Author Archives: cummingb

And the name of my microbe is… -Brianna

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What were the results of your 16S analysis? 

Four of my isolates had their 16S ribosomal DNA sequenced. Based on sequence identity, two (LB 2/ PDA 5) were determined to belong to the Pseudomonas genus and one (R2A 18) to the Bacillus genus. The fourth (PDA 2) had high percent identity match to a lot of different genera, so it’s family was determined to be Enterobacteriaceae.

Does your Gram stain agree? 
For reference, here is a table with my four isolates with their Gram stains and microscopic characterization:

Each of the Gram stains agree with what is typical for the determined genus/family; positive for Bacillus, and Gram negative for Pseudomonas and Enterobacteriaceae.

Pick one of your isolates and find out more about the genus (it is unlikely you will be able to determine the species).

I choose to research the Pseudomonas genus since two of my isolates belong to the genus, and they were the only isolates with working cell-free extracts.

a) General cellular and morphological characteristics of the genus (taxonomic classification, nutrition, cell shape, habitat).

Over 140 bacteria belong to the Pseudomonas genus, and are Gram negative and rod shaped. Pseudomonas are typically found in wet environments such as soil and water, but they are also commonly found in hospitals as well in locations such as respiratory equipment, sinks, and food. Additionally, they can be grown in a lab setting on most standard media types. (1)

Here is a Gram stain of a common Pseudomonas, Pseudomonas aeruginosa.(2)

b) Information regarding antibiotic production in this genus.

As for antibiotic production, papers have been published indicating specific species produce antibiotics. One in particular that I found interesting is about Pseudomonas fluorescens. This species has been isolated from cultivating soils which naturally suppress typical soil-based plant pathogens. In the study, they found and isolated four different antibiotics from Pseudomonas fluorescens: pyoluteorin, pyrrolnitrin, phenazine-1- carboxylic acid, and 2,4-diacetylphloroglucinol. (3)

Additionally, within the species Pseudomonas aeruginosa, 90 percent of strains produce a lethal toxin, called Toxin A. This toxin inhibits protein synthesis in other cells, effectively killing them. The lethal dose in mice is about 0.2 µg. (2)

I did a little more searching due to the very distinct orange pigment that my isolate produced to see if that could give any insight as to the species. However, I found that the pigments produced by pseudomonas are typically green/blue as in the case for Pseudomonas aeruginosa and Pseudomonas fluorescens. Which is interesting because one of my extracts was a green color. However, I could not find any Pseudomonas that produce a bright orange pigment.

References:

  1. Iglewski BH. Pseudomonas. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 27.
  2. http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab12/Psaeruginosa.html
  3.  Raaijmakers, J. M., Weller, D. M., & Thomashow, L. S. (1997). Frequency of antibiotic-producing Pseudomonas spp. in natural environments. Applied and Environmental Microbiology, 63(3), 881–887

 

Extract News! – Brianna

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I was able to obtain a total of 6 extracts between my 4 samples (there was a little bit of labeling issues, so I could not combine the duplicate sets). I chose to test these extracts against  #1 (Staphylococcus epideridis) and #4  (Enterococcus raffinosus) as the gram positive ESKAPE pathogens and #3 (Erwinia carotovura) and #7 (Enterobacter aerogenes) as the gram negative. These were chosen since my producers  showed inhibition against these pathogens (some showed against one or two, but not all three at once) with the exception of number 7 (which was chosen out of convenience).

Out of all the extracts, two showed inhibition against Staphylococcus epideridis, Enterococcus raffinosus and Erwinia carotovura . While the numbering/labeling was messed up from before, these were my two pigmented extracts. I believe these correspond to my samples, PDA #2 and LB #2.

 

Here is a table summarizing the cell free and colony inhibition tests.

 

 

Meet My Microbes! – Brianna

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Here are (some of ) my original plates from the soil sample extracts.

The first is my 10-3 dilution plated onto R2A , the second is a 10-2 dilution on PDA, and the third is 10-3 dilution on LB media. All are photographed on day three of incubation. For the most part, PDA showed the most diverse colonies with a variety of colors and shapes. From these and other dilution plates, 24 colonies (per plate type) were picked and selected to be further investigated.

Here are my master patch plates after four days of incubation, in the same order as before. A few interesting things occurred at this stage. The diversity of the colonies was much easier to identify. They went from being generic-off-white-round colonies to having slightly different pigments, shapes, and morphologies. Additionally, some of the picked colonies did not grow on the new plate (RIP), so who knows if I lost the answer to the antibiotic crisis.

 

Then, all of these colonies were tested for antibiotic producers. Below is my champion of my antibiotic producers, PDA #5. As you can see, a vary large halo formed around colony #5, clearing the tester ESKAPE #6, B. Subtillis.  I also had 3 other producers from other plates and colonies, but they produced only a small halo against the background.

From all the ESKAPE testing, I narrowed down my colonies to a total of 4 producers. These are the streak plates for each of these microbes. In order, they are: PDA #2, PDA #5, LB #2, and R2A #18.

These four microbes were then stained to determine if they were gram positive or negative. All images are taken under a 100X oil emersion microscope lens. For reference, here is the positive (Staphylococcus epidermidis) and negative (E. Coli) controls.

 

The following are of my isolates. The first image is of R2A #18. This microbe is gram positive, and appears in diploid rods. The second is LB#2, which is gram negative and appears to be a shortened more ovular rod, and tends to be in diploid clusters. The third, PDA #2,  is a gram negative cocci, and is generally diploid, but forms larger clusters as well. Lastly, PDA#5, is gram-negative, and the cells are shaped like a typical rod bacterium.

  

 

Meet the ESKAPE pathogens: Brianna Cummings

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  1. Assigned ESKAPE Pathogen

Staphylococcus Aureus

  1. Why is this ESKAPE Pathogen of interest (in brief)

Staphylococcus Aureus(S. aureus)is a fairly common bacteria, anywhere from 30 to 50 percent of humans have this bacteria growing on them (1, 2). Typically, S. aureus is found on human skin and mucous membranes, and is not of any concern until it crosses into the bloodstream or internal tissues (2). Infections typically are spread in clinical and hospital settings, but can also occur in non-clinical environments (2).  The major concern regarding S. aureusis a particular strain, methicillin-resistantS. aureus, or MRSA, which is a highly difficult form of S. aureus to treat due to resistance and has a very high morbidity rate (3).

  1. General Cellular and Morphological Characteristics of the Organism (taxonomic classification, nutrition, cell shape, habitat)

Staphylococcus aureusis a Gram+bacteria with a cocci shape (round, spherical shape) and is a member of the Staphylococcaceae family (2). Interestingly, they form clusters of about 1µm in diameter, which can look like grapes when stained (2). However, unstained, S. aureus present as golden colonies (2).

The optimal growing temperature for S. aureusis 37C, while it will grow between 7 and 48C and can survive in temperatures of below -20C (4). Unlike many other bacterium, S. aureus is resistant to high salt content(4). Additionally, S. aureus has the ability to grow under aerobic and anaerobic conditions, although aerobic conditions are preferred(4). Typical medias for colony growth include, blood agar. Tryptic soy agar, and heart infusion agar (5).

As mentioned earlier, S. aureus is typically found in the mucous membranes and on the skin of humans, and are found particularly in the nasal passage (5). However, S. aureuscan also be found in food, and due to its ability to survive in harsh conditions S. aureus is a source of food poisoning (4).

  1. Clinical Importance and Prevalence

S. aureus infections have stabilized in numbers since the 1990s, with about 10-30 cases per 100,000 people per year (1). However, the number of MRSA diagnosis has been rapidly increasing over the same time period (1). Both community and health care related epidemics have contributed to the increasing number of MRSA outbreaks (1). Newer more intense infection control procedures have helped to reduce the number of outbreaks in recent years (1).

  1. Infection (How does the infection occur and where is it localized?)

Infection is most common in people at the “extremes of life,” i.e. infants and the older populations (1). Additionally, humans working in the health professions, hospitalized people, and individuals that are immunocompromised are at a greater risk of contracting the infection (2). S. aureusis transmitted via direct contact or fomites (objects that have the bacteria on them such as clothing, tools, furniture) from person to person (2). Infections can occur in virtually every part of the body, however are most common in skin and soft tissues.

  1. Pathology (What disease is caused? What are the symptoms?)

Many different diseases can be the result of  S. aureus. These include, endocarditis, osteomyelitis, septic arthritis, gastroenteritis, meningitis, toxic shock syndrome, urinary tract infections, pneumonia, but the most common being skin and soft tissue infections (2). Depending on the strain and location of the infection as well as the resulting disease, symptoms vary greatly. However, for ease of answering the question, the symptoms for some of the diseases are as follows:

Skin infections (abscess and cellulitis)  / MRSA :  Swollen, painful bumps that are warm and full of pus, and associated with a fever. Untreated, these lead to deeper more painful infections and can burrow into the skin (6).

Osteomyelitis: fever/chills, swelling of the infected limb with redness, and eventually stiffness and inability to maneuver infected limb. Diagnosis involves blood tests, and potentially a bone scan (7).

Pneumonia: Difficulty breathing, fever, chills, cough. This is diagnosed via chest x-ray and usually involves treatment with antibiotics and hospitalization (8). (Ironic because S. aureus pneumonia is usually transmitted in hospital settings)

  1. Ineffective Antibiotics (Antibiotics to which the organism has acquired resistance)

Depending on the strain, S. aureus is resistant to a variety of antibiotics (9). The emergence of antibiotic strains began in the 1950s with penicillin resistance (9). Then, in the 1960s, the first strains of methicillin resistant S. aureusemerged (9). And more recently, vancomycin resistant strains emerged (9). Thus, S. aureuscan be resistant to the majority of antibiotics, and discovery of new treatments is ever so important.

  1. Effective Antibiotics (Antibiotics known to inhibit the organism)

Treatment of S. aureusis usually done with penicillinase-resistant beta-lactams (5). Penicillin is the typical antibiotic of choice for sensitive strains (2). However, due to the many different strains ofS. aureus, characterization of the bacteria may be necessary before treatment. For example, the MRSA strains are resistant to beta lactams and are typically treated with vancomycin. Additionally, other treatments may coincide with the antibiotics such as fluid replacement.

  1. Corresponding Safe Relative

Staphylococcus epidermidis

Reference:

  1. Tong, S. Y. C., Davis, J. S., Eichenberger, E., Holland, T. L., & Fowler, V. G. (2015). Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management.Clinical Microbiology Reviews, 28(3), 603–661. http://doi.org/10.1128/CMR.00134-14
  2. Taylor TA, Unakal CG. Staphylococcus Aureus. [Updated 2017 Oct 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2018 Jan-.Available from: https://www.ncbi.nlm.nih.gov/books/NBK441868/
  3. Green, B. N., Johnson, C. D., Egan, J. T., Rosenthal, M., Griffith, E. A., & Evans, M. W. (2012). Methicillin-resistantStaphylococcus aureus: an overview for manual therapists. Journal of Chiropractic Medicine, 11(1), 64–76. http://doi.org/10.1016/j.jcm.2011.12.001
  4. https://www.foodstandards.gov.au/publications/Documents/Staphylococcus%20aureus.pdf
  5. https://www.ncbi.nlm.nih.gov/books/NBK8448/
  6. Chambers, H. F., & DeLeo, F. R. (2009). Waves of Resistance:Staphylococcus aureus in the Antibiotic Era. Nature Reviews. Microbiology, 7(9), 629–641. http://doi.org/10.1038/nrmicro2200
  7. https://www.mayoclinic.org/diseases-conditions/mrsa/symptoms-causes/syc-20375336
  8. https://www.healthline.com/health/osteomyelitis
  9. http://www.health.state.mn.us/divs/idepc/diseases/staph/basics.html

2. Fun with Soil – Brianna Cummings

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Where did you obtain your soil sample?Why did you choose this location?

I collected my soil sample between the pyramid and the bank of the Hans Groot Kill.  I dug about an inch down, and tried to avoid the grass and roots that were in the soil.  I chose this location for two reasons. The first being out of sheer convenience. The location was right on the way to class, so I was able to just wake up 15 min early and be set with collecting my sample. I also chose this location because I thought there would be some interesting bacteria found there due to the activities that happen on and near the pyramid.

Do you expect a lot of isolates? Why or why not? Have you initial observations supported this?

I expected to get a fair amount of isolates due to the location and soil type. The soil was clay-like and as mentioned in class this tends to support more anaerobic microbes. However, since my sample was collected from the top layer of soil, I figured there would still be a good amount of aerobic microbes. I also thought due to the highly trafficked area (and activities that go on there), there would be a lot of different microbes. Also the sample was collected near the creek, and after hearing how there’s lots of stuff in the water during class, I again thought there would be plenty of microbes in my sample. Initial results have proven that my sample has a good amount of microbes in it, yet the diversity is lacking (the majority of colonies are off-white and round with very similar morphologies).

What media did you choose? What dilutions? Will you need to redo any? How did your sample differ on the different media?

I chose LB, R2A, and PDA media. Since I thought there would be lots of microbes in my sample I chose to do two of the less rich media and only the LB rich media. For similar reasons, I used the higher dilutions of 10^-1 through 10^-3 (numbering system in book) for each of the medias. Interestingly, the R2A had very rapid growth of both colonies and mycoides, and needed to be diluted to 10^-4 to obtain accurate counts. As for the different media, it mostly affected the diversity of the colonies rather than the number of colonies. For instance, the R2A tended to have mostly white colonies that pretty much looked the same and mycoides, whereas the PDA had many different morphologies and colors. LB samples tended to be somewhere in the middle regarding morphologies- there were many similar ones, but there were also some unusual/unique looking ones.