28 April 2017
Bio 342 F01
All That Public Services Have To Offer: The Little Company of Microbes You Do Not See
There is a great dependency on public services: public schools, public bathrooms, and public transportation. Here, almost everyone is welcome to use the facilities provided. However, it is not only people that are clustering in these locations. Having so many different people coming together all in one place provides the perfect environment for microbes to gather. The definition of a microbe is still being debated today. However, for the sake of this research, we will go with the Brock Biology of Microorganisms definition: microorganisms are a microscopic organism consisting of a single cell or cell cluster, also including the viruses, which are not cellular (Madigan et al.).
As an avid user of public facilities, I chose to isolate, characterize, and identify a bacterium from the Fairbanks city bus. I retrieved my sample during the winter season when the common cold and flu was rampant through the city. Most bus users were the elderly or the working class or less common, college students like me. I was curious to find out what microbes we were all coming in contact with, I hypothesized it would be a bacterium or virus that could make you sick. After some observation, I realized most riders would use the poles in the bus either to help themselves on or off the bus. This bar is where I swabbed approximately two-inches with a damp cotton swab. I then streaked a TSA plate and kept my culture in a room temperature environment until further study which included: isolating my bacterium, running tests to find certain traits of my bacterium and finally obtaining its genome sequence.
The data of my bacterium brought me to the conclusion that I had Pseudomonas aeruginosa. Pseudomonas aeruginosa is described as an opportunistic pathogen found both inside and outside of the human body. It is an important bacterium for its multidrug resistance, antibiotic resistance, and association to diseases (Friedrich, 2016).
I chose my sample site on the Fairbanks, Alaska city bus. I used sterile swabs dipped in DI water to collect my sample from a one inch section of the hand bars near the front of the bus. I then streaked my TSA Plate and kept it in a dark and room temperature environment, approximately 75 degrees Fahrenheit. After five days, culture began growing on the plate. The plate had three different colonies growing in the same area. I chose an isolate in the middle of the colonies and streaked a plate using the four quadrant streak method. I followed the protocols of sterilization as provided in Lab 2 Handout. My plate was kept in an incubator set at 37 degrees Celsius. After 48 hours, I streaked another plate using the same method and repeated this process four times.
After obtaining a pure culture, I used the gram stain test to find if my bacteria were gram positive or gram negative. This test is the first test I ran to place my bacterium in a morphological group. I followed the gram stain protocol in Lab 4 Handout: Staining Techniques. After staining, viewing my gram stain slide under a microscope would also allow me to see the morphology of my cells.
I then began growing my pure culture in an agar slant tube. The slant was kept in an incubator at 37 degrees Celsius before being moved to the refrigerator at 4 degrees Celsius after the culture had visibly grown after a week. The next week and API 20E test was done on my bacterium. The API 20E test strip tested for twenty different characteristics of my bacterium. Some tests included GLU which tests if glucose is used which it usually is. The test trip protocol I followed was provided in Lab 6 Handout and I then incubated the strip at 37 degrees Celsius for 24 hours. Further tests included the fluid thiogylcellate test, the oxidase and catalase test, and the genome sequence test. The oxygen status whether it is aerobic or anaerobic is told by the fluid thioglycellate test. The oxidase test I ran was would tell me if there is cytochrome c oxidase was present or absent. It would also tell me if my bacterium can use oxygen as a terminal electron acceptor in respiration. The catalase test would tell me if this enzyme is present, the presence of this enzyme means my bacterium is protected from oxidative damage.
Ian Herriott, a technician in the Institute of Arctic Biology DNA Core Lab, performed the whole genome sequencing on the genomic DNA of my isolate on Ilumina MiSeq. Through a series of steps reported in Lab 7 Handout, my sequence is provided and analyzed in a computer program called “BaseSpace’. Through the app “SPAdes Genome Assembler’ I was given information on my bacterium’s contigs and the app “Kraken Metagenomics’ gave me its taxonomic classification.
The final test done on my bacterium was to test its susceptibility or resistance to antibiotics. I chose six antibiotic discs at random to test. I split two TSA plates in sections of three. I followed the protocols in Lab 9 Handout to test my bacterium for antibiotics.
Figure 1. A quadrant streak of my experimental culture. This shows a pure culture on a TSA Plate.
Figure 2. A 1000X light microscopic view of my experimental bacterium after a gram stain test.
Figure 3. An API 20E Strip results after 24 hours. From left to right the tests are: ONPG, ADH, LDC, ODC, CIT, H2S, URE, TDA, IND, VP, GEL, GLU, MAN, INO, SOR, RHA, SAC, MEL, AMY, ARA.
The gram stain test’s outcome was gram positive. In addition to my bacterium being gram positive, I saw the morphology of my bacterium was rods where some were coupled and some were in clusters. The cell size was roughly 0.6 by 1.5 micrometers.
The API 20E test had negative results in every section: ONPG, ADH, LDC, ODC, CIT, H2S, URE, TDA, IND, VP, GEL, GLU, MAN, INO, SOR, RHA, SAC, MEL, AMY, ARA. The API20E test appeared invalid as some of the wells appeared to have dried out. The first and second test of the fluid thioglycellate did not yield any result for my microbe. There was no bacterial growth to be seen in the broth.
The results of the catalase and oxidase tests were both positive: Bubbles forms when Hydrogen Peroxide was dropped on my bacterium sample for the catalase test. The genomic sequencing software gave a species name, Pseudomonas Aeruginosa with a 99.15% reads classified species level. Finally, my bacterium did not grown on the TSA plate for this and therefore yielded no results.
Apart from the inconclusive tests, all tests corresponded to the literature on Pseudomonas aeruginosa. Pseudomonas aeruginosa is a very common bacterium often characterized as an opportunistic pathogen (Juhas et. al, 2005). Though I did not find my isolate in a mucous or water environment, it is not unusual for my bacterium to adhere to metal surfaces in a biofilm. Finding my bacterium on a public bus hand bar was consistent to its preferred environment. However, where my bacterium was finding its food source is still in question. This is a study I could go further into.
- aeruginosa is a gram-negative rod shaped bacterium which was analogous to my morphological tests. The expectation was that my bacterium needed oxygen considering the location of where it was found. The positive results from my catalase and oxidase tests were also conclusive with to my prediction. The one thing that was not correspondent was P.aeruginosa’s motility. However some sources said it could be both motile and immotile (Campbell, 1994). Since my isolate was not found in water, it is possible I sampled a colony in a biofilm state (Tiedje, 2007).
Pseudomonas aeruginosa proved capable of growing in all of the mediums used for the experiment except the agar used in the thioglycellate test. I still do not know why my bacterium did not grow the two times that I conducted this test. It is possible the culture I used to inoculate the broth for the test was no longer active. I should have made a fresh culture for the test. P. aeruginosa can grow in the Mueller-Hinton agar but may have been unsuccessful due to the poor preparation in growing the pathogen in the broth before applying it to the plate. However, after some research, I found my bacterium would have been resistant or partially resistant to all of the antibiotics (Friedrich, 2016).
Some wells on the API 20E test strip, such as those for mannitol fermentation and gelatin hydrolysis, should have been positive (Aryal, 2015). As was the case in my fluid thioglycellate test, it is possible my bacterium was not grown enough in the medium I used to fill the wells. As time went on during these tests, I may not have revived my culture enough and it could have gone dormant. In the future, it would be best if I prepared a fresh plate before every test. Further, I could do additional tests such as seeing how long my isolate would survive in various kinds of environments without a constant food source.
Though there were insufficient results from human error, through the genome sequencing results and successful tests, I was convinced that I did indeed have Pseudomonas aeruginosa. I had a high confidence interval on the “Kraken Metagenomics’ app with few other results coming close. P. aeruginosa is a common bacterium found in many locations and the probability of coming across it is plausible. However, P. aeruginosa is mostly found in hospitals, which does lower my credibility since I did not swab my sample from a hospital. It was exciting and weary possibly finding such an opportunistic pathogen in the location I did. Now that I am informed that microbes like these are ubiquitous, it gives a new light on the importance of washing your hands.