat. Sci 21.2 (2022): e2022024. https://www.researchgate.net/profile/Mohamed-Abdel-Latif-14/publication/358582245_Identifying_biofilm-forming_strains_of_Staphylococcus_epidermidis_isolated_from_intravascular-catheterized_patients_by_icaA_and_icaD_genes/links/620a3918cf7c2349ca13389c/Identifying-biofilm-forming-strains-of-Staphylococcus-epidermidis-isolated-from-intravascular-catheterized-patients-by-icaA-and-icaD-genes.pdf

Lee, Ezra, and Fatima Anjum. “Staphylococcus Epidermidis.” StatPearls [Internet]. StatPearls Publishing, 2021. https://www.ncbi.nlm.nih.gov/books/NBK563240/

Moraveji, Z., et al. “Characterization of hemolysins of Staphylococcus strains isolated from human and bovine, southern Iran.” Iranian journal of veterinary research 15.4 (2014): 326. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4789207/

Pinheiro, Luiza, et al. “Staphylococcus epidermidis and Staphylococcus haemolyticus: molecular detection of cytotoxin and enterotoxin genes.” Toxins 7.9 (2015): 3688-3699. https://doi.org/10.3390/toxins7093688

Tripathi, Nishant, and Amit Sapra. “Gram staining.” (2020). https://europepmc.org/article/NBK/nbk562156

Williams, Kim. “Online lectures and labs.” Biology 210. Great Bay Community College, New Hampshire. (2022).