Unknown Project Lab Report

Microbiology Lab 29 April 2019 Unknown Project Lab Report The overall goal of the Unknown Project was to isolate and identify three bacteria. A Gram positive and Gram negative were isolated off me, while a third known unknown was given to me by the professor, who knew its identity. This project provided the opportunity for us to use our knowledge of differential tests toward identifying unknown bacteria. An additional goal of the project was to learn about the human microbiota and its relevance to health and disease. In order to isolate a Gram positive and Gram negative from myself, I took samples from several different parts of my body, specifically the oral cavity, ear canal, armpit, gastrointestinal (GI) tract, and the back of the ear. The oral cavity and GI tract samples were the most promising, as those plates had lawns of bacteria, while the others only had a few colonies. After Gram staining colonies from each sample plate, I was able to find a Gram positive and a Gram negative that I would work with. After narrowing down my search to the oral cavity and GI tract sample plates, I needed to ensure that I had pure cultures of a Gram negative and a Gram positive. Once I had pure cultures, I was able to move on with the process of identifying each of my unknowns, including the known unknown that I was assigned. Using the diagnostic test flow chart packet that we were provided I performed a series of differential tests on each of my unknowns until I had narrowed them each down to one bacterial species. At this point, I consulted Bergey’s Manual of Determinative Bacteriology in order to find a final test that could I run to confirm my results. Once I had a preliminary identification for each of the three unknowns, I used Multi-test kits to confirm the results. In addition, I isolated the genomic DNA of each of the three unknowns, which was sent for sequencing in order to further confirm the results. This report includes a detailed account of the project as well as the results. Unknown Gram Positive Sample Source and Isolation Method The unknown Gram positive was isolated from my oral cavity. I chose to take a sample from this location because I knew it was a common location to find a diverse array of bacteria. In addition, I was feeling sick and had a sore throat when taking samples and suspected that may influence the bacteria in my oral cavity. The oral cavity is well suited for bacterial growth, as it provides host bacteria with a regular source of nutrients and water, all while maintaining a moderate temperature. However, in order for bacteria to survive in there, they must be able to adhere to the teeth and gums, so they do not get flushed down to the stomach with food and water. Bacteria in the oral cavity are known to form biofilms, microbial communities with cell-cell and cell-surface adhesion, which are known to have health implications (1). Factors that could influence the biodiversity of the oral cavity are dental hygiene, diet, and illness. In order to isolate a single bacterial species, I started with swabbing the back of my throat and plating that sample on a nutrient agar plate. From the resulting heterogeneous bacterial lawn. I used a sterile inoculating loop to take a small amount of bacteria and quadrant streak that onto another nutrient agar plate for isolation. From there, I made a broth culture using an isolated colony. I then used the broth culture to quadrant streak onto another nutrient agar plate, which became the plate I worked with for the rest of the project. I Gram stained the bacteria on the final plate to confirm that I had isolated a single species of Gram-positive bacteria. Morphological Description The colony morphology of the unknown bacteria isolated from the oral cavity was round with smooth margins, convex, opaque, white or cream colored. A single isolated colony can be seen in Figure 1. The cell morphology, as can be seen in Figure 2, is clustered cocci. The purple color of the stain indicates that the species is Gram positive. Figure 1- Single colony of Grampositive isolate on nutrient agar plate Figure 2- Gram stain image of unknown isolate (right) depicting Gram positive, clustered cocci. Control image (left) with S. aureus and E. coli Differential Tests and Multi Test Kit In order to determine the identity of the unknown Gram positive isolated from my oral cavity, I performed a series of differential tests as listed in the diagnostic test flow chart packet. Based on the Gram stain results, I knew I was working with a Gram-positive coccus. The first step was to perform a catalase test, which is generally used to differentiate Staphylococcus and Micrococcus species from Streptococcus based on the presence of the enzyme catalase. I performed the catalase test on the unknown Gram positive with S. epidermidis as a positive control and E. faecalis as a negative control. A catalase positive species is able to break down hydrogen peroxide into water and oxygen gas, so the test involves adding a drop of hydrogen peroxide to the bacteria and observing whether gas bubbles appear, indicating that catalase is present. Upon adding hydrogen peroxide, I saw bubbles form for both the unknown Gram positive and S. epidermidis, and there were no bubbles formed with E. faecalis. Based on those results, I concluded that my Gram-positive isolate was catalase positive, and was therefore either Staphylococcus or Micrococcus. The next test I performed was the mannitol fermentation test, which is used to isolate and differentiate S. aureus from other Staphylococcus species based on its ability to survive on the plate and ferment mannitol. For this test, I plated my unknown isolate on a mannitol salt agar (MSA) plate with S. aureus as a positive control and S . epidermidis as a negative control. Mannitol fermentation results in Figure 3- MSA plate with positive control (top left), unknown Gram positive (top right), negative control (bottom left) and known unknown (bottom right) yellow growth and a yellow halo around the growth. The results for the test can be seen in Figure 3. Both the positive control and the unknown Gram positive have yellow growth with a yellow halo, while the negative control has red growth indicating no mannitol fermentation. Since my unknown Gram-positive isolate was positive for mannitol fermentation, I went on the perform the coagulase test. This test is used to differentiate S. aureus from other Grampositive cocci based on its ability to produce coagulase enzymes. The coagulase test involves inoculating rabbit plasma and testing for coagulation. For this test, the positive control was S. aureus and the negative control was S. epidermidis. Both the positive control and the unknown Gram positive came back coagulase positive, while S. epidermidis was coagulase negative. At this point, I had reached the end of the flow chart, and my preliminary identification of the unknown Gram positive was S. aureus. I performed a urease test as confirmation, since S. aureus is known to be urease positive. I used P. mirabilis as a positive control and E. coli as a negative control. Both the unknown and P. mirabilis came back urease positive, and E. coli was urease negative; however, I was still unable to conclude that the unknown Gram positive was S. aureus. Upon consulting Bergey’s Manual, I found that there were several similar species that I could have had that would have had the same differential test results, including S. delphini, S. intermedius, and S. schleiferi. In order narrow down my options, I performed a trehalose fermentation test using phenol red trehalose broth. I knew that S. schleiferi did not ferment the sugar trehalose, and that S. aureus and S. intermedius did. I used S. aureus as the positive control and S. epidermidis as the negative control. Both the unknown and S. aureus came back positive for trehalose fermentation, while S. epidermidis came back negative. This allowed me to narrow down my results to either S. aureus or S. intermedius. After completing all of the differential tests, I used an API-Staph multi-test kit to confirm my results. Figure 4 shows the results of the multi-test kit. Even though the kit came back with a high percent identification of 97.7% for S. aureus, the result was listed as a doubtful profile with the note that It could also be S. intermedius if the bacteria was of veterinary origin. Research shows that S. intermedius infections in humans is most commonly associated with exposure to dogs (2). Since I had not been exposed to any dogs recently, I was able to rule out S. intermedius as my unknown Gram positive. Figure 4- API-Staph Multi-test kit results for unknown Gram- positive isolate. Genetic Identification The final step in confirming the identity of the unknown Gram positive was to sequence its genome. In order to do this, I first had to isolate DNA from the bacteria by lysing the cells. This was accomplished by subjecting the sample to a series of freeze-thaw cycles. A microcentrifuge tube containing bacterial growth suspended in DNA-free water was placed first on dry ice to freeze for three minutes, then transferred to a 95 C water bath for another three minutes. This freeze-thaw cycle was repeated twice more, after which the tube was centrifuged in order to separate the gDNA containing supernatant from the cellular debris pellet. The next step was to amplify the 16s RNA gene fragment, as this gene fragment is highly conserved and is present in almost all bacteria. In addition, since its function is the same in most bacteria, small changes in the gene among species are an accurate measure of evolutionary time that can be used to differentiate species (3). In order to amplify the 16s RNA gene fragment, I needed to prepare my sample to undergo PCR amplification. This was done by combining the PCR master mix with the gDNA sample and one of two primer sets each. After the PCR reaction was set up, the sample underwent one cycle at 95 C for three minutes, followed by forty cycles of denaturing, annealing, and extension for one minute each. After PCR amplification, I ran the sample in an agarose gel to determine which of the two primers had successfully amplified the gene. Once that was determined, the correct sample was then cleaned with the ExoSap-IT reagent, containing an exonuclease to degrade the remaining primers and Shrimp Alkaline Phosphatase to degrade the remaining dNTPs from the PCR reaction. Once the DNA sample was cleaned, it was sent off for sequencing. I was able to analyze the sequencing results by using the Basic BLAST function on the NCBI web page. My sample came back with a 99.76% match to S. warneri, a 99.29% match to S. pasteuri, and a 99.05% match to S. aureus. I was able to eliminate S. warneri and S. pasteuri as potential candidates because I knew they were both coagulase negative, while my Gram-positive isolate was coagulase positive. At this point I was able to confidently conclude that the Gram-positive bacteria that I isolated from my oral cavity was indeed S. aureus. I was interested to find that S. aureus is highly pathogenic. I was aware that some strains of the bacteria are becoming increasingly resistant to antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA). However, I found that S. aureus itself can cause severe food poising by creating seven different toxins. Some of these toxins are incredibly fast acting and can cause symptoms in as little as thirty minutes after ingestion. While most who suffer from Staphylococcal food poisoning will recover quickly, those with weak or compromised immune systems may require hospital attention (4). Unknown Gram Negative Sample Source and Isolation Method I was able to isolate a Gram- negative bacteria from the GI tract sample. After taking the sample, I immediately plated it on an eosin methylene blue (EMG) plate, which is selective for Gram negative bacteria. Since there was growth on the plate, I could confidently assume that I had isolated a Gram- negative; however, I did confirm this with a Gram stain. I chose to take a GI tract sample because I was struggling to find Gram-negative bacteria at other sample sites. I was aware that the most common place to find a Gram- negative was the GI tract, since much of the bacteria that inhabits the GI tract is Gram negative. Factors that could influence the biodiversity of GI bacteria include diet, illness, and antibiotic use. Bacteria found in the GI tract play an important role in human health. They are crucial to maintaining enterohepatic circulation of metabolites and are also involved in fiber digestion and the synthesis of certain vitamins (5). In order to isolate a pure culture of a single bacterial species, I first plated the sample swab onto an EMB plate. The resulting growth can be seen in Figure 5. I used an inoculating loop to take a small portion of the growth and quadrant streak it for isolation on a nutrient agar plate. From there, I Gram stained half of an isolated colony, and used the other half to quadrant streak the sample onto another plate, which was Figure 5- GI tract sample plated on an EMB plate. the pure sample that I worked with for the rest of the project. Morphological Description The colony morphology of the unknown bacteria isolated from the GI tract is round with smooth margins, convex, opaque, and white colored. Figure 6 shows an image of an isolated colony. The cell morphology can be seen in Figure 7. The unknown bacteria are rod shaped and clustered. The the Gram stain indicates that it is negative. Figure 6- single colony of Gram-negative isolate on NA plate. pink color of Gram- Figure 7- Gram stain image of the unknown isolate (right) depicting Gram negative, rod shaped, clustered bacteria. Control image (left) with S. aureus and E. coli Differential Tests and Multi Test Kit The first step for identifying my unknown Gram negative was to do an oxidase test. This tests for the presence of Cytochrome C oxidase, which can be used to differentiate Enterobacteriaceae from other Gram-negative rods such as Aeromonas, Pseudomonas, and Vibrio. The oxidase test involves adding the chromogenic reducing agent tetramethyl-pphenylenediamine to bacterial growth. If a blue color appears, then the organism is oxidase positive. I used P. putida as a positive control and E. coli as a negative control. The results were a blue color change for P. putida and no color change for the unknown Gram negative or the E. coli control. The negative oxidase test indicated that I had an Enterobacteriaceae. The next step on the flow chart was to test for lactose fermentation. I did this by spot inoculating my unknown Gram negative on a MacConkey agar plate along with E. coli as a positive control and P. stuartii as a negative control. Pink colored growth on a MacConkey agar plate indicates that there has been acid production from lactose fermentation. The results can be seen in Figure 8. Pink colored growth appeared for both the positive control and the unknown Gram negative, while the negative control had colorless growth. Based on these results, I concluded that the unknown bacteria was positive for lactose fermentation. The next step was to test for indole production. This could be done using SIM medium, which tests for sulfur reduction, indole production, and motility. I stab inoculated a SIM tube Figure 8- MacConkey Agar plate with positive control (top), Gram- negative isolate (bottom left) and negative control (bottom right) with my unknown isolate, along with E. coli as a positive control and M. luteus as a negative control. After allowing the bacteria to incubate, I added Kovac’s reagent to the medium. The reagent reacts with indole, produced by the breakdown of tryptophan by tryptophanase, and produces a red color. Both the positive control and the unknown Gram negative produced a red color upon the addition of Kovac’s reagent, and there was no color change for M. luteus, indicating that the unknown isolate was indole positive. The positive indole test allowed me to narrow down my results to Citrobacter diversus, Escherichia coli, Erwinia chrysanthemi, or Klebsiella ocytoca. The next step was to run a citrate test, which tests for citrate fermentation, specifically whether the organism can utilize citrate as its sole carbon source. Bacteria that survive on Simmons citrate agar create basic byproducts which change the medium from green to blue. For this test, I used E. aerogenes as the positive control and E. coli as the negative control. After incubation, the E. aerogenes tube was blue while the E. coli and the unknown Gram-negative tubes were still green. This indicated that the unknown Gram-negative bacteria was citrate negative. At this point, I was at the end of the flow chart, and was able to make a preliminary identification for my unknown Gram negative. I had narrowed down my results to E. coli. After consulting Bergey’s Manual, I saw that E. coli was motile. Since I had already run the SIM test for motility, I knew that my unknown was also motile, along with the E. coli control, while the negative control, M. luteus, was nonmotile. Therefore, I concluded that my unknown Gram negative from the GI tract was E. coli. In order to confirm this, I used the API 20E multi- test kit. The results from the multi- test kit are shown in Figure 9. Unlike with my unknown Gram positive, the results for this test came back with a very strong profile for E. coli, with a percent identification of 99.5% and no tests against. Genetic Identification I was fairly confident that my unknown Gram negative was E. coli; however, to know for certain, I was able to isolate and analyze its genomic DNA. Through the same procedure as the unknown Gram positive (see Unknown Gram Positive- Genetic Identification for methods) I was Figure 9- API 20E multi- test kit results able to obtain the 16s RNA gene sequence. After running the sequence through the BLAST analysis function on the NCBI page, I received confirmation that my unknown was E. coli with 100% match. E. coli is found in the GI tract of healthy individuals. While most strains of E. coli are harmless, there are some that can cause food poisoning. E. coli outbreaks linked to certain foods are commonly tracked by the CDC. Currently, the CDC is tracking an outbreak associated with ground beef that is caused by E. coli O103. The outbreak can be traced back to a single meat packing plant in Illinois (6). Known Unknown Morphological Description The third unknown organism that I was tasked with identifying was Known Unknown Number 11. A pure culture of this bacteria was given to me by the professor. The colony morphology of the known unknown was round with smooth margins, convex, opaque, and white in color. Figure 10 show an image of a single isolated colony of the known unknown. The cell morphology of the known unknown can be seen in Figure 11. It is a clustered coccus. The purple color of the Gram stain indicates that the known unknown is a Gram positive. Figure 10- Single colony of Known unknown on NA plate Figure 11- Gram stain image of known unknown (right) depicting Gram positive clustered cocci. Control image (left) with S. aureus and E. coli Differential Tests and Multi Test Kit Since my known unknown was a Gram-positive coccus, like my unknown Gram-positive isolate, I was able to follow the same flow chart to determine its identity. I performed the catalase test alongside the unknown Gram positive and found that it was catalase positive (see Unknown Gram Positive- Differential Tests and Multi Test Kit for methods and positive and negative controls and their results). From there, I ran the mannitol fermentation test. The results for that can be seen on Figure 3. I again ran the test alongside the unknown Gram positive (see Unknown Gram Positive- Differential Tests and Multi Test Kit for methods and positive and negative controls and their results). As can be seen in Figure 3, the results for this test were difficult to interpret. The growth was yellowish in color. However, since there was no yellow halo and since the growth did not match the positive control, I concluded that the known unknown was negative for mannitol fermentation. At this point, I went in a different direction on the flow chart from the unknown Gram positive. Since the known unknown was negative for mannitol fermentation, I knew I either had a Staphylococcus other than S. aureus or a Micrococcus. However, I concluded that I did not have a Micrococcus since those colonies are yellow in color, while my known unknown colony pigment was white. The next step on the flow chart was to test for novobiocin sensitivity, which is used to differentiate coagulase- negative Staphylococci and to identify Staphylococcus saprophyticus. For this test, I used S. epidermidis as a positive control, as it is sensitive to novobiocin, and S. saprophyticus as the negative control, since it is resistant to novobiocin. After incubation, the positive control had clearing around the diffusion disk while the negative control and the known unknown did not. Therefore, I was able to conclude that the known unknown was negative for Figure 12- API-Staph Multi-test kit results for known unknown. novobiocin sensitivity, which led me to the end of the flow chart. My preliminary identification for the known unknown was S. saprophyticus. To confirm this result, I checked Bergey’s manual and found that S. saprophyticus was urease positive. I was able to run a confirmation urease test alongside the unknown Gram positive (see Unknown Gram Positive- Differential Tests and Multi Test Kit for methods and positive and negative controls and their results). The known unknown was indeed urease positive, confirming my preliminary identification. In order to further confirm my result, I used the API- Staph multi- test kit. The results can be seen in Figure 12. The overall results showed a doubtful profile, with only a 78.4% match with S. saprophyticus. However, since no other organism had results that matched the profile or had any multi-test kit tests against, I was able to conclude that S. saprophyticus was the likely known unknown organism. Genetic Identification The final step in identifying my known unknown bacteria was analyzing its genomic sequence. Following the same procedure as with my unknown Gram positive and negative (see Unknown Gram Positive- Genetic Identification for methods), I was able to retrieve the genomic sequence of the 16s RNA gene and analyze it. My results came up with a 100% match with S. saprophyticus, as I had expected, along with a 100% match with S. edaphicus. However, upon doing some research, I found that S. edaphicus was one of two Gram- positive, coagulase negative staphylococcal strains that was recently (Jan 2018) isolated from stone fragments in sandy soil in James Ross Island, Antarctica. Its 16S RNA gene sequence is nearly identical to S. saprophyticus. The discovery of S. edaphicus is especially relevant because it can be used to compare multi-drug resistant Staphylococci from human and animal sources to those isolated from humans (7). In the fight against multi-drug resistant bacteria, this information is extremely useful. S. saprophyticus is relevant to human health on its own. It has been shown to be the second most common bacteria to cause urinary tract infections (UTIs) after E. coli. Research has shown that while it is part of the normal GT tract flora, it can cause up to 42% of all UTI’s (8). Conclusion Through the unknown project, I had the opportunity to learn a lot about the human microbiota. In addition, I had the chance to practice the skills that I learned in microbiology lab while performing the many differential tests needed to identify my three unknown bacteria. In the end, I was able to successfully identify a Gram- negative and a Gram-positive bacterium isolated off of me, as well as the known unknown that was given to me. 5. Gorbach SL. Microbiology of the Gastrointestinal Tract. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 95. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7670/ 6. Center for Disease Control and Prevention, “Outbreak of E. coli Infections Linked to Ground Beef,” 2019 Outbreaks, www.cdc.gov, Web. 2019 7. Pantůček, Roman et al. “Staphylococcus edaphicus sp. nov., Isolated in Antarctica, Harbors the mecC Gene and Genomic Islands with a Suspected Role in Adaptation to Extreme Environments.” Applied and environmental microbiology vol. 84,2 e01746-17. 2 Jan. 2018, doi:10.1128/AEM.01746-17 8. Ehlers S, Merrill SA. Staphylococcus Saprophyticus. [Updated 2019 Mar 27]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK482367/