Micrococcus luteus Isolated From a Fish Tank Filter

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Ariana Casey

BIOL 342 — Microbiology

Introduction

The human race predominantly lives on land and breathes in the air surrounding them, not giving much thought to the bodies of water that cover the majority of the earth’s surface.   In all these environments exists a common organism — bacteria.   One must look past the human —past what the naked eye can see- to understand the bacteria.   Many species exist, inhabiting a multitude of niches in the world, from the inside of soil clumps and the bottom of the ocean —devoid of oxygen- to the skin on our hands and the surfaces of plants —with aerobic respiration to sustain them.

With so many different species of microorganisms, it would be a shame to disregard the ones that live in water simply because it’s not the environment that people exist in.   With the human body and the surrounding environment being so dependent on water, is would make sense to examine this substance to find what microbes exist there.   Among one of the many uses for water that people have, the one that seemed it would have a good measure of microbiota was a fish tank filter.

The objective of the following research was to isolate a bacteria from a sampled location and to determine through physiological and genetic testing what bacteria was isolated from the sampling location.  Being that a fish tank seems a rich environment for microbiota, it is hypothesized that the bacteria will be unique, with the ability to live aerobically or anaerobically.

Methods

Sampling

The chosen location for sampling was the filter of a fish tank.   To properly sample, the filter was first removed from the water.   After removal from the fish tank, the filter was swabbed with a cotton swab, which was then streaked over the surface of the TSA plate (Lab Handout 1).   The plate was kept at room temperature for six days before being brought into lab.   At this point, a colony was pulled with a sterilized metal loop and put onto another TSA plate using a quad streak (Lab Handout 2).   As the bacteria grew, individual colonies were used to further isolate the bacteria through additional quad streaks.

At the fourth quad streak, the culture was determined to be fairly pure through microscopic observation (Lab Handout 4).  At this time, the culture was deemed pure enough to be physiologically and genetically tested to determine bacterial strain.

Physiological

One objective of obtaining a pure culture through quad streaking was to observe the colony morphology of the cells in isolate (Lab Handout 2).  To determine cell size, shape, arrangement, and wall-type, gram-staining was performed (Lab Handout 4).     Fluid thioglycollate test was performed and incubated at room temperature to determine oxygen class (Lab Handout 6).   Presence of cytochrome C oxidase was tested using an oxidase test strip with an indicator dye (Lab Handout 6).   A catalase test was used to determine if the strain of bacteria has the enzyme catalase (Lab Handout 6).   To determine which sugars the bacteria could ferment, as well as to determine other physiological characteristics which could help to characterize the isolated bacteria, an API 20E test strip was used (Lab Handout 6).   Later, due to the results with API 20E being semi-inconclusive, and with more information on the isolated bacteria, the API staph strip was used to test the bacteria.

To determine if the isolated bacteria had the ability to grow in the presence of methylene blue, and further to see if it could ferment lactose and/or sucrose, the isolate was plated onto Eosin Methylene Blue (EMB), following lab 6 protocol.   For determining if the isolate had the ability to grow in the presence of crystal violet and bile salts, and to further determine capability to ferment lactose, the isolate was plated on MacConkey (MAC) Agar (Lab Handout 6).

Additionally, to further determine the identity of the isolated bacteria, its susceptibility or resistance to various antibiotics was tested via the protocol of Lab Handout 9.  The isolated bacteria was suspended in TSB and then plated with a cotton swab onto two TSA plates, onto which antibiotic discs were placed evenly apart.  To determine susceptibility to antibiotics, zone diameter was measured once a bacterial lawn had time to form (approximately 48 hours past inoculation).  The tested antibiotics were Amakicin, Cefaperazone, Vancomycin, Trimethoprim, Oxacillin, Clindamycine, Piperacillin, and Gentamicin.

Genetic

An isolated pure culture bacteria colony was suspended into tryptic soy broth (TSB).   After a week, this TSB suspension of bacteria was used for genomic DNA extraction (Lab Handout 5).     DNA extraction was done using the PowerSoil DNA kit (MoBio), which was sequenced using Illumina MiSeq technology at the University of Alaska Fairbanks’s DNA Core Lab (Lab Handout 5).   The data procured from the Illumina Miseq sequence analysis will then be computed using BaseSpace (Lab Handout 7).  Assembly of the genome was done using the SPAdes Genome Assembler application (Lab Handout 7).  Using the Kraken Metagenomics application and the assembled genome, a taxonomic classification of the bacterial isolate was determined (Lab Handout 7).  The final application used on the data procured from the DNA Core Lab sequencing was the Prokka Genome Annotation to determine potential functional genes of the genes (Lab Handout 7).

To further analyze the results, the first node of the contigs.fasta file was pulled from the SPAdes Genome Assembly application and ran through the Nucleotide BLAST (Basic Local Alignment Search Tool) application provided by the U.S. National Library of Medicine.

Results

Physiological

The pure, isolated bacteria grew in yellow-pigmented, shiny, smooth, small, and roung colonies.  Gram-staining revealed the bacteria to Gram-positive in nature, with cocci approximately 1.5 um in diameter.  The cells were usually found in tetrads with the smallest aggregations being diplococci, though often the cells would clump further.   The fluid thioglycollate test determined the bacteria to be an obligate aerobe.   Hydrogen peroxide resulted in formation of bubbles, testing positive on the catalase test.   The oxidase test strip turned a light grey-purple in the presence of the bacterial suspension, testing positive on the oxidase test.   The API 20E tests came back predominantly negative, with the only reliable positive being the test for presence of gelatinase.   Additionally, the VP turned a slight pink within the 10 minutes after VP1 and VP2 reagents were both added.  This was inconclusive as to whether the test was positive or negative.  With the API Staph strip, there were also all negatives within 48 hours of starting the strip.   After addition of the VP and NIT reagents (there was no access to PAL reagents, so that test is inconclusive), the VP turned slight pink after the 10 minutes.  Using apiweb as a resource, I determined this to be a positive reaction.

Antibiotic testing results (Table 1) reveal this isolate to be fairly susceptible to antibiotics.

Table 1: Antibiotic Zone Diameters and Susceptibility
Antibiotic Zone Diameter (mm) Susceptible, Intermediate, or Resistant
Amakicin *radius: 22 Susceptible
Cefaperazone *radius: 18 Susceptible
Vancomycin 29 Susceptible
Trimethoprim 15 Intermediate
Oxacillin 15** Resistant**
Clindamycine 36 Susceptible
Piperacillin 50 Susceptible
Gentamicin 36 Susceptible
*Radius was used where the zones overlapped and the diameter was impossible to measure due to overlap.  The radius of each zone was greater than the determined diameter susceptibility range.

**Plated oxacillin disc was contaminated with a piece of agar from another lab mate’s bacterial isolate, and so would be inconclusive, but my bacterial isolate was used by another student that lost their isolate in a lab clean-up.  This student also used the Oxacillin disc and had a zone diameter in the resistance range.

 

Genetic

Basespace results indicate that the bacteria is likely Micrococcus luteus NCTC 2655 with 75.83% of the reads classified and 98.59% of the 73.91% analyzed reads were classified to the species level.   This is further supported by the BLAST results, which indicated a 97% identity match to Micrococcus luteus NCTC 2665.   The SPAdes Genome Assembler data revealed a genomic length of 2,615,526 base pairs with a GC content of 72.41%.   The bacteria also had 53 tRNAs and 2365 coding regions.   Probable functional genes were discovered through use of the Prokka Genome Annotation application.  Putative acetyl Co-A acetyltransferase, putative oxidoreductase, and NADH dehydrogenase-like protein SA0941 were all probable functional genes found in the genome of the isolate.

Discussion

The bacteria isolated from the fish tank filter was not a unique microbe, as was expected, but a ubiquitous microbe found in many niches.  Additionally, the bacteria was surprisingly an obligate aerobe, which was unexpected as with an environment comprised mainly of water.  The ability of the bacteria to survive in such an environment may be due to the ability of M. luteus to become quite dormant and awaken with the use of the Rpf gene (Mukamolova et al 2002; Young et al 2010).

Identification of the bacterial isolate through the genetic data alone is alright in cases where the said bacteria cannot be plated and isolated.  In the case of this study, looking at both the physiological and genetic aspects of the bacteria for being able to identify the bacteria as Micrococcus luteus NCTC 2665 strain is important.

Physiological

Micrococcus luteus NCTC 2665 “Fleming Strain’ have a distinct yellow-pigmentation to them, likely an adaptation to protect against radiation (Young et al 2010).  The bacteria is quite ubiquitous, in that it is usually found on mammalian skin, but can also be found in soil as well as fresh and marine water (Akayli et al 2016; Rickard et al 2002; Young et al 2010).  This aligns well with the sampling location being a fish tank filter, as the bacteria are found in water, plus the filter is something often handled by people, thus either could have been the source of the M. luteus at this particular location.  The bacteria is known to be coccoid, gram-positive, and form mainly tetrads (Akayli et al 2016).  With the tetrads, clumping happens, which makes sense as M. luteus are observed to create coaggregates in fresh water with some other bacteria to form a biofilm (Rickard et al 2002).  Additionally, M. luteus tests positive for both catalase and oxidase, all of which agrees with the testing done for the isolated bacteria.

The result of being an obligate aerobe from the fluid thioglycollate test also falls in line with the literature on M. luteus (Young et al 2010).  The physical test results of the API Staph test, when placed into the apiweb resource, come back with a 99.1% identity as a Micrococcus species, with an oxidase positive and yellow colony morphology leaning the test results towards Micrococcus luteus.

Genetic

With both the BaseSpace and BLAST results identifying the strain as M. luteus, especially with all the physiological evidence, it would be hard to refute that the isolated strain is, in fact, M. luteus NCTC 2665.  Beyond this, additional genetic evidence points to the identification of the strain obtained as M. luteus.  For one, the GC content of the isolated strain was 72.41%, while it is 73% for NCTC 2665 (Young et al 2010).  Strain NCTC 2665 has 2,501,097 bp, encodes 2,403 (of 2,458 total) genes to proteins, and has 48 tRNAs, which is all comparable to the isolated bacteria’s 2,615,526 bp, 2,365 coding genes, and 53tRNAs (Young et al 2010).  Of the genes, the oxidoreductase, CoA, and NADH-like protein mentioned beforehand are all important to M. luteus.  Cytochrome c oxidoreductase is a component of the respiratory chain and CoA is part of the citric acid cycle (Young et al 2010).

In discussing genes, it is important to relate some of them to physiological aspects that are seen.  A lack of glucokinase, for instance, results in M. luteus being unable to grow with glucose alone as a carbon source, which is what we witnessed through the API test strips having negative results for fermentation of glucose (Young et al 2010).  Additionally, the high susceptibility of M. luteus to antibiotics is possibly due to lack of the gene wblC, as it is a pleiotropic regulator in other bacteria that helps with resistance towards antibiotics (Young et al 2010).

Additional Discussion

The strain represented in this research is the “Fleming strain’ NCTC 2665, which is still known as Micrococcus luteus, but another strain of M. luteus —primarily ATCC 9341- has been reclassified as Kocuria rhizophila (Tang and Gillevet 2003).  In fact, the NCTC 2665 strain had, “recently’ (in accordance to a 2010 paper) been declared taxonomically separate from K. rhizophila (Young et al 2010).

Strain ATCC 9341 is different in a few ways, some being that it is oxidase negative compared to the oxidase positive nature of NCTC 2665, as well as that the strain ATCC 9431 forms acids from glucose and fructose, neither of which were positive tests on the API test strips performed, meaning that my bacteria isolate does not confer with the traits presented by the Micrococcus luteus ATCC 9431 strain that was reclassified as Kocuria rhizophila (Tang and Gillevet 2003).  It, indeed, appears that the isolated bacteria follows more closely with the “Fleming strain’ NCTC 2655 of M. luteus which has not been reclassified to Kocuria rhizophila.

 

References

Akayli, T., Albayrak, G., Ürkü, Ç, Çanak, Ö, & Yörük, E. (2015). Characterization of Micrococcus luteus and Bacillus marisflavi Recovered from Common Dentex (Dentex dentex) Larviculture System. Mediterranean Marine Science, 17(1). doi:10.12681/mms.1322

Mukamolova, G. V., Turapov, O. A., Young, D. I., Kaprelyants, A. S., Kell, D. B., & Young, M. (2002). A family of autocrine growth factors in Mycobacterium tuberculosis. Molecular Microbiology, 46(3), 623-635. doi:10.1046/j.1365-2958.2002.03184.x

Rickard, A., McBain, A., Ledder, R., Handley, P., & Gilbert, P. (2003). Coaggregation between freshwater bacteria within biofilm and planktonic communities. FEMS Microbiology Letters, 220(1), 133. doi:10.1016/S0378-1097(03)00094-6

Tang, J. S. and Gillevet, P. M. (2003). Reclassification of ATCC 9341 from Micrococcus luteus to Kocuria rhizophila. International Journal Of Systematic And Evolutionary Microbiology, 53(4), 995-997. doi:10.1099/ijs.0.02372-0

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. doi:10.1128/jb.01254-09

Microbes of Decomposition

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For the piece I have been working on, I decided to make   a woman with a dress of microbes laying on the forest bed.   I always find images with woman lying on the forest to be interesting (especially one series of photos I’ve seen that represents stages of fertility).   I first thought of a woman lying in some moss in the forest, and then decided to do a depiction of the bacteria that go behind the decomposition of a body to make up the dress of the person.

For the purpose of finding out which microbes are involved in decomposition, I ended up reading through a paper by Metcalf et al. that discussed use of microbes in forensics to better determine time of death.   I discovered which microbes played parts in different stages of decomposition and placed them throughout as such.   Many of the soil bacteria were Proteobacteria, which meant that they were all gram-negative.

Some of the interesting bacteria to find included Firmicutes that would stain gram-negative though they were gram-positive and serratia, which could form endospores.

Researchers discover ‘switch’ that allows microbes to recognize kin

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Researchers discover ‘switch’ that allows microbes to recognize kin

Published: March 27, 2017

https://phys.org/news/2017-03-microbes-kin.html

In our microbiology class, we learn a lot about how bacteria will interact with each other, but don’t really mention how they might be recognizing their own kind.   A plasmid shared among your species is much better than one shared with a competitor in the end.   And we also learned how beneficial a biofilm is, but not how a bacteria would know it can be a part of the biofilm.   In this article, we learn of TraA receptors —the cause of this recognition.   Digesting a study that looked at Mycococcus xanthus, this particular receptor was discovered to help recognition of M. xanthus cells for outer membrane exchange.   Additionally, different strains of bacteria have different TraA sequences, further supporting this receptor as a receptor to help with recognition of other bacteria of the same strain.   An interesting reason for the recognition receptor had to do with outer membrane exchange (OME).   The paper discussed that if different bacteria were to go through OME with each other, there might be toxins exchange that the recipient (being of a different bacterial strain) would then have no antidote to.

The article also discussed that the bacteria in question are phagocytes, and so they are of interest in the agriculture world.   So, as these would be great for getting rid of plants’ pathogens, it only makes sense that there would be interest in how they distinguish each other from other microbes.

One confusing part of the study is the way they present the ability to recognize different TraA sequences, saying one amino acid (AA) changed is enough to cause no recognition.   The way that the information was presented had me asking myself, “Is it a specific AA that when changed makes it so that the bacteria don’t recognize each other or is it that changing any AA would be enough?’

Waste-munching bacteria could make nuclear stores safer

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Waste-munching bacteria could make nuclear stores safer

Published: 11 April 2017

https://www.newscientist.com/article/2127588-waste-munching-bacteria-could-make-nuclear-stores-safer/

As we learned about in class before, through research shown to us by Dr. Leigh, that some microbes use Uranium or other such things as a food source.   For that reason, when I was this article, I thought it might be interesting to see it there was any progress made.

As part of a radioactive waste disposal plan, the UK is hoping to put the radioactive waste deep underground and to cover it with cement.   The problem with cement (as stated by the article) is that it will create conditions too alkaline for microbes to grow.   To test this theory, a research team studied a similarly-conditioned site.   It was seen that there were microbes that were able to withstand such conditions.   In alkaline conditions, there is a possibility of the uranium to form soluble complexes with isosaccharinic acid and to leak out, but with the presence of microbes, the isosaccharinic acid would be degraded by them, which would help to stop these leaks.   An additional benefit of the microbes is that some break down H2, which would stop the gas from building up pressure and causing a radioactive gas leak.

One nice thing about this study was seeing the use of the words “carbon source,’ as well as the author describing that the bacteria that break down uranium and other metals (such as neptunium) use it “in place of oxygen’ thereby representing the use of oxygen as an electron acceptor.   The way it is presented is very interesting and makes me thing that it does a good job of making the information accessible to the public.   The paper also expands on the findings, suggesting using them for decontamination of drinking water, such as we saw in the study presented by Dr. Leigh.

One thing that is lacking in this paper is differentiation of microbes, albeit this might make the article harder for the general public to consume.   For this reason though, I wonder, are the microbes mentioned through the paper the same microbes or are different ones used in different scenarios (I find the latter more likely)?   Additionally, I wonder, are the microbes ones that would naturally be present and persist in the given environment or are they being introduced?

Getting antibiotics as a baby may have lasting effects on brain, behavior

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Getting antibiotics as a baby may have lasting effects on brain, behavior

https://arstechnica.com/science/2017/04/getting-antibiotics-as-a-baby-may-have-lasting-effects-on-brain-behavior/

Published: April 5, 2017 at ArsTechnica

This article looks at a few different articles having to do with gut microbiome and their effects on the human brain in terms of behavior.   The main focus is a later paper in a series which looks as the exposure of baby mice to antibiotics given before and after birth.   The mice were split into two groups were one had penicillin introduced through the mother while in the womb and to themselves when they were born, with a later group added in which mice had the penicillin as well as were given a probiotic.   The group that had the penicillin had 42 percent of the population that were aggressive as opposed to the 9 percent in the control group.   Additionally, the antibiotic group appeared to be less social and a little less anxious.   The probiotic mice also had a thinner blood-brain barrier.   The group with the probiotics introduced had some of the effects of the antibiotic blocked.

Being that we talk a lot in class about how helpful a good gut microbiome is and how bad it is to overuse antibiotics —especially those that are more broad-spectrum, the contents of this article are no real surprise to me.   Even talk of cytokines being increased in the brains of exposed mice is a concept that I can understand thanks to learning about then with the immunology portion of class.

Overall, the article does a pretty good job at telling the story of the paper to the general public.   It goes as far as to include a section to discuss some of the limitation of the discussed study, such as the fact that the period of exposure to penicillin was quite long as well as the fact that the exposure before and after death was not differentiated in the study.   My only issue with the paper was that in one of their background statements, they say that, “gut microbes have been caught making most of the neurotransmitters our brains use to regulate themselves,’ but yet the link they provide leads to a paper that seems to more-so talk about the way that our neurotransmitters will influence the gut microbe, not vice versa (though the full paper was a paid paper and not free to view).

Question:   If there is a chance for the probiotics to help block the effects of the antibiotic use in infancy, would it be possible for probiotics to be used in a longer trial such that they eventually reverse the behavior issues that arise from the use of antibiotics?

Ariana: Painting with Microbes

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For the different media, I made 3 different animals.   In the end, the only one that I liked the most was this rabbit done on a TSA plate.   It was definitely the most simple design of all the designs I did, which made it a lot easier for me to draw, as well as not having to think of how the colors would change or not change.   In the end, the colors on the other two plates looked about right (with one or two things not really going according to plan, but they just were more complex and didn’t really look like the animals I was trying to draw.

With making my design, I also planned out which bacteria to use.   Seeing as most of the bacteria turn out cream/white or yellow on a TSA plate that doesn’t have indicator dye, the challenge was creating definition between my white rabbit and white snow.   I chose the flava for snow as the opacity was richer and the white more true white.   Then, I chose citrobacter for the rabbit, as the color was a nice translucent cream that seemed fur-like to me.   The part that I thought would really make this piece pop was the S. marcescens, which grows a pink/red.   Effectively, I made an albino rabbit.

For the pieces, I actually wish that I had done the rabbit design for each one and just changed strain type.   I feel that this would have made it easier to see where I messed up on placing bacteria versus how the bacteria interacted with the plate.

The biggest challenge with the dyed media was trying to have a light color, especially with the EMB media.   I found that I didn’t know what shades of red I would get and just hoped the shades would be different enough to reveal form, same with the MAC media, although that one was lighter, and thus easier to see some of the color and form on.

Introducing Myself (Assignment 1)

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Hello All,

My name is Ariana Casey and I am returning to classes after having graduated with a Bachelors of Science in Biological Science plus a minor in Japanese.   When I finished classes in May last year, I still had no idea what I wanted to do (well, still don’t fully) and it was a really stressful summer trying to find something I felt passionate about.   That’s when I rediscovered Radiological Technology as a possible career path.   I’m back in classes this semester to do Medical Terminology so that I can apply to the Rad Tech Program through a CTC and UAA partnership.   Since I’m back to school, I decided to take Microbiology just in case I go for Med School (not really my thing, but I don’t know if I might change my mind later in life).   To be in full-time student status (and also because I have loved art since my earliest memories), I am also in two art classes -Printmaking plus Color and Design.   Alongside classes, I am also working in the Institute of Arctic Biology as a Lab Safety Assistant and am planning my wedding.

This is me at my friends’ house, posing with their cute dog, Midnight.