Isolation and Identification of a Microbial Species from a College Dorm Dryer Vent
Introduction
Microbes are frighteningly fantastic creatures. They can grow practically anywhere and can do almost anything given enough time. Microbes can even grow in dry, hot, low-nutrient environments such as on a dryer vent. Laundry rooms are generally a place where things go to be cleaned, however the vents in a laundry room often get ignored and can become particularly dusty which provides an environment for microbes to grow. A previous study done by Barberán et al found that there is a lot of variety in microbial communities living on dust and since dryer vents accumulate a lot of dust I felt as though it would give a very diverse community of microbes that I could isolate from. This is why I set out to isolate, characterize, and identify a species of microbe that can grow on these vents.
I inoculated a culture of mixed microbes from the vents behind a dryer machine in a college dorm and over time created a pure culture of a single microbial species and performed various tests to help identify the species I isolated. I assumed that the microbial species living on a dryer vent would be interesting to isolate because it would need various adaptations to be able to grow on metal that frequently changes temperatures where there is low nutrient concentrations. I believed that the isolate would have been able to form endospores as it would be able to solve the issues with living in the inhospitable environment where it had been taken from.
I predicted my isolate would be aerobic to some extent and must have some mechanisms to cope with the relatively extreme environment that is a dryer vent. The microbe isolated must also cope with periods of time where it is cold (~5 °C after a period in which the dryer is left unused) and be able to withstand living for long periods of time without water since it lives where these coping mechanisms are a necessity.
Methods
The initial mixed culture was collected using a sterile, moist swab on the vent behind a dryer and streaking it over a tryptic soy agar plate. After swabbing the plate was left sealed in a dark, warm environment for a week before being brought to the lab. This was done because I could not culture the plate in a moist area and dark warm locations are fairly good for microbial growth. In the lab the microbial colony was chosen and isolated using the streaking method multiple times over two weeks. By streaking many times I managed to isolate the microbe and had a pure culture. When not in use, cultures were kept growing in an incubator kept at 37 °C or in a refrigerator after they had been grown for a few days.
After isolating the microbe I performed a Gram stain of the microbe since it is easier to classify the microbe based on whether it is Gram-positive or Gram-negative. A fresh culture of the isolate was then used for a DNA extraction. The kit used was the PowerSoil DNA kit manufactured by MoBio as it functions well for pure cultures (Lab 5 Handout). The DNA was then sent out and sequenced using the Illumina MiSeq DNA sequencer. I then performed 4 physiological tests on the isolate which consisted of a fluid thioglycolate test to determine oxygen class, an oxidase test to determine the presence/absence of cytochrome c oxidase, a catalase test to test for presence/absence of catalase for use in protection against reactive oxygen species, and an API 20E test strip that is a miniature test for 21 different physiological processes that works well for Gram-negative microbes. These tests were done to give me insight into the ability of the microbe to cope with various different situations, further narrowing the mystery that is the unknown isolate.
When I received the isolate’s sequence data I ran it through SPAdes genome assembler to assemble the genome of my isolate, Kraken Metagenomics which can be used to identify my isolate to the species or genus level, and Prokka genome annotation which is used to annotate the genes of my isolate and determine their function. The isolate was further tested using antibiotic discs on a fresh culture of the isolate spread over new plates. This was done to identify whether the isolate had any adaptations suited for common antibiotics.
Results
Performing a Gram stain revealed that the isolate is Gram positive. The isolate formed shiny, yellow, large colonies that smelled pretty foul. The isolate tested positive for catalase and was shown to be an obligate aerobe in a fluid thioglycolate test. In the API 20E test strip the isolate tested positive for oxidase only.
SPAdes genome assembler gave 337 contigs >=1000 bp with the largest contig of 47421, a total length of 2409415 and a GC % of 73.02%. This means there was an ample amount of contigs to be read by the next tests and that most of the genome of the isolate consisted of cytosine and guanine. Kraken metagenomics classified 99014 reads which was 82.26% of the total reads. Of the total reads, 81.23% were classified to species level and of the reads classified to species level 98.75% were classified as Micrococcus luteus. The results of Kraken also gave Figure 2 which identifies M. luteus as 80.21% of the analyzed reads. Prokka genome annotation gave 2177 coding genes for the isolate.
The results of antimicrobial testing revealed that the isolate was susceptible to Tetracycline, Tobramycin,Cefazolin, Trimethoprim, Amikacin, Gentamicin,Vancomycin, and Cefoperazone.
Fig 1. Colonies of the isolate after ~108 hours upper left portion of the large colony is how most fresh colonies looked.
Fig 2. Krona classification chart from Kraken metagenomics.
Discussion
Results from genome sequencing (Figure 2) alongside all the physiological tests performed, has lead me to believe that the isolate is indeed Micrococcus luteus. Given that the isolate had an 81.23% match to M. luteus with 18% of the DNA being unclassified, it is most likely that the isolate I found was M. luteus. The isolate also tested Gram positive, and as M. luteus is Gram-positive the results support my claim. The isolate also tested as an obligate aerobe, which is yet another reason why the isolate is likely M. luteus (Woodward and Douglas 1991).
The identity of my isolate is congruent with where I collected my sample and the literature supports how it existed in the environment of a dryer vent. M. luteus is known to live in dusty environments and has mechanisms to survive extreme changes in the environment that would come with living on a dryer vent (Kaprylants and Douglas 1993). Unlike most other bacteria M. luteus does not form spores, but instead can become dormant and return to normal when conditions are favorable (Greenblatt et al 2004). This adaptation would provide a way for the isolate to have lived with the changes that come from turning on the dryer and heating up the otherwise cool vent. The isolate would also have been fine without having methods to deal with chemicals as the vent behind the dryer is not normally cleaned and spends long periods of time without being exposed to chemicals that could kill M. luteus. As M. luteus, the isolate would be able to survive extended periods without food and various temperature changes that come from living on a dryer vent all while avoiding the need to survive harmful cleaning chemicals.
That being said, the isolate tested positive for catalase which may mean that even though the isolate would survive all aforementioned conditions the isolate still needed the catalase gene to prevent reactive oxygen species from harming it. That would make sense as reactive oxygen species would likely interfere with the survival of M. luteus as the oxygen species would likely interfere with cell structure and function if there was no catalase gene. This means that the catalase gene could be necessary for the extended dormancy period of M. luteus or that the isolate was exposed to enough reactive oxygen species existing on a dryer vent that catalase was an important gene to have. The isolate was also susceptible to the antibiotics I tested on it, which makes sense because the isolate lived in an environment that was likely free of antibiotics and would have no use for antibiotic resistance genes.
Works Cited
Barberán, Albert, et al. “The ecology of microscopic life in household dust.” Proc. R. Soc. B. Vol. 282. No. 1814. The Royal Society, 2015.
Greenblatt, C. L., et al. “Micrococcus luteus-survival in amber.” Microbial ecology 48.1 (2004): 120-127.
Kaprelyants, Arseny S., and Douglas B. Kell. “Dormancy in stationary- phase cultures of Micrococcus luteus: flow cytometric analysis of starvation and resuscitation.” Applied and Environmental Microbiology 59.10 (1993): 3187-3196.
Woodward, Andrew M., and Douglas B. Kell. “On the relationship between the nonlinear dielectric properties and respiratory activity of the obligately aerobic bacterium Micrococcus luteus.” Journal of Electroanalytical Chemistry and Interfacial Electrochemistry 321.3 (1991): 423-439.
