A1: Introduction Post

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Hello! My name is Brittany Wintter and I am a senior here at UAF in the Biological Sciences program. My goal is to attend Dental School then come back to Fairbanks to work in the public health sector.

I am a born and raised Fairbanksian but love traveling the world. A few of my life goals are to travel to all 50 states and as many countries across the world as possible with my son. The image included is from a cathedral in Austria that has over 100 faces and many full statues, taken in 2017.

I am particularly interested in Porphyromonas gingivalis which is the keystone bacterium in the formation of periodontal disease and also contributes to immune diseases; it is also intriguing that if present it generally consists of 1% or less of the oral microbiome.

I look forward to our semester!

Gurk, Carinthia

A1: Intro Post

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Hi, my name is Reed Thomas and I’m currently a freshman pursuing a BS in Biological Sciences.  I plan on using my degree to apply to medical school and, hopefully, come back to practice in Fairbanks.

I am Lathrop High School graduate from Fairbanks but enjoy traveling throughout the states. I have been to several interesting places, but most notably was Italy and San Marino where I played soccer for several weeks. In my free time, I enjoy playing soccer, watching soccer, and coaching it too! I look  forward to a fun semester.

A2: Microbes in the News – Post 1

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https://www.npr.org/sections/goatsandsoda/2018/04/07/598093165/could-you-fight-off-worms-depends-on-your-gut-microbes

Title: “Could You Fight Off Worms? Depends On Your Gut Microbes” – Nadia M. Whitehead

Source: NPR.org

Date: 4/7/18

Summary: This article discusses a discovery of individuals infected with parasites share common microbes even though the individuals live in different geographic locations. The article further discusses how certain bacteria known as  Lachnospiracae is associated with individuals who can fight off worms naturally.

Connections: This article is focused on the microbiome and the possibility of altering the human microbiome to fight parasites naturally without the use of drugs. We have discussed in class about the human microbiome and our bodies natural defenses over disease.

Critical Analysis: I liked how the author explains that 25% percent of the world’s population is infected with parasitic worms. The author further explains how these worms are contracted and that despite decades worth of deworming efforts to exterminate the world of worms, people in developing countries continue to be reinfected. I wish the author would have included graphs/charts on the research that was associated with the article. It would help further explain the research and engage the reader. The writer gives credit to Makedonka Mitreva, the lead researcher on the study reported in the article. Mitreva suggests a great way to rid the world of worms. She hopes to use fermented foods to plant “worm-defending” microbes inside of individuals to help fight worms.

Questions: Can we use the same plan of attack against worms to alter the human microbiome to fight other diseases, such as cancer or common infections?

 

Kocuria rhizophila- isolation from a free weight at UAF and classification using physiological and genomic testing.

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Introduction:

My original swab came from a dumbbell in the Patty Center at UAF. I chose a colony from the plate and streaked it. Throughout lab, a series of tests were conducted to indicate the strain of bacteria that was isolated from this free weight. The strain of this isolate was determined by looking at colony morphology, staining tests, cell morphology, API tests, and DNA sequencing. Through this series of tests, I determined that my isolate was Kocuria rhizophila. This bacteria can be found on human skin and in soil, so it is possible that it was transferred to a dumbbell through a person holding it in their hand (Savini et al). Kocuria rhizophila is not motile, so it would have to be transferred to a surface through contact or some other mean (Kovacs et al, Wood et al). Microbial communities on gym equipment tend to be shaped by the microbial flora of the hosts that use and come into contact with them (Wood et al). Micrococcus (Kocuria’s genus) was commonly found on free weights according to Wood et al.

Based on my bacterium’s original environment, I hypothesized that it was aerobic and could proliferate at normal temperatures. The isolated bacteria was found on the surface of an object; this could indicate that it had a good supply of air and would not need to conduct metabolic processes like anaerobic fermentation. Based on this, I did not expect it would be able to reduce nitrate. I can not say as to whether or not it could be a pathogen. Based upon it’s human commensal status, I would not expect it to be.

It is possible that the Kocuria rhizophila that was isolated was from the dumbbell on the gym. Since it is a human commensal, it could have easily ended up in the surface of the dumbbell (Savini et al). However, Kocuria rhizophila was also a common contaminant in our lab. It was used for other experiment and there were many cultures of it in the incubators where my isolate plates were kept. The Kocuria cultures were also used in lab either before or after we did a streak of our isolates. Kocuria rhizophila DNA was also present in many other isolate’s DNA samples.  It is possible that Kocuria rhizophila came from the gym, or that it was a contaminant in my plate that was mistaken for a colony from the original swab.

Methods:

Original Swab: I did not use my own original swab for my isolate, my Tryptic Soy Agar (TSA) plate didn’t grow colonies in time to start isolating bacteria colonies, so I used my lab partners. Her swab came from a dumbbell in the Patty center. I streaked a smooth yellow colony onto a TSA plate using aseptic technique. This plate was incubated for a week at 37 degrees Celsius. (Lab 1, Lab 2)

 

Isolating a pure culture: After a week, my culture was not pure. There were two kinds of colonies on the plate: white colonies and yellow colonies. I chose to isolate a large yellow colony with wavy edges growing near the center of the plate. Using aseptic technique, I streaked the colony onto a fresh TSA plate. This was done many times throughout the length of this experiment to ensure I had a fresh culture. Multiple streakings also ensured my culture didn’t die off and stayed as pure as possible. I also streaked my isolate onto a TSA tube. These isolate samples were incubated at 37 degrees Celsius for a week until the next lab. (Lab 3)

 

Staining: Using the Gram stain technique, I stained my isolate and viewed it under the microscope. Gram staining involved drying and heat fixing smears, applying crystal violet stains, applying Gram’s iodine, washing with water and ethanol, and then applying safranin to stain any Gram- negative bacteria so they would be visible. Under the microscope, It did not look like there were any other kinds of bacteria, so I assumed my culture was pure. I inoculated an agar slant to use as a backup culture. It was stored in the refrigerator.  (Lab 4)

 

DNA extraction and sequencing: Before I extracted my isolate DNA, I cultured my isolate in a fresh Tryptic Soy Broth (TSB) tube a few days before lab and incubated the sample at 37 degrees. I used aseptic technique while doing this to ensure my DNA sample would be as pure as possible. DNA extraction was conducted by the methods described in Lab Handout 5. Cell lysis, removing inhibitors and proteins, and obtaining a pure solution were all used to extract the DNA. The pure DNA was sent off to a lab at UAF for sequencing. After the sequences came back I used the BaseSpace site to analyze my sequenced genome. BaseSpace reported information on tRNAs, 16sRNA gene, CRISPRS, coding genes, the length of the isolate’s sequence, the number of contigs, taxonomy of my strain, and GC content. (Labs 5, Lab 7)

 

Physiological tests: I conducted a fluid thioglycolate test, a catalase test, oxidase test, and set up an API 20e test strip to test for various physiological characteristics in my isolate. Fluid thioglycollate tests were conducted using liquid agar contained in a test tube and stabbing an inoculated loop into it. Where the bacteria grew and revealed the oxygen class of the isolate. The catalase test involved applying hydrogen peroxide to my isolate on a microscope slide. Bubbles forming indicated gas being released as the enzyme catalase catalysed the release of oxygen from the hydrogen peroxide. The oxidase test assessed my isolate for cytochrome c oxidase. The API test strip involved a series of tests that tested for ß-galactosidase enzyme, Arginine Dihydrolase, Lysine Decarboxylase, Ornithine Decarboxylase, Citrate utilization, H2S production, urease, indole production, acetoin production, gelatinase production, fermentation of: glucose, mannitol, inositol, sorbitol, rhamnose, saccharose, melibiose, amygdalin,  and arabinose, cytochrome-oxidase production, and nitrate reduction. The test was conducted using the differential mediums in the API test strip to show positive or negative results for each of these tests. (Lab 6)

 

Physiological Testing for Fermentation: The bacteria was streaked onto MacConkey Agar (MAC) and Eosin Methylene Blue (EMB) plates to test for lactose fermentation and select for Gram-negative bacteria. (Lab 8)

 

Antibiotic Resistance Testing: Using the Kirby-Bauer Method, I tested my strain for susceptibility or resistance to the following antibiotics: piperacillin, erythromycin, gentamicin, tetracycline, cefazolin, oxacillin, trimethoprim, and amikacin. (Lab 9)

 

Additional Physiological Testing: Since the first API strip I used was for Gram-negative bacteria, I decided to do an API Staph test strip in addition to my first physiological test. This strip tests for different strains of Staphylococcus and a few strains of Kocuria. This test showed that the isolate was not Kocuria kristinae, Kocuria varians, or Kocuria rosea.

 

Results:

 

Colony Morphology of Pure Isolate: The colonies were yellow, convex, small, and had smooth edges. In fresh colonies, the margins of the colonies were even and circular as seen in Figure 1, but eventually they become wavy as they become larger (not pictured).

Figure 1. Colony Morphology of my isolate.

 

Staining Techniques and Physical Traits of my Isolate: Gram staining resulted in a deep purple color; this indicates the bacteria is Gram-positive. It is a coccus that can be observed in singles, tetrads, and packets.

.  

 

DNA Sequencing: BaseSpace could say with 76% confidence that my isolate was Kocuria rhizophila.

Figure 2. Taxonomic Classification of my isolate

 

The bacterial DNA sample was fairly short. The full assembly length was 1,743 base pairs and included 3 contigs. The SPAdes results gave a GC content of 58.69%. The DNA sample yielded no tRNAs, rRNAs, or CRISPRS. There were also no unique genes in my sample.

 

Physiological testing: My isolate tested positive for catalase; bubbles were formed when it was exposed to hydrogen peroxide. For the thioglycollate test, My isolate grew on the very top of the molten agar, and in the agar right below the surface. This would indicate that it was an aerobe. My isolate tested negative for cytochrome c oxidase. The API 20E test strip I originally used was for Gram-negative bacteria. With the original API tests strip conducted on 2/28, I got no positive results four days later. Checking back a week later in the next lab period showed that I had many positive results. This is why I decided to do another API test strip meant for Staphylococci and Kocuria bacteria.

 

Fermentation testing: The bacteria did not grow on either the MAC plate or the EMB plate. This is further indication of the fact that the bacteria is Gram-positive.

 

Additional API testing on my isolate: My isolate was a strain of Kocuria that this test was not specifically designed to identify. It tested negative for everything but acetoin production (Voges—Proskauer test) and nitrate reduction.

 

Antibiotic Testing: I found that my isolate was susceptible to piperacillin, gentamicin , tetracycline, cefazolin, oxacillin. It was intermediate towards erythromycin, and resistant to trimethoprim and amikacin.

Discussion:

 

The evidence from my research leads to the conclusion  that my isolate is Kocuria rhizophila. Kocuria  “belongs to the family Micrococcaceae, suborder Micrococcineae, order Actinomycetales, class Actinobacteria’ (Savini et al, Takarada et al).  Kocuria rhizophila lives on mammalian skin and in soil (Savini et al). It is a gram-positive coccus that can be found grouped together in a staphylococci “packet’ formation. This is consistent with Kovac et al’s findings. The yellow color of the colonies is similar to that of Kocuria rhizophila when streaked on a plate.

Kocuria rhizophila was a common contaminant in our lab. It is possible that my isolate was a contaminant from the lab, or that it was cultured from the dumbbell weight at the UAF Patty Center. My second TSA plate had Kocuria colonies growing on it after a week in the incubator where other Kocuria samples were being cultured. The first swab had yellow colonies that resembled Kocuria, but they were not necessarily Kocuria rhizophila. The results for where my bacteria exactly came from are inconclusive.

My hypothesis that my isolate would not be able to conduct fermentation were correct. The isolate did not grow on either that MAC or EMB plates, this is most likely because they select for Gram-negative bacteria. Kocuria rhizophila can grow under both aerobic and anaerobic conditions (Takarada et al, Moissenet et al). My isolate was shown in the thioglycollate test be an obligate aerobe.

The API 20E test strip’s unusual results were inconclusive. The strip was mostly negative after 4 days and then mostly positive after a week. These positive results after a long period could have been due to my isolate not growing quickly enough, or it could have been due to a contaminant microbe in my strip. API 20e strips also are meant for testing on Gram – bacteria, this affected th results as well.

The second API test strip performed, API Staph, came back completely negative other than the VP test and the nitrate reducing capabilities. The VP result is unusual because Kocuria rhizophila normally tests negative for this (Savini et al). It should be noted that the VP test on the API 20E test strip was negative, so the positive result could have been due to contamination. With regards to nitrate reduction, Kocuria has been shown to have genes similar to E. coli that could code for the ability to reduce nitrate under anaerobic conditions (Takarada et al). K. rhizophila tested positive for nitrate reduction all the way to nitrogen gas on both the API 20E test strip and the API staph test. This could speak to it’s ability to proliferate aerobically as found by Takarada et al. Since it is a soil dwelling microbe, the nitrate reduction could be useful in anoxic conditions soils sometimes creates (John Martinko et al).

Antibiotic testing of my isolate yielded interesting results. There have been records of Kocuria rhizophila being susceptible to amikacin, yet my isolate was resistant (Moissenet et al, Shashikala et al). It could have developed resistance to the antibiotic through it’s efflux pumps (Takarada et al).  This shows that Kocuria rhizophila has the ability develop antibiotic resistance, which could pose problems due to its emergence as a pathogen (Becker et al).

My hypothesis about my isolate’s ability to be pathogenic was correct. The Kocuria genus is slowly becoming more recognized as a pathogen, where it had previously been identified as a contaminant from human skin (Becker et al, Savini et al). Kocuria rhizophila has verified as a pathogen that contaminates venous catheters, leading to septic episodes in those using the catheters (Moissenet et al) . In both early cases of this, the patients had previous genetic disease cases and were being given slightly damaged catheters (Moissenet et al, Becker et al). Kocuria rhizophila was able to grow at low and normal temperature, and it’s growing temperatures range between 10 and 40 degrees Celsius (Kovacs et al, Savini et al).

In the future, more specific test could be done on this isolate to verify it’s exact strain.

Being able to re-do DNA sequencing for better results on special sequences, tRNAs, and CRISPRS could help tell me more about this bacteria’s genome. Repeating API strips would also tell me more about my isolate. Finding an API strip that could specifically help identify Kocuria rhizophila would be helpful to very what my strain is, rather than disqualifying a few kinds of Kocuria that it isn’t. Since Kocuria can reduce nitrate, it could be considered an important part of the nitrogen cycle in soil (John Martinko et al). With it’s emergence as a pathogen, Kocuria rhizophila is sure to be studied more in the future.

 

Works Cited:

Becker, K., F. Rutsch, A. Uekotter, F. Kipp, J. Konig, T. Marquardt, G. Peters, and C. Von Eiff. “Kocuria Rhizophila Adds to the Emerging Spectrum of Micrococcal Species Involved in Human Infections.” Journal of Clinical Microbiology 46.10 (2008): 3537-539. Web. 15 Apr. 2017.

Kovacs, G., J. Burghardt, S. Pradella, P. Schumann, E. Stackebrandt, and K. Marialigeti. “Kocuria Palustris Sp. Nov. and Kocuria Rhizophila Sp. Nov., Isolated from the Rhizoplane of the Narrow-leaved Cattail (Typha Angustifolia).” International Journal of Systematic Bacteriology 49.1 (1999): 167-73. Web. 9 Apr. 2017.

Martinko, John, Kelly Bender, Daniel Buckley, and David Stahl. “The Nitrogen Cycle.” Brock Biology of Microorganisms. By Michael Madigan. 14th ed. London: Pearson Education, 2015. 660-62. Print.

Moissenet, D., K. Becker, A. Merens, A. Ferroni, B. Dubern, and H. Vu-Thien. “Persistent Bloodstream Infection with Kocuria Rhizophila Related to a Damaged Central Catheter.” Journal of Clinical Microbiology 50.4 (2012): 1495-498. Web. 15 Apr. 2017.

Purty, Shashikala, Rajagopalan Saranathan, K. Prashanth, K. Narayanan, Johny Asir, Chandrakesan Sheela Devi, and Satish Kumar Amarnath. “The Expanding Spectrum of Human Infections Caused by Kocuria Species: A Case Report and Literature Review.” Emerging Microbes & Infections 2.12 (2013): n. pag. Web. 20 Apr. 2017.

Savini, V., C. Catavitello, G. Masciarelli, D. Astolfi, A. Balbinot, A. Bianco, F. Febbo, C. D’amario, and D. D’antonio. “Drug Sensitivity and Clinical Impact of Members of the Genus Kocuria.” Journal of Medical Microbiology 59.12 (2010): 1395-402. Web. 10 Apr. 2017.

Takarada, H., M. Sekine, H. Kosugi, Y. Matsuo, T. Fujisawa, S. Omata, E. Kishi, A. Shimizu, N. Tsukatani, S. Tanikawa, N. Fujita, and S. Harayama. “Complete Genome Sequence of the Soil Actinomycete Kocuria Rhizophila.” Journal of Bacteriology 190.12 (2008): 4139-146. Web. 10 Apr. 2017.

Wood, M., S. M. Gibbons, S. Lax, T. W. Eshoo-Anton, S. M. Owens, S. Kennedy, J. A. Gilbert, and J. T. Hampton-Marcell. “Athletic Equipment Microbiota Are Shaped by Interactions with Human Skin.” Microbiome 3.1 (2015): n. pag. Web. 24 Apr. 2017.

Planococcus sp. Isolated from sub-artic decomposing wood

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Planococcus sp. Isolated from sub-artic decomposing wood.

Morgen Southwood April 26, 2017

 

Introduction

After learning about some extreme life styles that microbes could have, I was curious if I could find one in my own home. I sampled two places, a piece of wood from my fire wood stack (which is exposed to the extreme cold temperatures of Alaskan winters) and the ashes from my stove. If any microbes grew from the firewood it would mean that they were able to withstand temperatures below -50 degrees Celsius and be psycrophiles, if any microbes grew in my wood stove they would have had to be thermophiles. I was unable to grow any microbes from the wood stove but the growth from the previously frozen wood was abundant. Each of those colonies represented a microbe that had the physiological capability of surviving those conditions, and I was interested in observing them in the lab.  The identification of this microbe would add to the depth of knowledge of what bacteria are and can be found on decomposing wood in the sub-artic.

The firewood pile was slowly decomposing, and it is likely that the microbe that was isolated from it was a part of the community that was utilizing the wood as an energy source. Decomposing communities frequently have a mix of fungi and bacteria. These communities emit extracellular enzymes to decay the wood, this results in an environment with high acidity and enzymes that are producing free radical oxygen species7. To thrive in this community it would be helpful to have mechanisms to process radical oxygen species. One such mechanism is the use of Catalase to catalyze the transformation of the reactive species hydrogen peroxide into O2. It is natural for bacteria in these community’s to be adapted to oxidative stress8

Throughout the course of this study I isolated one colony and performed many physiological and genomic tests to identify what species my isolate was. Considering the environment in which it was found, there were a few expected results: such as the microbe’s oxygen class and catalase test results. Once identified, I could then research the species and compare it’s physiology and genomic data to current literature to confirm the taxonomic assignment.

 

Methods

Collecting sample and isolation process

The sample was collected from a dry piece of wood. Sterile water was used to moisten the wood and a sterile swab was rolled along the wood’s surface.     The sample was streaked using the quadratic method to grow pure isolated colonies onto a tryptic soy agar (TSA) plate. To control growth rates the plates were grown in incubators for 2-4 days at 37 degrees Celsius then moved to a refrigerator to ensure colonies did not grow large enough to touch. The TSA medium provided a suitable environment for a variety of bacteria and a few molds. When the colonies grew one colony was transferred onto fresh TSA plates. The quadratic streak was used 4 times to ensure a pure culture. At this time the culture was transferred into an agar slant. Tests were performed on the isolate by obtaining cells from either the TSA plates or from the slant agar.

 

Physiological Characteristics

The gram stain was performed to assess the thickness of the peptidoglycan wall. The sample was stained using the provided protocol, gram-positives and negative controls were compared to the isolate to determine its gram-state (lab 4). While the cells were prepped in slides some characteristics were observed with the microscope, such as cell alignment shape and length. Colony morphology including color, size, margin and elevation was observed from growth on the TSA agar.

Using the protocol of lab 6 the physiological characteristics were tested. A suspension of the microbe in broth was tested simultaneously in all 21 of the tests included in the API 20E test strip. Additionally the isolate underwent the thioglycolate test- for oxygen class, oxidase test- for the presence of cytochrome c-oxidase, and lastly the catalase test- for the presence of catalase enzyme.

The cells inoculated onto Eosin Methylene Blue (EMB) and MacConkey Agar (MAC) plates. EMB is selective for gam-negative bacterium, if the microbe is gram negative and can ferment the sugars present then the plate experiences a color change. MAC plates are also selective for Gram-negative microbes and differentiates between lactose fermenters (agar turns pink) and non-lactose-fermenters (agar turns colorless).

The susceptibility of the isolate to 8 different antibiotics was tested using the Kirby-Bauer Method agar and the disk diffusion test per the protocol of Lab 9. The antibiotics tested were Amikacin, Cefazolin, Cefoperazone, Gentamicin, Piperacillin, Tobramycin, Trimethoprim and Vancomycin. After a two-day waiting period while the microbe was given the opportunity to develop where it could, the diameter of prohibited growth was measured and compared to standard antibiotic specific diameters and then classified as either susceptible, intermediate or resistant.

 

Bioinformatics

                      Dna was extracted and purified from the isolates cells using protocol from lab 5. Cell lysis was accomplished through adding sodium dodecyl sulfate (SDS), which is a surfactant that chemically weakened the cell walls, and the use of PowerBeads and the centrifuge to mechanically break the cell walls. A series of patented solutions C1-4 were added following the protocol to purify and remove inhibitors and proteins until all that remained was a pure solution of DNA. The DNA was then sent to the UAF’s DNA Core Lab sequenced using the Illumina MiSeq DNA sequencer which utilized the next-generation sequencing methodology.

Using Illumina’s BaseSpace dashboard to handle the data, the isolate’s genome was analyzed and compared to an existing database to search for similar sequences of DNA. BaseSpace has many apps that provide different information about the genomic data. The SPAdes Genome Assembler provided the overall result of the genomes assembly. Of this information, only the number of contigs that were greater than 1000 base pairs long [# contigs (>=1000bp)], The total length of the assembled genome, the longest contig and the guanine and cytosine percentage in the genome (GC%) were observed. The next app that was used was the Kraken Metagenomics, which provided taxonomic assignment. This app provided sample information such as the total number of reads that were used for classification and the percent of reads that were classified. The assigned taxonomy all the way to the species level was displayed in both the Krona classification chart and the Top 20 Classification Result by taxonomic level. Both sources have specific classification percentages that confer certainty. The final app Prokka Genome Annotation assigned gene regions that likely code for either tRNAs, rRNAs, CRISPRs and coding genes (CDs) and the abundance of them. The APP provides an extensive list of regions that were specifically identified as the above elements.

Further analysis of the genomic information was necessary. The contig.fasta file from the SPAdes Genome Assembly produced by Illumina’s BaseSpace was analyzed by BLAST. BLAST had a different database and could possibly have the genome of the isolates species if base space did not.     BLAST suggested Genus and species with associated query cover percentage and identity percentage.

 

Results

Physiological Characteristics

The Isolate appeared to be a pure culture by the third quadratic streak; a fourth was performed for certainty. The Microbes transfer to the agar slant was successful: Image 1, and was successfully re-cultured from the slant when necessary.

Image 1: Microbe growing on TSA agar

Image 2: Gram-stained microbe

The gram-stain was repeated three times before a successful stain was accomplished. The gram-stain revealed that the isolate was a gram-positive coccid: Image 2. While under the microscope it was observed that the cells could be aligned linearly, in pairs or quads but not in a longer strep chain. The purity of the isolate was confirmed with the microscopic observation of a mono-culture. The cells were measured to be 1 micrometer in diameter.  Colony morphology on the TSA agar revealed a yellow colony that’s size increased with time, a smooth round margin and the colony was an elevated domed.


Image 3: API 20E test results

The API 20e tests had negative results for all for the 21 tests: Image 3. Considering that the API 20e test is designed for enteric gram negative microbes these results are confirmation that the microbe is gram-positive. The thioglycolate test had growth throughout the agar with thicker growth at the surface, which revealed that the microbe’s oxygen class was facultative aerobe. The catalase test produced bubbles and the oxidase test produced a blue color change on the strip indicating that the microbe was positive in both tests. These results reveal that the cells contained both cytochrome c-oxidase and the catalase enzyme respectively. The isolate failed to grow on EMB or MAC plates, considering that both plates are selective for gram negative, these results confirm the results of the gram positive test: Image 4.

 

 

Physiological Tests Results
Gram Positive
Cell morphology 1 micrometer diameter, coccus, cells divide linearly
Colony Morphology Yellow colonies that’s size increases with time, a smooth round margin, elevated dome.
API 20e All negative results
Fluid thyioglycolate Facultative anaerobe
Oxidase Positive result
Catalase Positive result
MAC No growth
EMB No growth

Table 1: Physiological Test Results

The microbe was susceptible to all eight antibiotics tested. The Inhibition Zones exceed the antibiotic specific threshold diameter for susceptibility see table 2 for details.

Antibiotic Name Minimum Inhibition zone for susceptibility (mm) Microbe exceed inhibition zone

(yes/no)
Amakicin >=17 Yes
Cefazolin >=18 Yes
Cefoperazone >=21 Yes
Gentamicin >=15 Yes
Piperacillin

    Enterobacteriaeae

    S. pneumoniae

  

>=21

>=18

  

Yes

Yes
Tobramycin >=15 Yes
Trimethoprim >=16 Yes
Vancomycin >=17 Yes

Table 2: Antibiotic Susceptibility Results

Bionformatics

The analysis of the DNA with BaseSpace revealed unreliable data. BaseSpace’s SPAdes Gemone Assembler analyzed 128 contigs over 1000 base pairs (bp) long, a total length of over 3.7 million bp, the longest contig  was 196 thousand bp, and the GC% was 44.4%.     Kraken Metagenomics app was only able to classify 2.67% of reads, and of those reads only 17% of those reads identified the microbe as Lysinibacillus sphaericus. The Prokka Genome Annotation app identified 64 tRNAs, 0 rRNAs, 1 CRISPR, and 3739 CDS.     Image 5 and 6 are from the Kraken app.

Image 5: Krona Classification Chart from BaseSpace displays the large portion of unclassified reads.

Image 6 To 20 Classification Results by Taxonomic Level from BaseSpace

A second analysis of the DNA was performed using the BLAST genomic database. BLAST classified the organism as Planoccus donghaensis with a query cover of 77% and a 78% identity. Several other species had similar query cover, including Planococcus antarticus: Image 7.

Image 7: BLAST Genomic Results

Discussion

The genotypic test results for this microbe were vague, the results of BaseSpace had a low number of reads that could be classified and of those reads there was little consensus. The results of the SPAdes Genome Assembler were above required values; # contigs >=1000 bp should be in the hundreds, total length should be at least a million bp and the longest contig should be at least 100000 bp. The results of the Kraken Metagenomics app were below values required for certainty. The percent of contigs read needed to be greater than 80% and the percent of those reads that needed to agree upon a classification to trust that classification to the genus level was 80% and to the genus level was 60%. The results did not meet these thresholds, and this is the reason that the BaseSpace indentified taxanomy of Lysinibacillus sphaericus was rejected.

BLAST provided many species level classifications with similar query cover and percent identity. The species classification with the highest query cover is poorly documented in literature. The first publication of a species under the genus Planococcus was P. citreus. It was identified in 1894, and was only approved in 19806. This genus is relatively young and is currently growing with many of the species being described within the last ten years. Of the few documents that relate to Planococcus donhaensis, there is one that notes the origin of the sample, the South Korean Sea. There are some qualities that are consistent between my sample and P. donghaensis, they are both, gram-positive, aerobic, coccus and oxidase positive 1. However these similarities are not enough to convince me that they are the same species. A hallmark of the genus Planococcus is that they are usually halo-tolerant gram-positive bacteria that frequently inhabit Antartica4. The isolates holotolerance was not tested but the similar cellular membrane composition and habitat conditions indicate that this genus is likely correct.

The negative results and lack of growth in the API 20e, MAC, and EMB are all consistent with the microbe’s gram-positive cell wall. These tests reveal nothing more than a conformation that it is indeed a gram-positive microbe. The oxygen class determined by the fluid thioglycolate test and the positive results of the Oxidase and Catalase test are both consistent with the oxygenic environment that the microbe was isolated from. These tests proved that the microbe could thrive in an oxygenated as well as an anoxic environment, that it contained cytochrome c oxidase which is a part of the electron transport chain found in microbes that utilize oxygen, and that it contained the catalase mechanism for dealing with oxidative damage, respectively.

At the genus level, morphology can vary greatly. Many of the morphological and physiological analyses made on the isolate are not held by every species in the Planoccocus genus. The morphological characteristics that are consistent with the isolate and across the genus are: gram-positive membranes, cocci cell shape, colonies are yellow orange in color, catalase positive and an aerobic oxygen class5. My isolate was not just aerobic but a facultative aerobe, which is inconsistent with two articles that state the genus is strictly aerobic5. The isolate was found to be susceptible to all antibiotics tested with the disk diffusion test. This is consistent with a study that found, to the level of the genus, that Planococcus’ were susceptible to all antibiotics tested3.

The second most likely species suggested by was P. antarticus, at least this species shares a similar environment. P. antarticus thrived in an Antarctica, my sample would likely have similar mechanisms for surviving temperatures around -45 degrees Celsius in Faribanks winters. P. donghaensis may have had the ability to deal with these temperatures in its genome, but it is not certain since it’s environment doesn’t select for such characteristics.

The literature on the Planococcus species like donghaensis, kocurii, halocryophilus and antarticus often notes how closely related the species are and how further analysis like G-C content, fatty acid strains, DNA-DNA hybridization etc1,2 are needed to differentiate the species. To conclusively Identify the taxonomy of this isolated microbe, it would be advisable to repeat DNA isolation and genomic analysis that was preformed in this project, and to additionally perform other genotypic analyses like, DNA-DNA hybridization and 16s rRNA analysis and fatty acid identity. Considering the limit of species identified to this date, there is the potential that this microbe could be a new species.

 

 

 

Work Cited

  1. Jeong-Hwa C, Wan-Taek I, Qing-Mei L, Jae-Soo Y, Jae-Ho S, Sung-Keun R, Dong-Hyun R. Planococcus donghaensis nov, a starch-degrading bacterium isolated from the East Sea, South Korea. Int J Syst Evol Microbiol. 2007;57:2645—2650. doi: 10.1099/ijs.0.65036-0.

 

  1. Reddy G, Prakash J, Vairamani M, Prabhakar S, Matsumoto G. Shivaji S. Planococcus antarcticus and Planococcus psychrophilus nov. isolated from cyanobacterial mat samples collected from ponds in Antarctica. Extremophiles. 2002;6:253—261.

 

  1. Tuncer, I. (2016). Antibiotic resistance of bacterial isolates from sediments of eastern Mediterranean Sea in association with environmental parameters. Journal of Bacteriology and Parasitology, 7(6), 51. https://doi.org/10.4172/2155-9597.C1.025

 

  1. See-Too, W. S., Ee, R., Lim, Y.-L., Convey, P., Pearce, D. A., Yin, W.-F., & Chan, K.-G. (2017). AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus Scientific Reports, 7, 42968. https://doi.org/10.1038/srep42968

 

5.         Fackrell, H. (n.d.). Planococcus. Retrieved April 11, 2017, from Uwindsor.ca website: https://web2.uwindsor.ca/courses/biology/fackrell/Microbes/17375.htm

  1. Euzeby, J. P., & Parte, A. C. (2017, April 1). Genus planococcus. Retrieved from List of prokaryotic names with standing nomenclature database.

 

  1. Valášková V., de Boer W., Gunnewiek P. J. K., Pospíšek M., Baldrian P. (2009).  Phylogenetic composition and properties of bacteria coexisting with the fungus  Hypholoma fascicularein decaying wood.  ISME J.  3  1218—1221. 10.1038/ismej.2009.64
  2. de Boer W., van der Wal A. (2008).  “Interactions between saprotrophic basidiomycetes and bacteria,’ in  British Mycological Society Symposia Series 8  eds Lynne Boddy J. C. F., van Pieter W., editors. (Cambridge, MA: Academic Press; )  28  143—153.

 

 

 

 

Art Project: The Lytic Cycle

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The Lytic Cycle Shown Through Dance

I knew from the beginning I wanted to incorporate my microbiology project with pole dance however, none of the topics seemed to easily transition into a dance. When we learned about viruses, I knew I had found my topic. I came up with a routine and asked a few girls at my studio to do it with me. Fortunately, they agreed! Unfortunately, I was asking them to give up their time and work without pay. However, they were very generous and in the short amount of time we had, I was able to show them the routine and this was only take two!

The dance starts with the “cell” being the first two dancers on the pole. Then the virus shows up on the pole (attachment). The scarf dropped down from the girls portraying the virus to the girls on the floor portraying the cell represents the DNA (DNA entry). The girls that then come into the routine is representing the virus duplicating and the floor work of the cell on the floor is its slow demise (synthesis and assembly). Finally, the virus bursts through the cell (release), ending the cycle.

https://www.youtube.com/watch?v=ZMn5DJOr9-k&list=PLIYwFhjuQVccevadgAxsl4UgMycMGyu6l

Art Project

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I designed this fabric on my computer and uploaded it to a website to have it custom printed. Microbes were the inspiration for the design. I was inspired by green algae cells and how different and complex they can be. Using the fabric, I created pillows that mimic the shape of some green algae cells. They are connected by Velcro in emulate the shape and design of green algae cells in chains.

What Kind of Life Wood You See in Fairbanks?

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This is my abstract interpretation of the decomposition of spruce and the interacting systems in a subarctic climate such as Fairbanks, with a focus on the fungi you may find on a piece of decomposing (as well as  living) spruce (left to right: lichens, slime mold, and turkey tails). The array of color in the background serves as a reminder of the diversity of the interacting systems, both biotic and abiotic. The plants on this piece represent the living features in an  healthy ecosystem, such as the different plants, animals, fungi, bacteria, archaea, and viruses that  inhabit that ecosystem. The soil on this  piece  is in reference  to  nutrient cycling within a system of decomposition, as well as the soil the wood will ultimately become part of and that many decomposing microbes  inhabit.    The symmetry and mixture of media serve to show the balance between the biotic/abiotic factors in a healthy subarctic habitat.

I chose this as my project because the there are so many components that go into the decomposition [of wood] that I had previously underestimated. Also, when you typically  envision   “nature” in Fairbanks, you may think: birch, spruce, squirrels, ravens, fireweed, etc., but the   great diversity of microbes within  the environment is typically less prominent. I felt it was important to highlight the interactions and results of microbes in a forest system.

I picked these fungi because as I was choosing a log from my wood pile to cut for this project, I saw 2/3 of these fungi on some of the logs. All of the plants in this piece are were found outside of my house.

**After painting this I found out slime molds(middle log)  are no longer categorized as fungi, but eukaryotes… so it’s really the interactions of fungi and eukaryotes.