Micrococcus Luteus

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

 

 

An exploration into a pharmacist’s microbes

Title:

An exploration into a pharmacist’s microbes

 

Description:

As an area of intrest, I isolated a bacteria from the contents of my father’s pocket, a practicing pharmacist as from the options available around me it presented itself as the most microbially interesting area available, both from having possible unusual outliers and from being socially relevant. This paper details my efforts in this endeavour and what I found (Spoiler: as can be seen in the picture for this post, I found a nice example of Staphylococcus epidermidis).

 

Google Drive Link:

https://drive.google.com/file/d/1PtbwWfIOt7I5WrKXzG5DTiWuih7B7LGw/view?usp=sharing

 

A2: Microbes in the News – Scientists Discover Nearly 200,000 Kinds of Ocean Viruses

Article:

https://www.quantamagazine.org/scientists-discover-nearly-200000-kinds-of-ocean-viruses-20190425/

Summary:

Researchers have assembled data from a global sampling expedition using genomic analysis and have increased the number of known oceanic viruses twelvefold.

Connections:

This is a story of people using the tools which we have been using to study the viruses we have been studying and improve the body of knowledge we have in this field.

Critical Analysis:

While microbiology has been studied in some manner since the early days of science the changes in the accuracy or our tools and the price of using them can dramatically change what we are able to accomplish. This article is just one example of how much more there is to learn in this field.

Question:

With a single study able to make such a large impact on the amount known, the question becomes how much more is there to know? I would not be at all surprised to see another twelvefold increase with the next study and another after that. If there is one thing that I have learned from this class it is that the microscopic world holds a multitude of secrets yet to be discovered.

Microbes in the News #3 – A Changing Ocean (Sage Robine)

Article:  Study: Much of the surface ocean will shift in color by end of 21st century

(https://news.mit.edu/2019/study-ocean-color-change-phytoplankton-climate-0204) –> MIT News, February 4, 2019

Summary:  Researchers at MIT have developed a model that simulates how the colours of the ocean may change over the next 100 years due to climate change. Their model looks at the colours of the ocean as seen from a satellite, where green hues indicate a greater concentration of algae and phytoplankton while dark blue hues indicate a lack of significant algae growth. Their model predicted that the subtropics will turn a deeper blue colour, indicating less phytoplankton growth and therefore less life in general. Meanwhile, the poles may turn a darker shade of green from increased algae blooms due to warming temperatures. These changes in levels of algal growth mean that entire food webs could be significantly altered by the end of the century, which would have significant impacts worldwide.

Connections:  This article is a good reflection of how microbial ecology can have a big impact on our world. Changes in microbial activity in the oceans over the next century could be big enough to be seen from space! And since life in the ocean is very dependent on levels of microbial growth (microbes make up the base of most ocean food webs) these changes could have dramatic impacts on all domains of life.

Critical Analysis:  I really enjoyed reading this article. It was easy to read and seemed to summarize the MIT study really well. I am somewhat cautious about this model, because I have not seen proof of its face and predictive validity, but I am sure if I read the entire peer-reviewed paper in depth it would prove to be a fairly accurate model for ocean colour change. Overall, this article was well-written, informative, accurate and easy to understand.

Question:  If the algal growth in our oceans changes as much as it is supposed to, how can we model these impacts on ocean food webs and what would those models show us?

Microbes in the News (#3)

Emily Werner

Title: Microbes in the human body swap genes, even across tissue boundaries: study

Summary: A team of researchers from the University of Illinois developed a method that helped to identify when HGT is taking place in the human biome. They concluded that microbes with similar DNA will readily carry out HGT with each other regardless of their location in the body. Microbes with similar DNA will swap genes with each other even if one is in the large intestine and the other is on the skin. They perform HGT more than with microbes in their same location with less similar DNA (i.e. mouth, GI tract, skin.)

 

The shortcomings of this method don’t help to determine the ancient HGT that occurred between species millions of years ago but they do help to determine the more recent gene transfers.

 

Connection: We’ve learned a lot about HGT in class and that this is a method that microbes use to transfer antibiotic resistance genes from one another. This article tells about how microbes in the human gut will do the same.

 

Critical Analysis: This article is general and ideal for anyone wanting to learn more about what is going on in research who don’t have a deep background in natural science. They discuss that this method they created for determining HGT is best for looking at the genes that were recently inherited, but they don’t specify that timeline. I think that since they specify ancient inheritance of genes, they should also do the same to give reference.  

 

Question: If antibiotic resistance is such a “scary’ thing today, how were we able to survive for millions of years before modern-day soaps and antibiotics?

In the simplified version, if antibiotics were critical to our survival, we should’ve gone extinct long ago why didn’t we?

 

Link: https://phys.org/news/2019-04-microbes-human-body-swap-genes.html

Civilization is Born – Matt Andrews

Title:

“Civilization is Born” by Matt Andrews

Artists Statement:

A virus is in some respects a pure expression of information made manifest. It is like a book, it exists and anything that happens because of what it contains is dependent on the life which reads it.

The only difference between humanity now and humanity a hundred thousand years ago is the information, be it science, culture, or other knowledge which we have accumulated and spread among ourselves.

In this piece I have drawn inspiration from viruses, to that end I havbe created a scene in a 3D program where I modeled the delivery protein structure of a virus bacteriophage including the icosohedral head, tail, base plate, and tail fibers, all of which I have scaled up to a size closer to that of a human, the DNA has been replaced with a book.

 

Some larger renders of this scene:

Civilization is Born

Biofilm Formation

Biofilm formation

Biofilms allow microbes to live close to one another, participate in genetic exchange, and take advantage of a nutrient rich area. They provide protection against predators, toxins, and are resistant to many antimicrobial chemicals and antibiotics that the cell alone are not.

Cells have the ability of quorum sensing, which allow them to sense the presence of others and communicate with one another.   Through quorum sensing they can stop producing flagella, make more polysaccharide, and increase the efficiency of nutrient transportation.

Common locations of biofilms are teeth, intestines, rocks, soil, plants, medical implants, and pipes used for things like oil and water.

Here I have depicted cells using flagella to swim.   Here they have come together and using their multitude of sticky fimbria they are able to adhere to a surface and produce a polysaccharide biofilm.   I have also depicted sells using their pili to exchange genetic information. The holes in the biofilm are water channels which allow for water and nutrients to flow through increasing the amount of nutrients that is able to be taken in.

Some common bacteria that produce biofilms are Streptococcus sanguinis with the oral cavity, Neisseria gonorrhoeae, Salmonella typhinurium.

Art Project – Zenteria

Title:  Zenteria

Medium: Zentangle using black markers on paper

My piece is a type of art called zentangling. It uses fine point black markers to draw small details to create a picture. I drew a facultative aerobic gram-positive bacterium with two flagella that I named Zenteria. I also incorporated other microbes into my overall drawing. The cell wall is made of cyanobacteria, each lipid in the plasma membrane is a bacterium with two flagella, and the transmembrane/membrane associated proteins are made of viruses. I drew ebola viruses, bacteriophages, rhinoviruses, herpesviruses, rabies viruses and hepatitis B to represent the different proteins you might find in the plasma membrane. For the flagella, I drew fungi with the fruiting bodies at the base of the flagellum (embedded in the cell) and the mycelia extending down the length of the flagellum. Within the mycelia you can see the nuclei of the cells. In the interior of the cell, you can see the chromosome attached to DNA organizing proteins as well as three additional plasmids. I have also drawn some inclusions in the cell. The large circles are poly- β -hydroxybutyrate (PHB) inclusions which are lipids that are stored by many bacteria for use as a carbon source under anaerobic conditions. The smaller granules that are clumped together are sulfur granules which are used by bacteria found in hydrogen sulphide rich environments.  Zenteria  are bacteria that can metabolize both carbon compounds and sulphur compounds.  The small circles scattered throughout the cell are ribosomes, and the large black circle is a group of photoreceptor proteins which allows the bacterium to be light sensitive (needed for photosynthesis using sulphur).

Of course, my representation of a bacterium is not 100% accurate, but I really enjoyed drawing the small details and incorporating other microbes into the overall picture.

A2: Microbes in the News(#3)

Ebola Toll Tops 700 in DR Congo

Image result for Ebola in DR Congo

Summary
Since last August, the second deadliest Ebola outbreak in history (the first being the the epidemic of 2014 that resulted in the deaths of over 10,000 people in Africa and Europe) has pervaded the Democratic Republic of the Congo. On April 6, the Congolese government confirmed that the deaths due to the most recent outbreak has surpassed 700. Furthermore, 100 of those deaths occurred within the first week of April.

In an attempt to control the spread of the disease, government officials have began vaccinating their citizens on the grand scale. With over 95,000 receiving the newly-developed vaccines by Merck to prevent the spread of the disease, a bright hope looms over the people of Congo.

All is not well for the government of Congo on the contrary; armed insurgency from Guerilla groups as well as rural communities’ resistance to receive medical treatment and vaccines have posed a serious risk to the exacerbated spread of the disease. In fact, because of public distrust for the government, more than a quarter of the people living within the cities of Beni and Butembo believe that Ebola doesn’t even exist!

Connection
In class, we learned a lot about pathogenicity, virulence, and even immunity.

Ebola is a pathogen with a high level of virulence. With such a high death rate from previous outbreak data, there is a high priority for such a population like that within the DR Congo to become vaccinated.
Furthermore, the concepts discussed in this article regarding mass vaccinations contributes to the idea of heard immunity. Since the government is stepping in to provide its people with the first ever effective vaccines for the disease, it is in the best interest to vaccinate as many people as possible against the virus for the sake of providing herd immunity and benefiting those who haven’t received the vaccine.

Critical Analysis
While Ebola may be contained within the northern region of DR Congo, it’s only a matter of time before it spreads elsewhere. Even more so, an effective vaccine has been developed for the virus and people within a country affected by multiple outbreaks of the disease are not even interested in what could be a solution to their future Ebola problems.

Although most resentment towards vaccination is stemmed towards distrust of the government of Congo, people should also be informed of the effects of herd immunity and how it could not only protect the people and their loved ones against the spread of diseases not limited to Ebola but potentially other harmful pathogens as well.

Question
Now that there is an effective vaccine against Ebola, should the government of Congo mandate that its people receive it?

Article can be found here.

A2: Microbes in the News (#2)

How electricity-eating microbes use electrons to fix carbon dioxide (Science Daily)

Find the article  here.

Summary:   The bacterium, Rhodopseudomonas palustris, has been identified to have the ability to metabolize electricity. It transfers electrons to fix CO2 to fuel its growth. Essentially, it enjoys feasting on rust and uses the electrons in a process called extracellular electron uptake. The research team at Washington University are using this knowledge to understand the microbe’s role in carbon cycling and has helped connect some unknown areas of basic concepts.

An understanding of how these microbes store the electrons could potentially lead to the production of alternative biofuels.

Connection: The electron tower helped to visualize which compounds are metabolized by a microbe in question. We’ve also talked about the use of microbes in our everyday lives (probiotics, waste water treatment, immunizations, etc.), and could potentially lead to an alternative energy source if researchers discover the mechanisms to harness this microbe for bioplastics or biofuel (as mentioned in the article).

Critical Analysis: This could potentially lead to a great alternative source for fuels in the future.   It’s in its exploratory stages currently and much more about specific mechanisms needs to be learned before researchers are ready to turn it into biofuel.

The draw back of this type of approach poses the concern about how this application will alter the microbial world around us. Other fuels accumulate in the atmosphere so how will the accumulation of an organism affect our environment?

 

Question: How could the artificial abundance of this microbe affect the ecosystems around it?