Characterizing and Identifying a Microbe Isolate from a Cheek Sample

Characterizing and Identifying a Microbe Isolate from a Cheek Sample

Introduction

                      Microbes are everywhere, in fact they are the most abundant life form on earth. Microbes have been around for 3.8 billion years and were the first life forms on our planet. Bacteria, archaea, and eukarya are some examples of microorganisms. Some microbes are very beneficial to humans, as they are used to produce vaccinations, antibiotics, and food. They can also assist in physiological functions of the human body. Some pathogenic microbes can be very harmful and can cause diseases and infections.

The microbe that was investigated in this study was isolated from a cheek sample. Since some of the most common bacteria present in the mouth is streptococcus and staphylococcus I hypothesize that my isolate will be one of these two strains of bacteria. The bacteria was classified as Streptococcus pneumoniae I-G2. According to the CDC this strain is usually found on the skin or in the mouth and can cause infections in young children, senior citizens, and adults with weakened immune systems. Streptococcus sp. can cause sinus and ear infections, which can be treated with antibiotics. However more serious infections can also be caused such as pneumonia and meningitis. A Pneumonia vaccine can prevent these infections.

Just like how microbes are both beneficial and harmful in the surrounding world, they are also beneficial and harmful in people’s mouths. There are 700 known microbe species that live in a person’s mouth and scientist are still finding more (Burton et al. 2011). Microbes vary from surface to surface. The microbes found on the teeth, cheek, and tongue are all different and have their own individual communities. For example biofilms are present on the surface of teeth and can cause cavities and periodontal disease if they are not physically removed. Biofilm can’t be removed chemically, and this is why the brushing of teeth is more effective in preventing cavities than just using mouthwash. However this same biofilm is not present on the surface of the cheek, gums or tongue. (Gurenlian 2007). Microbes in the mouth can be beneficial because they positively affect mouth and gum health by outcompeting other harmful microbes for resources. Microbes can also be problematic because they can cause cavities and serious infections.

The purpose of this research was to characterize and identify a microbe that was isolated from a cheek swab. After isolating the sample to a pure culture a series of tests were run on the isolate to help gather information about it to identify what it was and how it worked. These tests included gram staining, fluid thyioglycollate test, oxidase test, catalase test, API E test, API strep test and DNA sequencing.

Methods

         The sample was collected from a cheek with a sterile cotton swab. The sample was then transferred to a tryptic soy agar (TSA) via quadrant streaking and stored at room temperature to grow for 6 days. After 3 days the TSA plate had little to no growth, however on the 5th day the bacteria scattered the plate. A single, medium sized, orange colony on the TSA plate was transferred to a new TSA plate in order to isolate a bacteria. The process of isolating the bacteria was repeated multiple times until the bacteria on the plate was uniform.

Once isolated, to gather more information about the bacteria, it underwent Gram staining. Gram staining reveals information about the bacteria’s cell wall. After going through the Gram staining process, if the bacteria turns dark purple it is Gram positive, if it turns the bacteria a light pink it is Gram negative. Gram positive bacteria have a large peptidoglycan layer and lacks a lipopolysaccharide layer. Gram negative have a thinner peptidoglycan layer and a complicated lipopolysaccharide layer.

The Gram staining information was helpful, however it doesn’t give enough information to positively identify the bacterium and so a series of physiological tests were performed including: a fluid thioglycollate test an oxidase test, a catalase test, and an API 20E test. The fluid thioglycollate test determines whether or not the bacteria is aerobic, anaerobic, or can be facultative and live in both environments. The oxidase test reveals whether or not the bacteria has cytochrome c oxidase. If the test changes color than it is positive, however if there is no color change than it is negative.  Cytochrome c is involved in the electron transport chain in the cells mitochondria. The catalase test shows if the bacteria contains a catalase enzyme. If the enzyme is present in the bacteria the test will yield oxygen bubbles when introduced to hydrogen peroxide. Lastly, the API 20E test will help identify if the bacteria has any pathogens and if so, what kind, through 21 different physiological tests.

While these physiological tests were also helpful in provided valuable information about the bacteria, to be absolutely certain about the identity of the bacteria, a DNA sample was sent to the DNA Core lab to be sequenced using an Illumina Miseq. DNA sequencing identifies what strain of bacteria the isolate is, as well as gives information about the isolate’s genome, how long it is, the nucleotide sequence, as well as information about the genes. An antibiotic resistance test was also carried out through a disk diffusion test. During this test the bacterium was introduced to various antibiotics and the amount of growth was monitored. If the bacterial growth is uninhibited and grows right up to or under the antibiotic disc then it is resistant to the antibiotic, if there is little to no growth near the disc then the bacteria is susceptible to the antibiotic, and if there is a small amount of growth near the disc, then the bacteria is slightly susceptible to the antibiotic.

Results

         The results of the genotypic and phenotypic tests that were performed during the course of this project are what ultimately helped identify the isolated bacteria. The Gram staining procedure revealed that the bacteria was Gram positive, shown in Figure 1. The fluid thioglycollate test showed bacterial growth at the top of the medium, concluding that the isolate is a strict aerobe. The oxidase test showed no color on the test strip and was therefore negative. The catalase test, when the bacteria came in contact with hydrogen peroxide, bubbles were formed indicating a positive test result. When the API 20E physiological tests were run, the results showed no color change, which means that the results were all negative (Figure 2). When the API 20 Strep/Gram + physiological tests were fun there were observed color changes in some wells, meaning that the tests were positive. The positive result for the VP well indicates that the bacteria contains a pyruvate substrate which helps with acetoin production. The positive ESC well means that the bacteria contains esculin which is a glucosidase enzyme. The ADH well also had a positive result, which shows that arginine is present which is involved in arginine dihydrolase. LAC, AMD and TRE also had a positive result, this means that the enzymes lactose, starch and trehalose are both present and are involved in acidification.

The DNA sequencing results provided a large amount of information about the bacteria including the functional genes present and how they function in the cell. The bacterial strain was Streptococcus sp. and three functional genes present were ATP dependent helicase DinG homolog, metalo-hydrolase M6_Spy0554, and phosphate M6_Spy0533.

The first Antibiotic resistance test (Figure 5) showed that the bacteria was either susceptible to the antibiotics tested (Erythromycin, Piperacillin, Oxacillin, Tobramycin, Cefazolin, Cefotaxime, Cefoperazone, and Amikacin) or the bacterium was not properly inoculated on the Mueller-hinton agar plate as there was no bacterial growth after 3 days. The second test (Figure 6) performed with the same antibiotics showed the same results, no growth on either of the plates. This concludes that the bacterium was susceptible to all antibiotics tested.

Figure 1. Results of Gram Staining

Figure 2. Results of API 20 E

Figure 3. Results for API 20 Strep/Gram +

 

Test Color Substrate Reaction/enzymes Results
VP Pink after 10 min, brown after 24 hours Pyruvate Acetoin production Positive
HIP Chemicals not available to perform this test n/a n/a n/a
ESC Black Esculin Glucosidase Positive
Pyra Clear Pyrrolidonyl-2-naphthylamide Pyrrolidonly arylamidase Negative
αGAL Clear 6-Bromo-2-naphthyl-D-galactopyranoside Galactosidase Negative
βGUR Clear Naphthol AS-BI-D-Glucuronate Glucuronidase Negative
βGAL Clear 2-naphthyl-D-galactopyranoside Galactosidase Negative
PAL Clear 2-naphthyl phosphate Alkaline phosphatase Negative
LAP Chemicals not available to perform this test n/a n/a n/a
ADH Yellow Arginine Arginine dihydrolase Positive
RIB Pink Ribose Acidification Negative
ARA Pink L-Arabinose Acidification Negative
MAN Pink Mannitol Acidification Negative
SOR Pink Sorbitol Acidification Negative
LAC Yellow Lactose Acidification Positive
TRE Yellow Trehalose Acidification Positive
INU Pink Inulin Acidification Negative
RAF Pink Raffinose Acidification Negative
AMD Yellow Starch Acidification Positive
GLYG Pink Glycogen Acidification Negative

Figure 4. Interpretation of API 20 Strep/Gram +

Figure 5. First antibiotic susceptibility tests result after 3 days of incubation at 37áµ’C.

Figure 6. Second antibiotic susceptibility test results after 2 days of incubation at 37áµ’C.

 

Discussion

         Each of the results gathered from the various tests that were run during the course of this study, indicate that the bacterial isolate is a Streptococcus sp. The results of these test not only help to identify the isolate but also provide information about the bacterium and how it functions. The fact that the Gram staining showed that the bacteria was Gram positive means that it has a thick peptidoglycan layer in the cell wall. This layer is crucial to the cell because it provides structural support and a protective layer. Characteristics of Gram positive bacteria include not having an outer membrane, lipopolysaccharides and are generally more susceptible to antibiotics.

The fluid thioglycollate test provided information about the ideal environment for the isolate. Since there was growth at the top of the test tube, the bacteria needs an oxic environment to thrive. This test not only shows what the optimal oxygenic environment is for the bacteria, but also shows where the bacterium can’t grow.  This makes sense considering the sample was taken from the inside of a cheek, where oxygen is readily available.

The oxidase test determines if cytochrome c oxidase enzyme is present in the bacteria sample. This enzyme is involved in ATP production in the mitochondria (Ow et al. 2008). Since the oxidase test was negative, because there was no color change, the bacteria does not produce cytochrome c oxidase enzyme. Just because this enzyme isn’t produced doesn’t mean that the cell doesn’t produce ATP since there are multiple systems that produce ATP.

The catalase test is performed to determine if the bacterium produces catalase enzymes. If the bacteria releases oxygen bubbles when it comes into contact with hydrogen peroxide, then the test is positive from the presence of the catalase enzyme. The catalase enzyme is involved in providing the cell with protection against oxidative toxins that your body produces or comes in contact with. One toxin is hydrogen peroxide which is why the bacteria reacts when they are combined turning it into water and oxygen. If this was not present in the bacteria the toxins that it fights against could affect the bacteria’s DNA. This enzyme is predominantly found in mammalian cells it is reasonable that the enzyme is present in the isolate since the sample site was from a person’s cheek (Scibior et al. 2006). You don’t say if your microbe was positive for catalase

The API 20E test is a physiological test that tests 20 different metabolic processes and provide more information about the bacterium. Since the API 20E test is for Gram negative bacteria the results were all negative for the isolate. This is plausible considering that the bacteria was Gram positive. A second API test was done that is limited to Gram positive, Streptococcus strains. The API 20 Strep strip showed positive results for the VP, ESC, ADH, LAC, TRE, and AMD tests. Each of these positive results contributes to understanding how the isolate functions. The positive result for the VP well means that a pyruvate substrate is present that is involved in acetoin production, which is used in the cell to store energy.  The positive ESC result means that esculin produces glucosidase which helps with breaking down carbohydrates. The positive ADH means that arginine dihydrolase is involved in helping the cell generate energy. The positive LAC test shows that a lactose operon helps transport and metabolize lactose inside the cell. The TRE results reveals that trehalose is present and provide storage and protects the cell when in stressful situations such as if the cell experiences a low growth period. The last positive test, AMD determines that the cell is able to process starch by converting it into an acid.

The DNA sequencing process identified the bacteria strain as Streptococcus pneumoniae I-G2.  This result is justifiable considering bacterial strains of Strep are very common in human mouths, Strep is a Gram positive bacteria, and the isolate showed positive results for an API strip that is geared towards Strep bacterial strains. The DNA sequencing test not only provided an identification for the bacterium, but also gave information on the genes that are present in the bacterium. There was an incredibly large amount of genes that are present, some include ATP dependent helicase DinG homolog, metallo-hydrolas M6_Spy0554, and phosphate M6_Spy0533. ATP dependent helicase DinG homolog is involved in DNA repair and replication. Metallo-hydrolase M6_Spy0554 catalyzes metal hydrolysis of substrates. Phosphate M6_Spy0533 is a ribose phosphate. Ribose phosphate generates pyrophosphate that helps with nucleic acid synthesis.

The last test performed on the bacterial isolate investigated potential antibiotic resistance to eight different antibiotics using the Kirby-Bauer disk technique. This test had two trials done with the same eight antibiotic disks and there was no growth of any kind after both tests were performed which means that the Streptococcus sp. is susceptible to all eight of the antibiotics tests. The antibiotics include Erythromycin, Piperacillin, Oxacillin, Tobramycin, Cefazolin, Cefotaxime, Cefoperazone, and Amikacin. This result of extreme susceptibility is reasonable due to the fact Gram positive bacteria are generally more susceptible to antibiotics. These results match data from studies done on the susceptibility of Streptococcus to the same antibiotics. Erythromycin was found to have been 59% susceptible, Piperacillin 98.2%, Oxacillin 87.8%, Cefotaxime 98.6% (Traub et al. 1997). Tobramycin has no effect on Streptococcus sp. so it has a 0% susceptibility (Brogden et al. 1976). One explanation for why there was no growth on the Tobramycin quadrant is that the other effective antibiotics spread into the quadrant, preventing bacterial growth.

The research for this bacterial isolate, with a multitude of tests performed to gain as much information as possible over the course of two months, was successful. Most of these test were crucial in gaining information to aid in identifying the bacterial strain. If this project were to be performed again, one improvement that could be made is to continue culturing the bacteria multiple times until the DNA sequencing indicates that it is a pure sample before doing the physiological tests. This would eliminate any skewed results that may appear from other bacteria that may be present in the sample.

 

Citations

 

Burton, J.P., P.A. Wescombe, P.A. Cadieux, J.R. Tagg. 2011. Beneficial microbes for the oral cavity: time to harness the oral streptococci? US National Library of Medicine national Institutes of Health 22:93-101.

 

Ow, Y.P., D.R. Green, Z. Hao, T.W Mak. 2008. Cytochrome c: functions beyond respiration. Nature Reviews Molecular Cell Biology 9:532-542.

 

Scibior, D., H. Czeczot. 2006. Catalase: structure, properties, functions. Postepy Hig Med Dosw (Online) 60:170-180.

 

Traub, W.H., B. Leonhard. 1997. Antibiotic Susceptibility of α- and Nonhemolytic Streptococci from Patients and Healthy Adults to 24 Antimicrobial Drugs. Chemotherapy 43:123-131.

 

Brogden, R.N., R.M. Rinder, P.R. Sawyer, T.M. Speight, G.S. Avery. Tobramycin: a review of its antibacterial and pharmacokinetic properties and therapeutic use. The Journal of Drug Delivery Science and Technology 12: 166-200.

 

“Travelers’ Health.”  Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 05 Aug. 2014. Web. 28 Apr. 2017.

 

 

Gurenlian, JoAnn R. 2007. The Role of Dental Plaque Biofilm in Oral Health. Journal of Dental Hygiene 81, No. 5.

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