Introduction:

Microbes are too small to be seen by the naked eye; they can survive in conditions that many would think are unlivable like the anaerobic environment in the rumen of cows, hot springs, and cold Antarctic waters (What are microbes, 2010). Millions of microbes live both on and in the human body and can both make help us survive or make us sick, less than 1% of bacteria cause disease (What are microbes, 2010).

The nasal cavity microbiome primarily consists of the phyla Actinobacteria, Firmicutes and Proteobacteria (Bassis et al. 2014).  The microbiome of the nasal cavity can also change in response to environmental factors such as geographic location, and hygiene (Rawis et al. 2019).

Micrococcus luteus is found in lots of places including skin, soil, dust, water, air, mouth, mucosae, oropharynx, and upper respiratory tract of humans (Wikipedia, Micrococcus luteus, 2019). It is a gram positive, coccus shaped microbe, and contains catalase. This microbe forms large, round colonies. It can be easily be mistaken for staphylococci, as they are very similar morphologically and physiologically (Wikipedia, Staphlyococcus Aureus).

My goal in this experiment was to isolate, characterize and identify a bacterial colony that arose from a sample taken from my roommate’s nose. I hypothesized that it would be a bacteria commonly found in the nasal cavities and likely from the aforementioned phyla, so it would likely do best living in an aerobic, humid, and warm environment.

Methods:

I chose to sample bacteria from inside my roommate’s nose. To sample, I used sterile cotton swabs and streaked them on TSA plates. I kept the plate at room temperature for 7 days, and then selected a colony to purify using the pure culture streak plate method. I repeated this process three more times to further purify the colony. Once the culture was deemed pure enough, I inoculated a slant tube.

I performed many tests to find out the colony morphology and physiology. In order to determine physiological characteristics of the culture such as cell shape, arrangement, and whether it was gram positive or negative, which helps determine the cell wall type of the microbe, I performed a gram stain. I used an oxidase test strip and water to determine if cytochrome C oxidase was present, and performed a catalase test to determine if catalase was present. I also did a fluid thioglycolate test to determine the bacteria’s oxygen class. I grew my bacteria on an Eosin Methylene Blue (EMB) plate to see if it could ferment lactose and if it could grow with methylene blue which selects for gram negative bacteria. I also grew it in a MacConkey (MAC) plate to see if it could ferment lactose and if it could grow with both crystal violet and bile salts to further confirm if it was gram negative or positive. I used an API Strep test to determine more of the sugars the bacteria could ferment.

I grew my isolated in Tryptic Soy Broth (TSB) for a week to prepare for DNA extraction. I extracted the DNA using the PowerSoil DNA kit (manufactured by Qiagen) following manufacturer instructions. The sample was then sequenced using the Illumina MiSeq technology in UAF’s DNA Core Lab. I used the PATRIC software to perform a metagenome binning and to assign a taxonomy to the bacteria.

Results:

The colony took 16 days to be purified. The gram stain of this microbe showed that it is gram positive because it stained purple. This microbe is coccus shaped and forms in tetrads. The colony forms as a yellow, shiny round blob. The catalase and the oxidase tests came up negative, because the catalase test did not form bubbles, and the oxidase test did not see a color change. The oxidase test tests to see if the microbe contains cytochrome c oxidase. The catalase test tests to see if the microbe contains catalase. The fluid thioglycallate test showed that the bacteria was an obligate aerobe because the growth was concentrated at the top of the tube in the pink region. The MacConkey agar showed very little growth, and did not have a change in color, indicating that the microbe was gram positive and not a fermenter. The EMB agar showed no growth or change in color, also indicating the microbe was gram positive and a non-fermenter.

The API 20 Strep test I used came up with no conclusive results. This test had VP, HIP, ESC, PYRA, aGAL, bGUR, bGAL, PAL, LAP, ADH, RIB, ARA, MAN, SOR, LAC, TRE, INU, RAF, AMD, and GLYG tests. The PYRA, PAL, LAP, RIB, ARA, MAN, and TRE tests came up as positive.

The taxonomic assignment of this microbe was micrococcus luteus because it was the only bin that PATRIC gave. It had 27,372 contigs in assembly. It has multiple antibiotic resistance genes including dihydropteroate synthase, glycerophosphoryl diester phosphodiesterase, and SSU ribosomal proteins.

Figure 1. Krona chart of microbe shows bacterial classes thought to be present in the sample.

Figure 2. Kaiju webserver metagenome binning analysis chart. It shows that the sample contains bacteria from the Terrabacteria group. It is mostly Actinobacteria, but some Proteobacteria and Firmicules are in the sample as well.

The kaiju metagenome binning shows that the microbe sample is not completely pure (Figure 2). It shows that it is mostly Actinobacteria, with some firmicules ,and proteobacteria mixed in (Figure 2). This matches up with the PATRIC metagenome binning which also showed some impurities (Figure 1).

 

Discussion:

As the microbe is gram positive this means that it has a large peptidoglycan layer and lacks a lipopolysaccharide layer. The MacConkey agar is selective for gram-negative which is why my microbe didn’t show much growth on it, and because it didn’t change colors it means it didn’t ferment the lactose. The EMB plate is also selective for gram-negative bacteria which is probably why the bacteria didn’t grow on it. The oxygen class of the microbe, obligate aerobe, matches up with the predictions I had made about it because the bacteria was originally sourced in a nostril. Wikipedia also says that Micrococcus luteus is an obligate aerobe, backing up what my results show (2019).

The oxidase test results suggest that the microbe does not contain oxidase, despite what the metagenome binning test showed. The catalase test also indicated that the microbe does not have catalase, despite the metagenomic binning test suggesting it. These discrepancies could be due to human error, unpure culture, or an old agar plate. The API test strips’ lack of results suggests that the I used   the wrong test strip, I probably needed to use the Staph test instead of the Strep test, because the Strep test is for when Catalase is absent, but there could have been catalase present. The conflicting results of the metagenome binning and the catalase test influenced this mistake. I think based on all this information, that my microbe is in fact micrococcus luteus as suggested by the PATRIC metagenome binning test, and the krona (Figure 1).

In conclusion, some of my results were inconclusive and conflicting. This is likely either a cause of human error, unpure cultures, or not using agar plates that are fresh enough for the test. I think that this culture was mostly Micrococcus luteus based on the Kaiju and metagenome binning results. The oxygen class and the gram positiveness of the microbe also matches up with that of Micrococcus luteus. In future works with this microbe, I probably would want to purify the culture more and redo the tests.

 

References:

Bassis CM, AL Tang, VB Young, and MA Pynnonen (2014). The nasal cavity microbiota of healthy adults. Microbiome 2(27).

Rawis M, and AK Ellis (2019). The microbiome of the nose. Annals of Allergy, Asthma and Immunology 122(1):17-24.

(2010) What are microbes? Institute for Quality and Efficiency in Health Care.

Wikipedia contributors. (2019, March 14). Micrococcus luteus. In  Wikipedia, The Free Encyclopedia. Retrieved 06:20, April 16, 2019, from  https://en.wikipedia.org/w/index.php?title=Micrococcus_luteus&oldid=887698104

 

Wikipedia contributors. (2019, April 4). Staphylococcus aureus. In  Wikipedia, The Free Encyclopedia. Retrieved 22:17, April 16, 2019, from  https://en.wikipedia.org/w/index.php?title=Staphylococcus_aureus&oldid=890960280