Journal of Microbiology

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Isolation and characterization of Micrococcus luteus from Modern Technology

Introduction:

The abundance of microorganisms in our world cannot be overstated. They are everywhere, from the sulfur vents at the bottom of the oceans, to our skin, and to the inanimate objects we interact with on a daily basis. The variety of environments in which microbes survive and thrive demonstrates just how diverse they are in their metabolic capabilities. Microbial diversity can be beneficial for our environment and our health, although many microbes can also be pathogenic and have detrimental effects.

Our hands are especially effective at picking up and transmitting bacteria (Lax, 2017). What does this mean for bacterial transmission in an era of increased hands-on technology? Research is beginning to find that cell phones in particular are efficient vectors for transmitting pathogenic bacteria, as well as mechanisms for increasing antibiotic resistance (Loyola et al., 2016).

With this in mind, the main goal of this experiment was to isolate and determine one of the several types of microbes found on cell phones. Gram-staining, physiological tests, and genome sequencing were used to identify and characterize the isolate. Antibiotic susceptibility and resistance was also tested. The results from these tests determined the microbe isolated from the cell phone surface was Micrococcus luteus. M. luteus is typically a harmless bacteria commonly found on human skin (Brock et al., 2015).

Methods:

The sample was obtained using a sterile swab moistened with sterile water and swabbing the surface of a cellular device. The swab was wiped against a cell phone case and streaked on a tryptic soy agar (TSA) plate. The plate was sealed and left for several days at room temperature, with an additional day in an incubator at 37 °C before colonies formed. A single colony was selected to isolate and the streak plate method was used (Leigh, 2017a). Approximately three streak plates were done to obtain a pure isolate.

Once the colony was pure, observations regarding size, shape, morphology, and color were conducted, as well as a series of physiological tests. First, Gram staining was done to determine whether the isolated bacterial colony was Gram-positive or Gram-negative. Following this, a fluid thiogylcollate test was done to determine oxygen class, as well as oxidase and catalase tests, to determine if the strain had cytochrome c oxidase and the enzyme catalase, respectively. An API 20E test strip, which contains 21 miniature physiological tests including the nitrate reduction test, was conducted to determine a variety of physiological and metabolic properties (Leigh, 2017c). Additionally, antibiotic susceptibility was tested using the disk diffusion test (Leigh, 2017d).

In order to extract DNA from the bacteria, a fresh sample of the isolate was grown in liquid tryptic soy broth (TSB) culture and its genomic DNA was extracted using the PowerSoil DNA Isolation Kit (Leigh, 2017b). Extracted DNA was sent to the UAF Core Lab for genome sequencing using Illumina MiSeq technology (Leigh, 2017b). Sequence analysis was done using the BaseSpace and BLAST programs.

Results:

Once bacterium was isolated, it was noted that the pure culture was yellow and relatively flat. The isolate was determined to be Gram-positive, round (cocci) and clustered in groups of varying numbers, including tetrads. The fluid thioglycollate test was consistent with that of a facultative anaerobe, as there was growth throughout the tube, but slightly more so at the surface. The oxidase and catalase tests were both positive, meaning the isolate has cytochrome c oxidase and catalase enzymes, which indicate the presence of an aerobic electron transport chain and the ability to convert hydrogen peroxide into oxygen and water, respectively (Table 1). The API 20E test strip revealed the microbe’s ability to completely reduce nitrate, as well as other physiological traits (Table 1). This microbe also tested positive for arginine dihydrolase, which allows it to use the amino acid arginine as a carbon and energy source, in addition to testing positive for gelatinase, an enzyme that allows the organism to break down gelatin, a collagen derivative (Table 1). Those were the only positive test results. Given the microbe is Gram-positive, it is not surprising to see few positive reactions because the API 20e test strip is designed for Gram-negative Enterobacteriaceaea. A follow up API Staph strip was done, however most tests were negative, resulting in a 99.6% match with Micrococcus luteus.

Once sequencing was complete, it was discovered that of the 3,340 reads analyzed by BaseSpace, 2,666 were classified; of the classified reads, approximately 97.80% were identified as Micrococcus luteus (Fig. 2). When run through Basic Local Alignment Search Tool, or BLAST, the genome was determined to belong to Micrococcus luteus as well.

In analyzing antibiotic susceptibility, this microbe was found to be highly susceptible to a variety of broad-spectrum antibiotics, particularly Cefazolin, Tetracycline, Gentamycin, Amikacin, and Cefoperazone (Table 2). Cefazolin and Cefoperazone both interfere with cell wall synthesis by binding to penicillin binding proteins (PBP’s), while Tetracycline, Amikacin, and Gentamycin both interfere with translation by binding to the bacterial 30S subunit.

Figure 1. The Krona from BaseSpace is shaded based on the number of reads. Although BaseSpace could not classify 20% of the reads, BLAST was able to identify most of these unknowns belonging to Micrococcus luteus.

 

Test Enzyme/Reaction Result
Catalase test Catalase Positive
Oxidase test Cytochrome c oxidase Positive
Fluid thioglycollate test Oxygen class Facultative anaerobe
ADH Arginine Dihydrolase Positive
GEL Gelatinase Positive
GLU Fermentation/oxidation (glucose) Negative
Nitrate reduction NO2 production

 

Reduction to N2 gas

 Positive (complete denitrification)

Table 1. Physiological test results from general tests and API 20 E test strip. API 20 E results not shown were negative.

 

Antibiotic Isolate Zone of Inhibition (mm) Susceptibility Conclusion
Cefazolin 35 ≥18 Susceptible
Tetracycline 39 ≥23 Susceptible
Vancomycin 25 ≥17 Susceptible
Gentamycin 30 ≥15 Susceptible
Amikacin 30 ≥17 Susceptible
Trimethoprim 20 ≥16 Susceptible
Cefoperazone 32 ≥18 Susceptible
Tobramycin 22 ≥15 Susceptible

Table 2. Common and broad-spectrum antibiotics proved to be effective against this bacterial isolate.

Discussion:

The majority of morphological observations and physiological tests were consistent with Micrococcus luteus, except the thioglycollate test, which showed the isolate as a facultative anaerobe. Micrococcus luteus is actually an obligate aerobe though (Kooken et al., 2012). This conflicting result could be explained by poor technique, perhaps allowing oxygen into the anoxic zone of the tube. However, the yellow pigment, smooth morphology, tetrads and varying clusters, Gram-Positive, oxidase and catalase positive results that were observed throughout this study align well with established research (Kloos, 1974). Many Micrococci species, as well as Staphylococcus and Kocuria species share many of these traits though. One distinguishing physiological test is the glucose test, as many species within these genera are able to utilize glucose (Kooken, 2012). M. luteus is unable to use glucose as a sole carbon source, which was observed in the results of the API 20E test (Table 1). Compared to other Actinobacteria, M. luteus has one of the smallest genomes at approximately 2,501,097 base pairs and appears to lack the genes that would allow it to metabolize glucose (Young, 2010).

As noted previously, it had been difficult for scientists to distinguish Micrococcus species from those of Staphylococcus with only morphological observations (Kooken, 2012). While Micrococcus species are not usually considered pathogenic, it can be an opportunistic pathogen, albeit rarely, in the cases of immunocompromised patients such as those with HIV or acute lymphoblastic leukemia (Smith, 1999; Payne, 2003). It is for this reason physiological tests such as the API Staph test strips and genomic tests are important in the differentiation between the two bacteria, especially because as useful as the glucose test is, it does not always provide accurate results. Additionally, when considering that Staphylococcus aureus can be a potentially dangerous pathogen and has a much higher rate of antibiotic resistance, it is important to be sure that a strain is or is not M. luteus, which is generally less harmful, is shown to be susceptible to a broad range of antibiotics (Table 2), and is known throughout the scientific community as being a highly sensitive bacteria (Young, 2010).

While little DNA was successfully extracted from the sample, I can confidently identify my isolate as Micrococcus luteus, as the DNA sequences that could be analyzed had anywhere from 97% to 100% query cover with known M. luteus sequences. Closely related species, such as those of the genus Kocuria, which used to be part of the Micrococcus genus, were eliminated with the glucose test, as previously mentioned. As M. luteus is most commonly found on human skin and makes up a portion of the normal skin microflora, it makes sense that it can be isolated from a cell phone, since it comes into contact with human skin on a daily basis (Brock et al., 2015).

Works Cited

Kloos, W. E., Tornabene, T. G., & Schleifer, K. H. (1974). Isolation and Characterization of Micrococci From Human Skin, Including Two New Species: Micrococcus lylae and Micrococcus kristinae.  International Journal of Systematic Bacteriology,24(1), 79-101.

Kooken, J. M., Fox, K. F., & Fox, A. (2012). Characterization of Micrococcus strains isolated from indoor air.  Molecular and Cellular Probes,26(1), 1-5.

Lax, S. (2017, February 10). Our microbial interaction with built environments [Video file]. Retrieved from https://www.iab.uaf.edu/events/lsss.php?event_id=1829

Leigh, M.B. 2017-a. Pure culture techniques and isolating your bacterium. University of Alaska Fairbanks.

Leigh, M.B. 2017-b. Genomic DNA extraction of your isolate and creating BaseSpace account for genome sequence analysis. University of Alaska Fairbanks.

Leigh, M.B. 2017-c. Physiological testing of your isolate. University of Alaska Fairbanks.

Leigh, M.B. 2017-d. Antibiotic susceptibility of your isolate. University of Alaska Fairbanks.

Loyola, S., Gutierrez, L. R., Horna, G., Peterson, K., Agapito, J., Osada, J., . . . Tamariz, J. (2016). Extended-spectrum β-lactamase—producing Enterobacteriaceae in cell phones of health care workers from Peruvian pediatric and neonatal intensive care units.  American Journal of Infection Control,44(8), 910-916.

Payne, J. H., Welch, J. C., & Vora, A. J. (2003). Fatal Pulmonary Hemorrhage Associated With Micrococcal Infection in Two Children With Acute Lymphoblastic Leukemia.  Journal of Pediatric Hematology/Oncology,25(12), 969-974.

 Smith, Neafie, Yeager, & Skelton. (1999). Micrococcusfolliculitis in HIV-1 disease.  British Journal of Dermatology,141(3), 558-561.

Young, M., Artsatbanov, V., Beller, H. R., Chandra, G., Chater, K. F., Dover, L. G., . . . Greenblatt, C. L. (2009). Genome Sequence of the Fleming Strain of Micrococcus luteus, a Simple Free-Living Actinobacterium.  Journal of Bacteriology,192(3), 841-860.

“Big Bang Theory Theme ‘ Microbiology Parody

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The song I wrote for my art project was inspired by the Barenaked Ladies song from Big Bang Theory, one of my favorite TV comedies. I tried to incorporate a variety of material from throughout this semester, from before the atmosphere had oxygen to the power of our tears. If you want to listen to the actual song that inspired this work, here’s the link:  https://www.youtube.com/watch?v=CMSYv_Z4SI8

Below are the lyrics to my song (I’ll be presenting in class on Wednesday), I hope you enjoy it!

Microbiology Art Project Big Bang Theme Microbiology Parody

A2: Microbes in the News (Post 3)

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Genetically engineered microbes make their own fertilizer, could feed the world’s poorest

Source: Science Magazine

Date: April 4th, 2017

Link:  https://www.sciencemag.org/news/2017/04/genetically-engineered-microbes-make-their-own-fertilizer-could-feed-world-s-poorest

Summary: Currently, big chemical  plants use nitrogen and methane to make ammonia (i.e fertilizer), which is not typically a viable option for developing countries, not only because they are expensive to run and maintain, but also because they lack the resources to distribute the produced fertilizer.  While we know of some microbes that are capable of nitrogen fixation, researchers from Harvard have genetically engineered a bacterium to be able to convert nitrogen (N2) to ammonia or other forms that plants can use, with the hope it could be used on a widespread commercial scale, which India has started to work on.

Connections: Throughout the semester, we have learned about and tested the physiological capabilities of microbes. We did test to see if our microbial isolates were capable of denitrification (whether it be partial or complete).

Critical Analysis:  I do think this article was scientifically accurate and it did answer some of my questions (how/where does the energy for this come from? what enzymes are involved in the process?). However, the article does fail to discuss the fact that there are existing microbes that convert N2 to ammonia or nitrates/nitrites and does not explain why this specific genetically engineered bacterium is better than any of the preexisting microbes capable of this same process. It conveys a message that this is somehow a new concept, even though it is not. It also discusses the scientific conclusions  that the genetically engineered bacterium,  Xanthobacter autotrophicus,  works outside of the lab because  the researchers put it in solution,  watered  a sample of radishes with it, and noted  the radishes grew 150% more than the controls. They did this in the lab though. I’m not saying this conclusion isn’t valid, just that it doesn’t sound as if anyone has attempted to replicate this, or test this in an environment with several other factors that a lab cannot account for. They have a ways to go before this could lead to feeding the worlds poor. Overall, I think the author means well and conveys the science itself fairly well, but misleads the public as to what exactly the science means in the grand scheme of things.

Question(s):  If we’re wanting to commercialize nitrogen fixating bacteria, why not use one that does not require genetic modifications? Do  Xanthobacter autotrophicus  have greater diversity in the environments in which they can survive/thrive (i.e a variety of climates)?

Extra Credit: Microbial Worlds

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1. Water is Life by Jennifer Moss

This piece really caught my eye and I found it to be really well done. I love that the artist incorporated crude oil into the piece. While the concept behind the art is not wonderful, I think this piece is beautiful and does successfully embody the concept of microorganisms playing a role in oil degradation. Jennifer Moss used a variety of different colors to represent different microbes throughout the spill, with some being more concentrated in certain parts and others being scattered all over. This class has discussed how different microbes play different roles in the midst of an oil spill and I think Moss does a good job of demonstrating that.

2. Mycorrhizae by Gail Priday

This piece is based on the symbiotic relationship between the mycelium of a fungus and the roots of vascular plants. Mycelium help the plants increase their water and nutrient uptake, while the plants provide the fungus with carbohydrates. The piece provides an effective visual, aiding to explain just how interconnected this relationship is. I personally feel this piece is more attractive verbally, as the artist discusses the science in a simplistic, but accurate way, in addition to explaining why he chose this particular topic. Priday says in the statement that it’s a beautiful example for what humanity should strive for, but I don’t see that conveyed in the visual piece. I think it’s a wonderful piece of art, but I read the statement and just did not see it show in the painting. I think if the artist had done something with that idea, incorporated a way for us to learn from it, it would be even better.

3. Connections: Veiled Unveiled by Mariah Henderson and Eric Henderson

This piece touches on the fact many microbes are invisible, even on a microscope, and this is one of the reasons why staining methods are so helpful. We can unveil the formerly veiled microbes because of modern staining techniques (the one mentioned in this piece was crystal violet).  In this class, we have used staining methods to not only see our bacterial isolates, but to also determine a variety of characteristics, including whether they were Gram-Positive or Gram-Negative.

4. What I Would Have Created

Had I been an artist in this show, I probably would have created a piece about the skin microbiome. Assuming I had the skills, I would have liked to have created a painting of the human body, using different colors and shapes to demonstrate that our skin has a wide variety of microorganisms on it, from our feet to our face, due to different temperatures, moisture, etc.

 

 

A2: (Post 2) Microbes in the News

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New CRISPR tool can detect tiny amounts of viruses

Source: Science  Magazine

Date: April 13th, 2017

Link:  https://www.sciencemag.org/news/2017/04/new-crispr-tool-can-detect-tiny-amounts-viruses

Summary:  SHERLOCK, or specific high sensitivity enzymatic reporter unlocking, has been found to be a highly effective diagnostic tool. While the CRISPR genome editing applications that we hear so much about uses a DNA cutting enzyme known as Cas9, SHERLOCK uses an RNA cutting enzyme called Cas13a. The researchers have shown CRISPR-Cas13a to detect viral and bacterial infections, cancer mutations, and SNP’s. It is said to be 1 million times more effective than ELISA, as well as faster because it detects to the attomolar level (10^-18), and it can be done outside of the lab, which is useful for poorer countries.

Connections:  In the most recent lab, we used ELISA to test and see who was spreading a specific disease. ELISA is currently the most widely used diagnostic tool.

Critical Analysis:  I find the potential for this to be quite interesting. Being able to take a sample somebody’s blood, saliva, or urine, have it transcribed into RNA, and within a few hours know if someone has an infectious disease or severe mutation like cancer, is exciting (and perhaps a little frightening). While we have methods such as ELISA to detect the presence of infectious diseases, this new method is so much more sensitive (detects diseases and mutations to the attomolar level) and accurate. Not to mention, it doesn’t require a lot of laboratory technology, meaning it can be used in resource poor areas where infectious disease is rampant. This specific article did a decent job of explaining the scientific concepts behind Cas13a, other than perhaps  oversimplifying some of the science (“jiggered to make it work with DNA”, for example). I understand that sometimes this can help the general population  better understand the concept though. Although, it does assume that one has a general idea of  what CRISPR is and the general history behind it, which might be confusing for some people who’ve never heard of it. Overall, I think it is effective in helping people gain a better idea of what CRISPR-Cas13a can do though.

Question:  What are some other potential uses for CRISPR-Case13a? Also, the targets for the enzyme have to be incredibly specific, so how exactly does Cas13a work in finding mutations like SNP’s? I’m a little lost in how that mechanism works in that regard.

*I did some additional research after reading this article and kind of answered one of my own questions (although I am sure there are plenty more applications to be discovered!). If you want to know more, I recommend the reading following article (it has a longer list of discovered potential uses):

https://news.mit.edu/2017/scientists-unveil-crispr-based-diagnostic-platform-0413

A2: Microbes in the News

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Gut microbes contribute to age-associated inflammation, mouse study shows

Source: ScienceDaily

Date: April 12th, 2017

https://www.sciencedaily.com/releases/2017/04/170412132332.htm

Summary: Currently, the only ways for us to reduce the severity and risk of age-associated inflammation is to eat healthy and exercise. If you suffer from chronic inflammation, consulting your doctor is also helpful. As we age, we usually have an increase in tumor necrosis factor (TNF) and other pro-inflammatory cytokines. It turns out this may be related to the fact our intestinal walls become much more permeable as we age due to changes in the gut microbiome, and as a result, bacterial products  that shouldn’t “leak through” do (or at least, this is what they are discovering in mice). This escape of microbial products out of the GI tract triggers an immune response (inflammation) and weakens the immune system. Researchers believe if we can maintain a healthy gut microbiome, we can reduce age-associated inflammation and prevent this cascade events before they start. However, which bacteria in the gut lead its increased permeability is unknown, as is when in life the gut microbiome changes enough for this to occur.

Connections: Just the other day, we covered the pros and cons of inflammatory responses triggered by the immune system. While localized inflammation is usually a good thing, systematic inflammation is not, as it often causes high fever, a massive drop in blood pressure, and results in death for 30% of people affected.

Critical Analysis:  It continually amazes me how greatly our microbiome impacts our health. It doesn’t surprise me that it plays a crucial role in the aging process, but I had never considered it before. The health of our immune system is obviously important as we age, and most know its efficacy declines as we get older, but the fact that this decline could partially stem from changes in our gut microbiome is fascinating! This could mean that probiotics, assuming they determine which bacteria are at fault for breaking down the intestinal barriers, could enhance our immune system as we age. I do think this article is scientifically accurate and overall, does a good job of describing complex scientific principles to the general public, as it explains most of the science in general terms and makes it clear why this could have important implications as we age.

Question:  This team of researchers said they are setting out to identify the “bad” bacteria that induce the breakdown of intestinal barriers, but I was wondering if the scientific community already knows of some particularly useful or harmful bacteria in our guts that could play a role in this process? Or is this such a brand new idea that nobody has any hypotheses? If no specific bacteria are known, what kind of chemical products would these “bad” bacteria release to weaken our intestinal walls?

 

Painting with Microbes!

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I attempted to depict some of the topics we have discussed in lecture, but as you can see, it did not turn out as well as I had hoped! I tried to paint bacterial conjugation and triclosan (with a sad face!) with not much success. However, my virus turned out quite nicely. Doesn’t look so harmful from this perspective, does it?

Extra Credit Simon Lax Seminar

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The seminar’s major points pertained to how our built environment resembles its occupants and why this is important to study. Why should we care about this? One reason is that our microbiome and our surrounding microbiome play a big role in our health. The skin microbiome was the focus of Simon Lax’s research and he has found enormous diversity between people’s skin microbiome just on a day-to day basis. People’s built environment really does resemble what is on their skin. For instance, people’s feet and floor maintained relatively similar communities, but the bacterial communities found on people’s hands and therefore phones changed. With something changing so frequently (keeping in mind we shed millions of bacteria off of our skin onto a variety of surfaces that we touch), how does this affect health or help with forensics? These are questions he addresses.

I think overall it was a really fascinating seminar and provokes people to consider how interacting with the built environment impacts microbial diversity, human health, and how we approach forensics. It makes sense that the environments we interact with the most resemble much of the bacteria found on our skin, especially our phones, which change in their bacterial communities as our hands do.

I do wish he had been able to discuss the specific (or more specific) kind of bacteria found in the hospital setting though. As he mentioned (and as we have discussed in class), using 16S rRNA sequencing does not yield useful results in terms of identifying specific strains of bacteria or determining pathogenicity, which I feel in a hospital setting is important to determine. I did also wonder if antibiotic resistance genes increased over time in the hospital environment they studied, but he did address that this would take more time and research, seeing as the hospital they are studying has not been open for very long. I am also wondering if there is one factor related to the skin microbiome that contributes to an increase in antibiotic resistance more so than anything else in hospitals. Is it the staff skin microbiome, which has higher levels of microbial diversity, since they are interacting not only with the patients, but also with their environments? What are other ways the skin microbiome contributes to increased antibiotic resistance?