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

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.

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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.

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