All That Public Services Have To Offer: The Little Company of Microbes You Do Not See

 

Chelsea Brown

28 April 2017

Bio 342 F01

 

All That Public Services Have To Offer: The Little Company of Microbes You Do Not See

Introduction

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).

Methods

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.

 

Results                                    

 

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.

 

Discussion

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.

  1. 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.

 

 

References

1.             EHA. What is Pseudomonas aeruginosa?. 2017. https://www.ehagroup.com/resources/pathogens/pseudomonas-aeruginosa/

2.             L. Wiehlmann et. al. “Population structure of Pseudomonas aeruginosa.’ PNAS. 2007.

3.             M. Campbell. “Nonmotility and phagocytic resistance of Pseudomonas aeruginosa isolates from chronically colonized patients with cystic fibrosis.’Infect Immun. 1994.

4.             M. Friederich. “Pseudomonas aeruginosa Infections  Medication.’ MedScape. 2016.

5.             S. Aryal. “Biochemical Test and Identification of Pseudomonas aeruginosa.’ Microbiology Info. 2015.

Rhodococcus erythropolis: An Environmental Microbe Isolation Study

 

Microbes are an omnipresent living force found in every environment and habitat on the planet, from deep below the ocean in the crushing darkness at hydrothermal vents, to within human bodies and floating in the air around us. The diversity of microbes, especially bacteria, is staggering and constantly shifting through the tremendous evolutionary prowess and genetic flexibility of these organisms.

For the purposes of this research, I chose to investigate the microfauna of my aquarium biome. I have worked to culture a realistic and comfortable habitat for the organisms I keep, which has involved obtaining live plants, cultivating algae, using a heater to maintain constant temperature, and using a LED lamp to mimic sunlight. Nitrogen fixing and ammonia-oxidizing bacteria are necessary for a healthy aquarium environment (Urakawa et al., 2008)(Zehr et al., 2000), and bacteria considered pathogenic to fish and aquatic plants are perpetually present in the water (Baran et al., 2008)(Smith et al., 2012).

My goal was to isolate and identify a bacterial strain from my aquatic microbiome, then determine the likely role it plays within the system I have worked to cultivate. To do so, I sampled the aquarium water, allowed the bacteria to grow colonies from which to extract pure culture, and isolate a pure culture from which to run both genomic and physiological tests upon. I theorized the microbe I isolated is a form of nitrogen-fixing bacteria, as my tank is a healthy system with no diseased fish or plants, and has been thriving with minimal water changes, a sign of nitrogen cycling within the ecosystem.

However, the microbe I identified was Rhodococcus erythropolis, a versatile and occasionally pathogenic bacteria associated with aquatic and soil environments. This was somewhat surprising due to the predicted relative rarity of R. erythropolis in comparison to other microbial populations, such as nitrogen fixers in aquatic environments, but considering the methods I used, the bacteria I chose had already been through a selective process once I decided to isolate.

 

Methods

Sampling and Growth

The aquarium biome was sampled by submerging a sterile swab of cotton into the water and gently swiping it across a Betta splendens (Betta fish). The swab was then wiped across a plate of sterilized SA agar and left to incubate at room temperature for 6 days. Bacterial growth appeared after 72 hours in three small colonies. The SA plate had initially been intended to select for fungal growth, but I chose to isolate and study the acidophilic bacteria growing on the plate instead.

These bacterial colonies were isolated into pure culture by the quadrant streak method (Lab 2 Handout). Four successive streak plates were made over the course of 3 weeks, and were incubated at 37 °C during the growth phase of the bacterium.

 

Identification

When the isolate was sufficiently purified, a Gram stain was conducted to determine if the strain is Gram-positive or Gram-negative. This test was conducted according to the methods of Lab Handout 4.

The isolate was also tested for a variety of physiological abilities. A fluid thioglycollate test was used to determine the strain’s oxygen class, a catalase test establishes whether a bacterium contains the catalase enzyme, and a oxidase test to see if the isolate contained cytochrome c oxidase. An API 20E test strip was additionally used to test the isolate’s capacity for fermentation of various sugars, the presence of specific enzymes, and decarboxylations of amino acids, with 20 tests in total. All physiological tests were completed according to the methods of Lab Handout 6.

To obtain a more specific idea of the bacterium’s identification, DNA extraction and whole genome sequencing was conducted on the organism. Extraction was completed with a freshly streaked isolate and the PowerSoil Isolation kit according to the methods of Lab Handout 5. Genomic sequencing was completed in the Illumina MiSeq DNA sequencer at the University of Alaska-Fairbank’s DNA Core Lab, and analyzed in BaseSpace with SPAdes Genome Assembler and Kraken metagenomics according to the instructions of Lab Handout 6. These tests would determine the closest genetic match to the extracted DNA, and how many contigs, functional genes, and tRNA regions are identified, as well as what each section coded for, when relevant.

Unfortunately, after genomic extraction, the original isolate was lost. I attempted to reisolate the same bacteria again through similar sampling methods and selection of a colony of visually identical microbes, but was unsuccessful. Therefore, any antibiotic resistance by my original strain remains unknown, as well as its ability to ferment lactose or sugars, as would have been tested in Labs 8 and 9.

 

Results

This isolate was observed to grow most quickly at 25 °C. Colonies were matte white and circular with defined edges and a convex shape. This bacteria was able to grow under both refrigeration and at 37 °C, though slowly.

Gram staining of the isolate showed the microbe as a gram positive bacillus. The rods most commonly appeared clustered in pairs (diplobacilli), bent at an angle so as from a distance they appear to be one spiral bacteria. The individual bacilli are approximately 0.5μm long.

 

Figure 1. Isolate gram-stained and magnified at 1000X. This organism is clearly gram-positive and is clustered in pairs with few exceptions.

 

In physiological tests, the isolate was positive in the catalase and oxidate tests. (Acharya, 2015). Complete results for the API 20E test strip are listed in Table 1. The ONPG test (tests for β-galactosidase enzyme) was inconclusive due to an unfixable air bubble that hindered results, and is not included.

 

Test Result
ADH- determines decarboxylation of the amino acid arginine by arginine dihydrolase Negative
LDC- determines decarboxylations of the amino acid lysine by lysine decarboxylase Negative
ODC- determines decarboxylations of the amino acid ornithine by ornithine decarboxylase Negative
CIT- detects utilization of citrate as only carbon source Negative
H2S- detects production of hydrogen sulfide Negative
URE- detection of enzyme urease Positive
TDA- detection of the enzyme tryptophan deaminase Positive
IND- detection of production of indole by the enzyme tryptophanase Positive
VP- determines if fermentation of glucose by bacteria utilizing the butylene glycol pathway is being utilized Positive
GEL- tests for the production of the enzyme gelatinase Negative
GLU- detects fermentation of glucose (a hexose sugar) Positive
MAN- detects fermentation of mannose (a hexose sugar) Positive
INO- detects fermentation of inositol (a cyclic polyalcohol) Positive
SOR- detects  fermentation of sorbitol (a alcohol sugar) Positive
RHA- detects fermentation of rhamnose (a methyl pentose sugar) Positive
SAC- detects fermentation of sucrose (a disaccharide) Negative
MEL- detects fermentation of melibiose (a disaccharide) Negative
AMY- detects fermentation of amygdalin (a glycoside) Negative
ARA- detects fermentation of arabinose (a pentose sugar) Negative

Table 1. A complete listing of the results from my API 20E test strip for my isolate (Acharya, 2015).

 

In the fluid thioglycollate test, the isolate proved able to survive in all areas of oxygenation, but preferred to grow at the surface, making it a facultative aerobe. Growth was thickly concentrated at the surface and directly below the surface of the agarose, but a thin line of microbial growth was visible extending from the surface to the base of the tube.

Genomic tests indicated only approximately 70% of genetic data was usable, as the other data was not even classified as bacteria in the analysis. However, within the usable information, the confidence level of the identified sequences were 95% or greater. Complete percentages of taxonomic rank are illustrated in Table 2, indicating a high level of precision in the identification of the isolate as Rhodococcus erythropolis (BaseSpace, 2017).

 

Kingdom Phylum Class Order Family Genus Species
Bacteria Actinobacteria Actinobacteria Actinomycetales Nocardiceae Rhodococcus erythropolis
99.11% 98.41% 98.41% 98.34% 95.99% 96.05% 95.91%

Table 2. The confidence levels of identified sequences within the bacterial genome and the identified classifications. Note all of these values are derived from ∽70% of total data, but the remaining 30%  of data was completely unidentifiable, even to the kingdom level, and as such has been disregarded in this interpretation.

 

Only one contig of 1420bp was identified. Additionally, only one tRNA was observed, and 169 coding regions.

Lastly, BLAST analysis was used as a secondary classification system to check the accuracy of results from Kraken metagenomics. In BLAST, the result for nucleotide analysis on contigs came back as 97-94% match for R. erythropolis, confirming the Kraken results (National Center for Biotechnology Information, 2017).

 

Discussion

The results of the genomic tests led me to believe the strain I isolated was a variety of R. erythropolis, despite having relatively small amounts of genetic data to work with. The lack of contigs, functional genes, and tRNA leaves room for there to be a considerable amount of error in the identification, but the certainty of identification for what is present from the sample is considerably confident.

Additionally, if we are to analyze the characteristics of R. erythropolis, it is a Gram-positive, mesophilic, aerobic rod bacteria. Interestingly, it was recently observed to be a human pathogen under specific circumstances, causing septicemia in immunocompromised individuals (Park et al., 2011). Ironically, a fish in the tank I sampled died of septicemia earlier this year, though there is no means to determine if R. erythropolis was responsible for the death.

These organisms are commonly found in both soil and aquatic environments, as well as eukaryotic cells. In wild environments, they are known for utilizing a wide variety of organic compounds. Different strains have demonstrated the ability to undergo oxidations, dehydrogenations, epoxidations, hydrolysis, hydroxylations, dehalogenations and desulfurization in order to process the resources of their habitat (de Carvalho, 2005). Due to this diversity, Rhodococcus sp. are considered important industrial organisms, utilized in production of bioactive steroids and fossil fuel biodesulfurization, and is also regarded as the most commercially successful microbial biocatalyst (McLeod, 2006).

Comparatively, my isolate demonstrated several traits associated with Rhodococcus species. My isolate was gram-positive and grew in rods. The diplobacilli arrangement noted in my isolate was never mentioned as a distinguishing factor of R. erythropolis or other Rhodococcus species. I collected my sample from an aquatic environment, and though the environment I collected it from was warm, I found the bacterium grew much more successfully at room temperature. Additionally, my bacteria was established as tolerant of acidic environments from the initiation of the experiment. R. erythropolis is not characterized as an acidophile, so I believe it is an ability it possesses, but as demonstrated by growth on TSA plates and TSB, not the preferred environment of the organism.

Physiological tests revealed my bacteria sample can process urea, produces the enzymes tryptophan deaminase and tryptophanase, and can ferment a variety of simple sugars, but does not appear to have the dramatic abilities some R. erythropolis strains, though some were never tested. Although my isolate thrived in an aerobic environment, a characteristic of Rhodococcus sp., it also showed the capacity to persist in all oxygen zones, something not all Rhodococcus sp. can do.

Due to the genomic and physical evidence presented, I believe my isolate is a variety of Rhodococcus, though it may be debatable whether it is R. erythropolis, considering the lack of additional genomic data, and the diversity of the Rhodococcus genus. It is commonly acknowledged that Rhodococcus is environmentally widespread, and many more species may be present than currently documented in journals or databases (Park, 2011).

Several tests were conducted in lab research on other isolates which were not possible to do on my bacterium, due to its unfortunate and premature loss from the lab samples. As such, there can be no further research on this specific organism, though if there were, would like to investigate its abilities in several other ways.

I especially would have liked to analyze antibiotic resistance of my isolate. As it is an environmental microbe, especially one which can compete in the same pH zone as fungi, I would imagine it would have resistance to fungal antibacterials so as to better contend with fungi living in the same niche. Additionally, since it has potential to be pathogenic, it would have been beneficial to see what resistance or susceptibility it may have.

Though it would have been interesting and insightful to test my isolate’s ability to process petroleum, considering the use of R. erythropolis as a biodegradation, this was an opportunity not presented to me in this study. Another experiment I would have liked to complete in future research is analysis of the pathogenic potential of my specific strain. As there are multiple accounts of R. erythropolis causing septicemia in patients, perhaps analyzing growth on blood agar would show how quickly an infection of my isolate could become threatening.

In conclusion, my bacterium sampled was not a nitrifying organism, as I had predicted. Rather, it is a decomposer with the capacity to degrade a variety of environmental compounds, likely taking full advantage of the waste products and other organic matter present in the aquarium biome I took it from. There is a wide range of further experiments I would have liked to have conducted on this organism, but due to the follies of lab, I have, for the moment, lost the ability to further study this opportunistic organism. However, this entire study was a reinforcement of the staggering amount of diversity within the microbial world and the startlingly prolific world just out of our sight at all times.

 

References

Archarya, T. (2015). API 20E Test System: Introduction, Procedure Results and Interpretations. Retrieved from https://microbeonline.com/api-20e-test-system-introduction-procedure-results-interpretations/ on April 2, 2017.

 

Baran V., Dvorska L., Matlova L., Pavlik I., Svastova P. (2008). Distribution of mycobacteria in clinically healthy ornamental fish and their aquarium environment. Journal of Fish Diseases 29: 383—393.

 

de Carvalho, C.C.C.R., da Fonseca, M.M.R. (2005). The remarkable Rhodococcus erythropolis. Applied Microbiology and Biotechnology 67: 715—726.

Illumina BaseSpace. (2017). SequenceHub. Retrieved from https://basespace.illumina.com/dashboard on April 2, 2017.

 

McLeod, M.P., et al. (2006) The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proceedings of the National Academy of Sciences of the United States of America 103: 15582—15587.

 

National Center for Biotechnology Information. (2017). BLAST Nucleotide Sequence. Retrieved from  https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome on April 2, 2017.

 

Park, S.D., Uh, Y., Jang, I.H., Yoon, K.J., Kim, H.H., Bae, Y.J. (2011). Rhodococcus erythropolis septicaemia in a patient with acute lymphocytic leukaemia. Journal of Medical Microbiology 60: 252—255.

 

Smith K., Schmidt V., Rosen G.E., Amaral-Zettler L. (2012). Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water. PLOS One: https://dx.doi.org/10.1371/journal.pone.0039971.

Urakawa H., Tajima Y., Numata Y., Tsuneda S. (2008). Low Temperature Decreases the Phylogenetic Diversity of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofiltration Systems. Applied and Environmental Microbiology 74: 894-900.

 

Zehr J.P., Carpenter E.J., Villareal T.A. (2000). New perspectives on nitrogen-fixing microorganisms in tropical and subtropical oceans. Trends in Microbiology 8: 68-73.

 

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.

Micrococcus luteus and the Catahoula

 

Alex Frary

28APR2017

342 F03

Micrococcus luteus and the Catahoula

Introduction

The 21st century is an exciting time to be a microbiologist with all the new discoveries and advances in technology such as a portable, real-time DNA sequencer. In this new and exciting time, there has also been an increase in public notice and awareness of microbes like bacteria and viruses as well as an increase in access to advanced technology. The general population is now becoming curious as to what microorganisms surround them everyday such as those found in their fridge, on the floors, or even on their pets. As any owner knows, dogs are generally affectionate and have a tendency to express that affection through the transfer of their saliva when licking their owners. And so it leads one to wonder: what exactly is in dog’s saliva? After all, a dog’s mouth can be an ideal breeding ground for all kinds of microorganisms with its warm, moist climate and abundance of food being passed through.

In order to satisfy my curiosity as a dog owner and scientist, I swabbed the inside cheek and gums of my 21 month old Catahoula. One specimen of this sample was then isolated by streaking, characterized by several physiological tests and genome sequencing, and then identified. According to a similar study of a beagle, several genera of microorganisms were identified from the dental plaque of the animal to include Micrococcus, Pseudomonas, and Streptococcus (Wunder 1976). It is among these genera that the specimen taken from the Catahoula would more than likely fall under. My isolate was eventually identified as being Micrococcus luteus. This bacterium is commonly found not only in the human flora, but also in the microflora of skin, mucosa, and oropharynx of mammals (Engelkirk 2008). It is considered an opportunistic pathogen that is pretty much harmless and very common.

 

Methods

Collection and Isolation:

                      Using two sterile swabs, I lightly sampled the inner cheeks and gums of the Catahoula female canine and transferred the samples onto and a TSA plate. Over the next 48 hours, substantial growth of two different types of colonies could be seen on the Tryptic soy agar plate. Choosing the lighter colored bacteria, I used the Streak Plate Method to transfer to a new TSA plate, and over the course of the week, was able to isolate the strain. In between streaks, the plates were kept at 37 degree Celsius in an incubator.

Identification and Characterization:

                      After having isolated the strain, I utilized the Gram-staining process to determine whether the strain was Gram-positive or —negative (as per Lab 4 guidelines). Following this, I went on to use several physiological tests to further characterize the bacteria. The first of which was the fluid thioglycollate test, which I used to determine oxygen class. The next test was the Oxidase Test, which I used to determine if the strain has cytochrome c oxidase (which is helpful in differentiating between pseudomonas species and enteric species). The following test was then the catalase test, which I used to determine if the bacteria has the enzyme catalase. Lastly, the API test strip, which is used to identify enteric Gram negative rods, was then used. The exact methods used for the physiological tests can be found in the Lab 6 Manual.

In order to taxonomically identify the bacteria, I extracted DNA from my isolate using the PowerSoil DNA kit and using techniques described in Lab 5. The genome was then sequenced by the UAF DNA Core Lab Technician using Illumina MiSeq technology. Following the return of the data (bioinformatics) on BaseSpace, I was able to analyze genomic sequencing of my isolate following Lab 7 instructions. The applications used in BaseSpace were Genomic Assembly (to determine what genes are present and what their functions might be), Taxonomic Assignment (to identify isolate to the species/genus level), and the Functional Gene Annotation (to annotate genes and determine the function of those genes). One of the last tests I administered to the isolate was the antibiotic susceptibility test. In this test, I used the Amikacin, Cefazolin, Cefoperazone, Gentamicin, Oxacillin, Tobramycin, Trimethoprim, and Vancomycin disks to observe the zone of inhibition around each disk after being incubated at 37 degrees Celsius for 48 hours (Lab 9).

 

Results

After staining the isolate, the resulting images underneath the microscope showed purple, cocci bundles of bacteria, this lead to the conclusion that the isolate was Gram-Positive (Figure 1). During the physiological testing, the growth in the fluid thioglycollate tube revealed that the bacteria only grew on the top most layer of the tube, suggesting that the isolate is strictly aerobic. After performing the oxidase test, it was conclusive that there was color change on the oxidase test strip, which indicates the isolate contains cytochrome c oxidase. Next was the catalase test in which when hydrogen peroxide was added to the isolate, bubbles appeared. This reaction indicates that the isolate was a reactive oxygen species and contains the catalase enzyme. Following the catalase test, an API 20E test strip was utilized to further characterize the isolate. The results of the strip, however, came out to be negative for each test (Figure 2). It is because of the lack of results that an additional Staph API test strip was then inoculated. The results of this API test strip, however, came out to be negative for all sections just as the previous (Figure 3).

After using the PowerSoil DNA isolation kit to extract DNA from the isolate, the sample was sent off to the UAF DNA Core Lab for sequencing. The results on the BaseSpace application, Kraken Metagenomics, outlined the “Percent Reads Classified’ for “-species level identification-‘ were approximately 25.12%, suggesting that the classification revealed in the program has lower support and can be less certain of the result possibly due to contamination. Based on this data, however, the isolate was identified as (probably) being Micrococcus luteus.

Further testing of the isolate showed the strains antibiotic resistance (Figure 4). Among the antibiotics tested, Amikacin, Cefazolin, Cefoperazone, Gentamicin, and Vancomycin were the ones that the isolate was susceptible to. The isolate showed only intermediate susceptibility to Trimethoprim, and was resistant to Oxacillin and Tobramycin (Table 1).

Figure 1: Microscope view of Isolate

Figure 2: API Test Strip (negative results)

Figure 3: Staph API Test Strip (negative results)

Figure 4: Antibiotic Resistance Test

Antibiotic Diameter Result
Amikacin 38mm Susceptible
Cefazolin 30mm Susceptible
Cefoperazone 34mm Susceptible
Gentamicin 36mm Susceptible
Oxacillin 0mm Resistant
Tobramycin 8mm Resistant
Trimethoprim 12mm Intermediate
Vancomycin 20mm Susceptible

Table 1: Antibiotic Resistance Results

 

 

Discussion

Although the isolate sample sent in for genetic sequencing did not yield ideal results, due most likely to contamination , the physiological testing results were able to provide additional support that the isolate was indeed Micrococcus luteus. Micrococcus luteus is a Gram-positive, aerobic chemoorganotroph in the Genus: Micrococcus, Family: Micrococcaceae, Order: Antinomycetales, and Phylum: Actinobacteria (Slonczewski 2014).

Micrococcus luteus is also catalase positive, forms yellow colonies, oxidase positive, and also can occur in pairs, tetrads or clusters (Engelkirk 2008). The bacterium can be found in the microflora of skin, mucosa, and oropharynx of mammals, such as humans or Catahoulas as this study found (Engelkirk 2008). It is considered an opportunistic pathogen, meaning that certain strains of the bacteria have been linked to cases involving meningitis, pneumonia, and endocarditis (Whitman 2012).

Alexander Fleming discovered Micrococcus luteus in 1922 while studying an individual with a cold. He tried to find the specific cause of the individual’s runny nose. It was during this that he isolated Micrococcus lysodeikticus (now luteus), and it is now recognized as part of the normal flora of humans and that it is susceptible to lysozyme as Fleming discovered (Moticka 2015). I found the origin story particularly interesting as I found my sample in the saliva of my Catahoula.

In conclusion, I’ve found that the physiological and morphological results during the duration of the study provide strong evidence that my isolate is indeed Micrococcus luteus that I was able to isolate. My isolate was sampled from a yellow colony on the agar plate and is Gram-positive, strictly aerobic, catalase and oxidase positive, cocci clustered, and found in the saliva of a mammal. Based on the corresponding data available on the bacteria, my findings were supported. The end result, however, would have been more substantiated had I not possibly contaminated my DNA extract sample. Overall, proper laboratory technique was learned, and I believe that further studies into the possible pathogenic hazard of Micrococcus luteus should be considered given that it can be found on human skin and in the mouth of a pet and know to have caused illnesses such as meningitis.

 

 

 

REFERENCES

Engelkirk, P., Duben-Engelkirk J. 2008. Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology: p. 216.

Moticka, E. 2015. A Historical Perspective on Evidence-Based Immunology: p. 3.

Slonczewski, J., Foster, J. 2014. Microbiology: An Evolving Science, 3rd Edition.

Whitman, W., Goodfellow, M. 2012. Bergey’s Manual of Systematic Bacteriology, Vol 5: p. 572.

Wunder, J., Brinner, W. 1976. Identification of the cultivable bacteria in dental plaque from a Beagle dog. Journal of Dental Research.

Isolation and Characterization of an unusually small aquatic bacterium

Sam Dempster

Introduction:  

Humans like to think we are the top organisms on Earth; we live on every continent, we are numerous (~7 Billion individuals) and we can survive well in our environment. Humans for thousands of years had this superiority complex, but then something odd was discovered; microbes. These were found to outnumber humans billions to one. We had to accept that something that we can’t normally see with the unaided eye lives all around us, on us, and in us, and they’ve been doing it for billions of years before we showed up.

Microorganisms (or microbes for short) can be found in all domains of life; Bacteria, Archaea and Eukarya. I took Microbiology wanting to learn more about microbes and the world that they live in. For my semester long project of isolating and identifying a bacteria, I decided to sample the biofilm that forms in my aquarium and study a microbe that grew on my agar. I wanted to know if I could get a microbe that reduced ammonia or its byproducts like nitrate or nitrite. These bacteria are extremely important not just for natural bodies of water but for aquariums too, which reduce ammonia from fish metabolism to much less harmful nitrate.

Today, I will be presenting my data and try to identify my bacteria based on my physiological and genetic tests. I hypothesize that my bacteria will be a nitrogen reducer (known as a denitrifier), as these can be the most common microbe in many aquarium environments.

Methods:

To facilitate the tests for the semester, the bacteria had to be sampled from the environment. To do this, I used a sterile swab moistened with sterile water to take a sample of the biofilm that forms on top of my aquarium water. The sample was then streaked across a Tryptic Soy Agar (TSA) plate. These were sealed with parafilm and left to incubate in an open bag on top of the refrigerator upside down to prevent condensation.

A few days later, I observed a multitude of different bacteria colony growth on the TSA plate. During the second lab, I selected a small, fairly isolated yellow colony from the TSA plate and used the Quadrant Streak Method to try to isolate my bacteria for further tests. I let it incubate at 37oC in the sealed plate in between each streak. After 9 individual re-streaks, a pure isolate was finally achieved.

Once I believed I had a pure isolate, I performed a battery of physiological tests to profile my bacteria. These tests include: Gram Staining, fluid thioglycollate, streaking on MacConkey agar, catalase test strip and oxidase tests.

After physiological tests, I then performed whole genome sequencing to attempt to identify my isolate. I then extracted my bacteria’s DNA following the procedure laid out in Lab 5 using the PowerSoil ® DNA Isolation Kit. The DNA extract was then sent to the UAF Institute of Arctic Biology DNA Core Lab to be sequenced on an Illumina Miseq. I then performed two API test strips; the API 20E and API 20 STAPH. Interperating the results in Lab 6, my test strips gave me negative results across the board. I then analyzed the genomic data provided from the Illumina Miseq as outlined in Lab 7. This involved using applications on BaseSpace to assemble and analyze my data. Using SPAdes Genome Assembler, KRAKEN Metagenomics and Prokka Genome Annotation apps, I was able to narrow down the identification of bacterium as discussed below.

Results

I performed a battery of tests to try to identify my isolate. After performing the gram stain five times, my results point to a Gram-negative bacillus bacterium (Table 1). Oxidase and Catalayse tests both came back positive (Table 1). The fluid Thilloglycate tube tests suggested that my bacteria was a strict aerobe (Table 1). Genetic information points the identity of my bacteria towards Microbacterium sp., with the KRAKEN Metagenomics database giving an initial percentage of 92% unclassified and 2% Microbacterium (Figure 1). Of the 2% Microbacterium match, 62% of the DNA was attributed to Microbacterium testaceum (Figure 2).   Reanalysis of the 92% unclassified contigs from Figure 1 show that 47% was attributed to Microbacterium testaceum (Figure 3). I then used BLAST to analyze the 34% unclassified contigs from Figure 3 and the two top results that showed both a Query Coverage and Identification Confidence score of 90% were Microbacterium paraoxydans and Microbacterium sp.

I tested my isolates antibiotic susceptible as outlined in Lab 9. This included doing two disk diffusion tests testing Cefoperazone, Vancomycin, Amikacin, Trimethoprim, Gentamicin, Oxacillin, Tobramycin and Cefazolin (Table 4). My isolate was susceptible to all antibiotics tested except Tobramycin, which was intermediate (Table 4).

In the tables below, x# represents the number of times a successful result was recorded (example: Gram Stain x5 means the Gram Stain was preformed 5 individual times resulting in the same data).

Table 1. Physiological traits of isolate

Gram Stain x5 Negative (-)
Cell physiology Very Small, Rod Shaped (Bacillus)
Colony Phenotype Glossy, yellow, slightly raised colonies
Catalase Test Positive (+)
Oxidase Test x3                   Positive (+)
Fluid Thilloglycate Test Strict Aerobe

 

Table 2. Results of the API 20E test strip

API 20E Physiological Test ID API Test Result
ONPG Negative (-)
ADH Negative (-)
LDC Negative (-)
CIT Negative (-)
H2S Negative (-)
URE Negative (-)
TDA Negative (-)
IND Negative (-)
VP Negative (-)
GEL Negative (-)
GLU Negative (-)
MAN Negative (-)
INO Negative (-)
SOR Negative (-)
RHA Negative (-)
SAC Negative (-)
MEL Negative (-)
AMY Negative (-)
ARA Negative (-)

 

Table 3. Results of API 20 STAPH test strip

API 20 STAPH Physiological Test ID API Test Result
0 Negative (-)
GLU Negative (-)
FRU Negative (-)
MNE Negative (-)
MAL Negative (-)
LAC Negative (-)
TRE Negative (-)
MAN Negative (-)
XLT Negative (-)
MEL Negative (-)
NIT Negative (-)
PAL Negative (-)
VP Negative (-)
RAF Negative (-)
XYL Negative (-)
SAC Negative (-)
MDC Negative (-)
NAG Negative (-)
ADH Negative (-)
URE Negative (-)

 

 

 

Table 4. Results of Lab 9 antibiotic susceptibility tests

Antibiotic Zone of Inhibition (mm) Susceptibility (mm) Test conclusion
Cefoperazone 26 ≥18 Susceptible
Vancomycin 20 ≥17 Susceptible
Amikacin 29 ≥17 Susceptible
Trimethoprim 25 ≥16 Susceptible
Gentamicin 28 ≥15 Susceptible
Oxacillin 26 ≥(13,18,20) Susceptible
Tobramycin 14 ≥15 Intermediate
Cefazolin 22 ≥18 Susceptible

 

Figure 1 showing the results of analyzed genomic data. This shows that a great majority of the genome (~92%) is unclassified in the KRAKEN Metagenomics database. This shows that 2% of the genome can be attributed to Microbacterium sp.

Figure 2 shows the zoomed in porting of the previous figure (fig 1). This shows that out of the different species under the family Microbacteriuaceae, 4,169 reads were attributed to Microbacterium tesstaceum.

Figure 3 showing the identification results of the unidentified contigs from the previous (fig 1) KRAKEN Metagenomics analysis. Out of the 92% unclassified genome, 47% was attributed to Microbacterium testaceum.

 

Figure 4 shows the BLAST results of the unclassified data from the previous figure (fig 3). The highest matches confirm the KRAKEN Metagenomics analysis. The two highest matches tied at 90% Query cover and Identification certainty are Microbacterium paraoxydans and Microbacterium sp.

 

Discussion

According to research, Microbacterium sp. can be found in many places, including being isolated on a potato leaf (Wang et al., 2010). Microbacterium sp., under the phylum of Actinobacteria, are known to be ubiquitous in soil and water environments (Embley and Stackebrandt 1994), and have even been isolated from the livers of lab mice (Buczolits et al., 2008). They have many different lifestyles and metabolic pathways (Servin et al., 2007). In the end, my hypothesis of my isolate being a denitrifier was neither supported nor denied as I do not believe the API test strips were designed to test the metabolic functions of my genus of bacteria.

There was one very serious discrepancy between genomic analysis and physiological tests. This being the gram stain test, with Microbacterium sp. should always Gram stain positive. As referenced in Table 1, five individual Gram stains found the bacterium to be Gram negative. Microbacterium is in the phylum Actinobacteria, which a defining characteristic being that they are all gram-positive bacteria. A closely related bacterium in the same phylum, Mycobacteria, is known as acid-fast Gram positive. When a bacteria shows acid-fastness, this means the bacteria will stain abnormally due to the presence of one of many different factors. These can range from the presence of mycolic acid in their cell walls to other cellular inclusions which either resist the ethanol or acid based de-colorization process. This can result in a negative result when the cell cannot retain the stain (perhaps like my bacteria) and it washes out with the ethanol (Morello et al., 2006).

To better identify my bacterium, I would potentially perform several more tests. This includes an acid-fast stain, perhaps the Ziehl—Neelsen stain. This would allow me to see if my bacterium is acid-fast positive, meaning it would Gram stain negative but have a Gram-positive physiology. I would also like to perform several different API test strips to try to determine what metabolic pathways my isolate possesses.

 

References

Buczolits, S., Schumann, P., Valens, M., Rosselló-Mora, R., & Busse, H. (2008). Identification of a bacterial strain isolated from the liver of a laboratory mouse as Microbacterium paraoxydans and emended description of the species Microbacterium paraoxydans Laffineur et al 2003.  Indian Journal Of Microbiology,  48(2), 243-251. doi:10.1007/s12088-008-0035-0

Embley TM,   Stackebrandt E. The molecular phylogeny and systematics of the Actinomycetes, Annu Rev Microbiol , 1994, vol. 48 (pg. 257-289)

Morello, Josephine A., Paul A. Granato, Marion E. Wilson, and Verna Morton. Laboratory Manual and Workbook in Microbiology: Applications to Patient Care. 10th ed. Boston: McGraw-Hill Higher Education, 2006. Print.

 

Servin, J., Herbold, C., Skophammer, R., & Lake, J. (2007). Evidence Excluding the Root of the Tree of Life from the Actinobacteria.  Molecular Biology And Evolution,  25(1), 1-4. doi:10.1093/molbev/msm249

Wang, W., Morohoshi, T., Ikenoya, M., Someya, N., & Ikeda, T. (2010). AiiM, a Novel Class of N-Acylhomoserine Lactonase from the Leaf-Associated Bacterium Microbacterium testaceum.  Applied And Environmental Microbiology,  76(8), 2524-2530. doi:10.1128/aem.02738-09