Micrococcus Luteus

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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

 

 

A2: Microbes in the News Number 3

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“Scientists discover how ‘superbug’ E. coli clones take over human gut’

Phys.org

by: University of Birmingham (no author listed)

April 23rd

Link: https://phys.org/news/2019-04-scientists-superbug-coli-clones-human.html

Summary:

Research was done on a strain of E.Coli that is resistant to many drugs, and why it has become a source of infections related to the bacteria. The amount of E.Coli cases has risen 27% between 2012-2013 and 2017-2018. The researchers said that the reason it has not become completely dominant is because if there is only one strain of E.Coli and something happened to that strain then E.Coli would disappear. They said Negative frequency dependency selection keeps balance in E.Coli populations so this does not occur. It also mentioned that this strain of bacteria had a lot more variability genetically in genes that help colonize the gut than other strains.

Connections:

This connects to what we learned in class because it talks about drug resistance in bacterial species.

Critical Analysis:

I think that it is interesting that the amount of E.Coli cases has risen as much as it has. I think this article is credible as it cites from the authors of the paper it talks about. I think this article did an okay job at explaining to the public, there were a couple of terms they could have described further to make it make more sense such as negative frequency dependency selection.

Question:

How is it possible that one specific strain could become so much more dominant than other strains of E.Coli?

“Harry Potter and the Lesson in Latin”

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Savanna Ratky:

I created a short comic using crayons and pens to draw on paper. I made it Harry Potter themed, as that is a series that I enjoy, and that I thought most readers would relate to. I specifically made this about the iconic scene where Hermione, Ron, and Harry are trying out a new spell and Ron mispronounces it as LeviosA. Hermione then corrects him by saying “it’s LeviOsa not LeviosA’. The concept I chose to make the work about was specifically the use of “bacterium’ and “bacteria’, which are commonly missued by people. Bacterium is the singular version of bacteria.

Third Microbes in the News

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— Article and link: A Blazing Hot Coal Shows How Microbes Can Spring to Life   Source: Wired 04/21/19   Link: https://www.wired.com/story/a-blazing-hot-coal-seam-shows-how-microbes-can-spring-to-life/

— Summary: In Centralia many single-celled microbes live in the soil that is on top of the underground mine fire in the coal seam. Due to the fire it was initially thought nothing may have lived, but instead there are many microbes. The same amount of microbes have been found to be living in very hot areas including various thermophiles that microbes that live at geothermal hot springs.

— Connections: In class we have briefly studied thermophiles which are heat loving bacteria. We have also looked at the various species of microbes that live in soil, and I find it interesting how natural disasters can affect what microbes are in the soil. We studied factors that can cause microbes to go dormant, which is what can happen during a fire since there is no activity on the soil.

— Critical analysis: I thought this article was interesting because it involves microbe activity when a natural disaster occurs and using spores to regrow genomes. The article was well written and contained a large amount of detailed scientific information. It could be rather difficult for the general public to read the article and understand it all though, because of all the technical terms it contains.

— Question: What organisms do you think would be likely to live through a fire or other extreme natural disaster? Would they become dormant?

 

 

Bacteria Could be the Key to Biological ‘Wires’

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Article:  “Electricity-conducting bacteria yield secret to tiny batteries, big medical advances”

https://www.sciencedaily.com/releases/2019/04/190404143747.htm

Source:  Science Daily

Date Published:  April 4, 2019

Summary:  Scientists discovered that Geobacter sulfurreducens is conducting electricity through tiny fibers made of protein that “… surround a core of metal-containing molecules.”   It was previously thought that they were conducting electricity through pili, but new technology has made it possible to examine the smaller structures within the bacteria at a higher resolution.   These bacteria live in environments without oxygen, and they get rid of excess electrons almost as a way of ‘breathing.’   It is thought that this way of conducting electricity could eventually be harnessed and used in medical devices associated with human tissue.

Connections:  This article doesn’t relate exactly to the topics we have covered in class, but it does highlight how some bacteria have unique ways of processing molecules and electrons.   It mentions that these bacteria can also be used to clean up radioactive waste, which we have briefly talked about in class.

Critical Analysis:  I was not aware that bacteria could even conduct electricity, so the fact that they can do that, and people were able to figure out how they are doing it, is pretty cool.   It is also really interesting that the same technology the scientists used for this discovery was used to find a virus that was surviving in boiling acid.   The information seems scientifically accurate since they included quotes from the scientists that performed the research, and they included the citation for the article they were explaining.   I think the author did a good job of simplifying the material as best as they could so that any person could understand it.   It would be easier to understand if you were reading it with some background in biology, but it is not completely necessary to understand the basics of what they were talking about.

Question:  Will these proteins still function the same way if they are taken out of the bacteria and used for medical purposes?   Will the entire bacterium have to be used in the medical devices in order for them to work?

In India, a Renewed Fight Against Leprosy – post #3

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Article & Link: In India, a Renewed Fight Against Leprosy (The New York Times; April 17, 2019)

Summary: Leprosy, which is caused by Mycobacterium leprae has been eliminated throughout most of the world. However, it is still a huge problem in India, which currently has 60% of all leprosy cases in the world. This is largely due to the stigma against people with leprosy in India, who are not allowed to have jobs or even be in public if they have the disease. This means people with leprosy won’t seek help if they have leprosy, which causes nerve and muscle damage if it isn’t treated. However, there are some doctors in India who are trying to educate people better on leprosy to treat as many people as possible and hopefully prevent its further spread in India.

 

Connections: We were recently learning about pathogens in class. Leprosy, or Mycobacterium leprae causes a bacterial disease which affects nerves and muscles. The article mentioned that the bacterium is not culturable which prevents understanding of the disease. This explains why scientists don’t yet understand how the disease spreads. It sometimes spreads in a seemingly random way, infecting some people but not others. Furthermore, people can carry the disease for decades, spreading it to other people without realizing they are carriers.

 

Critical Analysis: I found the discussion about non-culturable pathogenic bacteria very interesting, because most of the examples we learned about in class were discovered and treated through culturing. It is also interesting to see how stigma and misinformation prevents treatable diseases from being eliminated. I learned a lot about how India’s culture regarding leprosy by reading this article. Many people hide it, potentially risking the health of those around them. Others go to leprosy colonies, or secluded communities, surviving by begging and helping each other. From what I could tell this article appeared to be scientifically accurate and not misleading. I feel like the writing is both accessible and informational to general audiences. It explains what leprosy is, the common symptoms, and why it’s such a big problem in India. I think it also did a good job explaining why leprosy is hard to study (mentioning its non-culturable, explaining it can’t be found by blood tests, and talking about how people can be carriers for decades) to people who may not have a lot of previous microbiology background.

 

Question: How should/do doctors and scientists study non-culturable bacterial species? Especially those species that are pathogenic?

A2: Microbes in the News – Post 2

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Title: “A Teenager Was Diagnosed With Schizophrenia – but it Turned Out to Be an Infection From HIs Cat”

By” Christina Oehlea

Healthy Living Newsletter

Link: https://www.health.com/condition/infectious-diseases/cat-scratch-schizophrenia

Summary:

A young Midwestern boy spent two years in and out of the hospital due to a bacterial infection.  The boy was first misdiagnosed with schizophrenia by two physicians, but later correctly diagnosed and treated for neurobartonellosis, which caused him to have psychiatric symptoms such as depression and suicidal thoughts, among other symptoms. After an antibiotic treatment, the boy made a full recovery.

Connections:

The content of this article relate to our study of the human immune system and antibiotics.

Critical Analysis:

I think that the article was easy and short, which is good for accessibility, however, I think that it should have provided more information about the bacteria strain, which carry Bartonella clarridgeiae.  But overall, I think that it covered the bases for to provide readers with preventive and informative facts. I also think that the article was from a reliable source, because the author cited that the original case was published in The Journal of Central Nervous System Disease, which is a peer-revised journal publication.

Question:

How did cats become a vector for Bartonella clarridgeiae and do they infect other hosts?

A2: Microbes in the News (#2)

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Bacteria in probiotics can evolve in your gut and turn nasty, study shows  (The Independent)

Link: https://www.independent.co.uk/news/health/probiotic-bacteria-gut-health-ibs-bowels-a8840636.html

Summary:  This article talks about a study performed at the University of Washington in Missouri where a probiotic evolved to attack the protective coating of the intestine in the mice they tested. Unhealthy mice with low gut microbial diversity were more likely to develop an evolved strain of the  E. coli Nissle bacteria that was used in the probiotic they studied. According to the researchers, their findings have implications for the development of safer probiotics in the future.

Connections: This article is very relevant to the human microbiome section that we covered in class. It involves the gut microbiome and the ways it can be more or less healthy, and more or less diverse. I think the way microbes can change and evolve right under our noses is fascinating!

Critical analysis: This was certainly an interesting piece and the writing style flowed well. However, this study is only one of many and might mislead readers to think that all probiotics can “turn bad.’ It could also be confusing to the regular reader, because the wording of the article makes it seem like probiotics are drugs that can change inside your body. Of course we all know that probiotics are made up of living bacterial cells that are supposed to help enhance the diversity of your gut microbiome. It was also unclear whether the  E. coli strain always evolved in a negative direction or if it was simply more prone to evolve in an unhealthy gut microbiome (toward good or bad characteristics, we don’t know). Overall, this was a well-written article, but I think the writer conveyed what he wanted the readers to believe and not necessarily the actual truth of the study.

Question:  What were the exact parameters of the evolution of  E. coli Nissle observed in this study?

A2 Microbes in the News: Post 2

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Title: New technique pinpoints milestones in the evolution of bacteria.

Jennifer Chu, February 7, 2019.

MIT news

https://news.mit.edu/2019/determine-bacteria-evolution-age-0208

Summary: A new technique has come up that will allow scientists to better determine when various species of bacteria evolved. This stemmed from a published paper that determined some groups of soil bacteria developed the ability to break down chitin 450-350 mya. They believe that this evolutionary change was caused by the changes occurring in other species, that were evolving and leaving behind chitin in the soils. Gregory Fournier claimed that tracing similar genes could allow them to find out more about animal history. There is no fossil record, so scientists have been using a molecular clock to determine when mutations were occurring. They also claim that they can use other species with clearer fossil records to determine when evolution of certain traits passed to the species they are interested in because of phenomena such as horizontal gene transfer. The scientists were specifically interested in learning about chitinase, because it is seen in most bacterial groups and fungi (whom apparently have a good fossil record). They created trees showing the relationship between all the species they chose based on genetic mutations, then used the molecular clock technique to determine when the species with chitinase diverged.

Connections:

This connects to what we’ve learned about in class because it discusses horizontal gene transfer.

Critical analysis:

I think it is interesting that this is considered a new technique, I thought they had already been doing this kind of research, but maybe they had just not found a way to do it in bacteria. I think the article was scientifically accurate, based on the fact that is cites an actual published paper that was funded by NSF.  I think it was written well, it seems to be in a language that most people might be able to understand if they have some sort of previous knowledge or interests in science, however if they don’t they might be confused by some of the terms used within the article.

Question: Would this research about the evolutionary history of microbes possibly be helpful in further understanding the concept of horizontal gene transfer, and maybe be helpful in fields such related to infectious diseases, so they could trace when the negative genes were transferred?

Pumping May Alter the Microbes in Breast Milk

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Article: “Pumping may be linked to an altered microbial mix in breast milk” by Laura Sanders

https://www.sciencenews.org/blog/growth-curve/pumping-may-be-linked-altered-microbial-mix-breast-milk

Source:  Science News

Date Published:  April 1, 2019

Summary:  A study compared the microbial makeup of breast milk from mothers who do not pump at all to breast milk from mothers who pump.   Although the microbes in breast milk is fairly unique from person to person, the researchers found two main differences.   The milk from women who pumped had more bacteria that could cause infections under optimal conditions and fewer bifidobacteria, which are thought to be beneficial bacteria.   It is still unknown as to what impact these differences have on the children, if any, and the origin for these bacteria is also under debate.   Some believe the bacteria from the gut travel to the breast and get into the milk, while others think that babies’ oral bacteria affects the bacteria in the breast.   There is some evidence that ‘baby backwash’ might trigger infection-fighting proteins in milk when the baby is fed directly from the breast, so this would not occur in milk that is pumped.

Connections:  This is related to what we were talking about with the human microbiome and how it can be affected by many different factors.   The way a mother feeds her baby not only affects the microbiome of the baby but the microbiome of her breast and breast milk, as well.

Critical Analysis:  I did not know that the way a baby receives breast milk can change the type of bacteria that occur within the breast milk itself.   Since the milk is made within the mother, I thought that only factors within the mother would affect it, so it is really interesting that babies play such a large role in that microbiome.   The information appears to be scientifically accurate since the author included some quotes from one of the scientists who did the research and did not add much of her own opinion when talking about the information from the research.   It was written well to relay the information from the scientists to people who may not have any science background.   It was easy to understand and follow.

Question:  Once the mother is done breast feeding, do any of the microbes remain in the breast?   Do they get flushed out with the milk or die?   If they do remain, are they there for the rest of her life or will they eventually go away?