ahhoveid Author

Introduction

Microorganisms consist of a single cell or cell cluster, and can also include viruses, which are considered to not be cellular (Madigan et al. 2015). The number of microorganisms in the world is too enormous to count or even comprehend, but if we look at a smaller sample, studying these microbes becomes somewhat easier. The sample in this study was taken from the mouth of a dog. It is known that a wide variety of microbes inhabit the mouth of both humans and dogs, but in a study done by Elliot et al. (2005), they found a significant difference in the cultivable oral microbes found in human and dog mouths. They found that the genera most frequently isolated from dog’s saliva were Actinomyces, Streptococcus, and Granulicatella, and the genera most frequently isolated from plaque were Porphyromonas, Actinomyces, and Neisseria (Elliot et al. 2005).

Micrococcus luteus has one of the smallest genomes of free-living actinobacteria sequenced to date (Young et al. 2010). It is a Gram-positive coccus shaped bacterium that belongs to the family Micrococcaceae, it is commonly found on human and animal skin as well as in water and soil. Because M. luteus is a part of the bacteria flora of humans it is not thought of as a pathogenic bacterium, but it is an opportunistic pathogen. In some rare cases, it has shown to give infections in immunocompromised patients (Smith et al. 1999). The genome of micrococcus lutes has shown many similarities with Kocuria rhizophila a closely related organism, and of late the ATCC 9341 strain known as M. luteus was reclassified as K. rhizophila (Tang et al. 2003).

In this experiment, I set out to isolate, characterize and identify a bacterium from an environmental sample. The sample was taken from a dog’s mouth and through a series of physiological test, genome sequencing, and antibiotic testing, characterization of the bacterial sample to the species level was achieved.

Methods

In order to identify our environmental sample, I started out with collecting my isolate from a dog’s mouth and placing it on TSA agar (Trypticase soy agar). The plate was inoculated at room temp, without light for five days until the first quadstreak was performed. After every quadstreak the samples were incubated at 37^oC for 2-3 days, until the next quadstreak was performed, and then placed in a refrigerator to inhibit growth. In order to try and isolate the bacterial strain a total of five quadstreaks were performed. After the bacterium was isolated it was Gram stained (Lab 4, Gram staining protocol) in order to classify the bacterium and to determine if the culture was pure. Following the Gram staining, genomic DNA was extracted from the latest quadstreak, and sent to sequencing. In order to extract the DNA from the sample we used the PowerSoil DNA Isolation Kit (Lab 5, DNA extraction protocol), and the samples were sequenced in the UAF Core Lab on the MiSeq Illumina Sequencer. In order to analyze our genome sequence datasets, I used BaseSpace Ilumina for the genomic assembly I used the SPAdes genome assembler, to determine taxonomic assignment we used Kraken metagenomics, and to look at the functional gene annotation I used Prokka genome annotation (Lab 7, Bioinformatics protocol).

In order to further characterize my sample several physiological tests (Lab 6, physiological test protocol) were performed. A fluid thioglycolate test in order to determine the oxygen class of the bacterium, an oxidase test to determine if the strain had cytochrome C oxidase, a catalase test to see if the strain had the enzyme catalase, and a API 20 E test strip was used to look at 21 different physiological processes. The API 20 E test strip was incubated for 3 days in 37^oC, while the test tubes from the fluid thioglycolate test were stored I room temp for 3 days.

The last procedure that was performed was an antibiotic disc diffusion test (Lab 9, Disk diffusion test protocol), to assess the susceptibility or resistance of our isolate to a variety of different antibiotics. Eight different antibiotics were tested: oxacillin, gentamicin, piperacillin, amikacin, clindamycin, trimethoprim, cefoperazone, and vancomycin.

Results
Most of the data were collected and analyzed over a span of several weeks. DNA analysis of the isolate gave 98.86 % confidence to the species level for Micrococcus luteus, and only 19% of the sample was unclassified (Figure 1). All the analysis performed by BaseSpace showed results that were either equal to or above the guideline.

Figure 1. Results from the genomic sequencing using Kraken Metagenomics and Krona, in BaseSpace Illumina.

 

The identification test performed on the isolate are summarized in table 1.   The isolate tested positive for both the catalase test and the oxidase test, indicating that the isolate contained the enzyme catalase and cytochrome c oxidase. The gram stain came out red, but because both rods and cocci were observed, the culture was considered to not yet be pure, and more isolation attempts were needed. From the 8 different antibiotics tested, the isolate was susceptible to 6 of them, it was resistant to oxacillin and intermediate resistant to trimethoprim. The zone measurement for oxacillin was 5 mm while the zone measurement for trimethoprim was 13 mm. The other antibiotics tested were gentamicin, piperacillin, amikacin, clindamycin, cefoperazone, and vancomycin.

Table 1. Results for various identification test for the isolate.

Identification test	Result
Gram Staining	Mixed culture (red)
Catalase test	Positive
Oxidase test	Positive
Fluid thioglycollate test	Facultative
Antibiotic resistance: Disk diffusion test	Resistant to oxacillin and intermediate resistance to trimethoprim.
API 20 E test strip	See Figure 2

From the last physiological test done, the API 20 E (Figure 2), 7 of the 23 tests performed were positive. The positive test was the LDC, URE, TDA, GEL, SOR, OX, and NO₂ tests (Figure 2).

Figure 2. Results from the API 20 E test strip

Discussion

M. luteus is described as the type species of the genus Micrococcus (Stackebrandt et. al. 1995), and is commonly found on human skin, and on animals, it can also be found in the mouth, and in the upper respiratory tract of animals and humans. The bacterium can likewise inhabit many other areas in the environment, like water, dust, and soil (Kocur et al. 2006).

In relation to the literature, the physiological test results were mostly consistent with information found. Nevertheless, there was one inconsistency found with the fluid thioglycollate test. I observed the bacterium to be facultative, although literature describes the bacteria as an obligate aerobe (Kocur et al. 2006). This may be due to human error or to the sample being impure.

The genus Micrococcus was first described more than a hundred years ago, but since then the description has been revised several times (Stackebrandt et. al. 1995). Today it is clear that this genus of bacteria is gram-positive, cocci shaped and catalase positive (Stackebrandt et. al. 1995). During our research, I found our isolate to be a mixed culture, and not gram positive as literature states. When observing the isolate under the microscope after gram staining it appeared red, with both rods and cocci present. Several isolation attempts were done, but the two bacteria observed seemed to live in symbiosis which complicated the isolation process. However, isolation was achieved after several quadrant streaks.

Numerous interesting studies have been done on the genus Micrococcus and their traits. Dib et al. (2013) discussed how genes present on the plasmids of Micrococcus bacteria can give advantageous features to their respective hosts like antibiotic and heavy metal resistances, the ability to degrade cholesterol, and osmotolerance. A study conducted by Greenblatt et al. (2004) looked at survival of Micrococcus in extreme environments. They found that M. luteus and other closely related cocci that are non-spore-forming seem well suited to extreme environments, all due to special individual factors (Greenblatt et al. 2013). Research has been done on M. luteus superior ability to absorb radiation through pigments that absorb long-wave UV radiation, between 350-475 nanometers, and the researchers hope to implement this into sunscreen and other cosmetic products (SINTEF 2013).

For further research, it would be interesting to look more closely at some of the traits that make M. luteus so good at surviving in extreme conditions, and absorb such long wavelengths of radiation. I only looked at some physiological traits of the bacterium, it would be interesting to see how these aid the bacterium in extreme settings, like the ones previously mentioned.

Our research looked at bacteria within our environment, and our sample was taken from a dog’s mouth where we found the bacterium M. luteus. We conducted several physiological and antibiotic tests, along with genome sequencing in order to understand and characterize the bacterium. Most of the test that we conducted were consistent with literature, but there were some variances within gram staining and the fluid thioglycollate test. However, the tests gave us important information about the bacterium and a better understanding of the environment it lives in.

 

Literature Cited

Dib, J., Liebl, W., Wagenknecht, M., Farías, M., & Meinhardt, F. (2013). Extrachromosomal genetic elements in Micrococcus. Applied Microbiology & Biotechnology, 97(1), 63-75.

Elliott, D. R., Wilson, M., Buckley, C. M., & Spratt, D. A. (2005). Cultivable Oral Microbiota of Domestic Dogs. Journal of Clinical Microbiology, 43(11), 5470-5476.

Greenblatt, C. L., Baum, I., Klein, B. Y., Nachshon, S., Koltunov, V., & Carlo, R. J. (2004). Micrococcus luteus – Survival in Amber. Microbial Ecology, 48(1), 120-127.

Kocur, M., Klosss, W. E., & Schliefer, K. (2006). The Genus Micrococcus. Prokaryotes, 3, 961-971.

Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., & Stahl, D. A. (2015). Brock biology of microorganisms (Fourteenth edition.). Boston: Pearson.

SINTEF. (2013). Super sunscreen from fjord bacteria. ScienceDaily. Retrieved April 17, 2017 from www.sciencedaily.com/releases/2013/08/130806091556.htm

Smith, K. J., Neafie, R., Yeager, J., & Skelton, H. G. (1999). Micrococcus folliculitis in HIV-1 disease. British Journal of Dermatology, 141(3), 558-561.

Stackebrandt, E., Koch, C., Gvozdiak, O., & Schumann, P. (1995). Taxonomic Dissection of the Genus Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov., and Micrococcus Cohn 1872 gen. emend. International Journal of Systematic Bacteriology, 45(4), 682-692.

Tang, J. S., & Gillevet, P. M. (2003). Reclassification of ATCC 9341 from Micrococcus luteus to Kocuria rhizophila. International Journal Of Systematic And Evolutionary Microbiology, 53(4), 995-997.

Young, M., Artsatbanov, V., Beller, H. R., Chandra, G., Chater, K. F., Dover, L. G., … Greenblatt, C. L. (2010). Genome Sequence of the Fleming Strain of Micrococcus luteus, a Simple Free-Living Actinobacterium . Journal of Bacteriology, 192(3), 841—860.