What were the results of your 16S analysis?

I ended up getting two of my samples sequenced, #2 and #4.  After analyzing the sequences from BLAST I found that #2 was from the Klebsiella genus with 1062 bp and 98% query cover (4). Sample #4 was from the Acinetobacter genus with 790 bp and 99% query cover (4).

Does your Gram stain test agree?

The Gram stain test for #2 matched the results from BLAST of the Klebsiella genus (2). It was a Gram negative bacteria with oval shaped bacteria.

The Gram stain test for #4 did not match with the Acinetobacter genus because this genus is Gram negative (1). I got a Gram positive bacteria with a rod shape from the Gram stain test.

General cellular and morphological characteristics of the genus

Klebsiella is a Gram negative bacteria that is rod shaped (3).   It is from the Enterobacterales order and the Enterobacteriaceae family (4).  It normally lives in the mouth and gut flora but can be pathogenic (3).  It can also be found in soil, water, and plants (3).  Almost all Klebsiella can be grown on minimal media with ammonium ions or nitrate (5).  Klebsiella grow as yellow, dome-shaped colonies that are also often mucoid (5).  They are an anaerobic, non-motile species (3).  The Klebsiella genus is resistant to many commonly used antibiotics and is responsible for some nosocomial infections (3).  The most common infection is pneumonia which is caused by the species Klebsiella pneumonia, a community acquired pulmonary infection (5).

Picture from Medical-Labs: Medical Laboratories Portal.

Gram-negative Bacilli of Klebsiella pneumoniae

Information regarding antibiotic production in this genus

There is not a lot of information about the antibiotics produced by species of Klebsiella.  There is, however, a lot of information on the antibiotics they are resistant to, including aminopenicillins and carboxypenicillins (5). Many Klebsiella bacteria produce beta-lactamase which is an enzyme that gives them resistance to certain antibiotics (5).

References

1. Nemec, A. et al. “Genotypic and phenotypic characterization of the Acinetobacter
   calcoaceticuse Acinetobacter baumannii complex with the proposal of
   Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3)
   and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter
   genomic species 13TU)”. Research in Microbiology, vol. 162, 2011, pp. 393-404.
2. Diene, S. et al. “The Rhizome of the Multidrug-Resistant Enterobacter aerogenes Genome Reveals How New “Killer Bugs” Are Created because of a Sympatric Lifestyle”. Molecular Biology and Evolution, vol. 30, no. 2, 2013, pp. 369–383.
3. Buckle, Jane. Clinical Aromatherapy. Elsevier, 2015.
4. BLAST, NCBI, 2018. Accessed 11 November 2018.
5. Brisse, S. Grimont, F., & P. Grimont. The Prokaryotes. Springer, 2006, pp. 159-196.

Neither of my bacteria had a successful 16S rRNA analysis. While mine look sort of like Streptomyces under the microscope, I’m going to call v13 Sheila, and v19 Manuel. Those are good names.

The two DNA sequences you gave me are either Burkholderia or the really closely related Paraburkholderia (the only difference between these two geneses is that Paraburkholderia is never pathogenic and just lives quietly in dirt. In fact, people realized that they are distinct clades only very recently).

When looking at the data for the 08E isolate, the most related species was Paraburkholderia phenazinium, though of course there are other really similar species. I can’t find morphology through the links provided by BLAST, but a brief Google search says that bacteria in the Paraburkholderia genus are Gm-, slightly curved rods. They are soil bacteria, but a group grew it on what looks to be a rich media because there are 3 grams of meat. The bacteria can create Iodinin, which induces apoptosis by possibly breaking the DNA. While that is technically an antibiotic, I wouldn’t recommend using it.

What were the results of your 16S analysis?
All four of my isolates, #13, 14, 15, and 17, did have PCR products, but I was only able to obtain the 16S rRNA gene sequence for #13, 14, and 15. When we looked at the PCR products on a gel, #17 did produce a band, but it was faint which could explain why it wasn’t able to be sequenced. BLAST showed that #13 has a 99% match with the Bacillus genus. Isolate #14 had a 99% match with the Pseudomonas genus, and #15 had a 99% match with the Staphylococcus genus.

Does your gram stain agree?
My gram stain agreed for all three of my isolates. The gram stain for isolate #13 showed it was a gram-positive, rod shaped bacteria. This matches the description for the bacteria in the Bacillus genus (1). The gram stain for isolate #14 showed it was a gram-negative, rod shaped bacteria. This also matches the description for bacteria in the Pseudomonas genus (2). The gram stain for #15 showed it was a gram-positive, coccus shaped bacteria which clumped together in random numbers. This matches the description for bacteria in the Staphylococcus genus (3).

a) General cellular and morphological characteristics of the genus (taxonomic classification, nutrition, cell shape, habitat).
I further researched the characteristics of the Pseudomonas genus which matched my isolate #14. The taxonomic classification for this is Bacteria kingdom, Proteobacteria phylum, Gammaproteobacteria class, Pseudomonadales order, Pseudomonadacae family, and Pseudomonas genus (4). They are gram-negative, straight or slightly curved rods (2). They are typically aerobic, although sometimes nitrate can be used as an alternate electron acceptor which allows it to grow anaerobically (2). Most species can not grow under acidic conditions and do not require organic growth factors (2). This bacteria is widely distributed in nature and some species within this genus are pathogenic for humans, animals, or plants (2).

b) Information regarding antibiotic production in this genus.
Pseudomonas have been found to produce a wide variety of antibiotics including mupirocin (a topical antibiotic), fosfadecin, fosfocytosin, and xantholysins (4). Xantholysins have been discovered more recently and have shown anti-fungal activity and antibiotic activity, specifically against gram positive bacteria. Pseudomonas fluorescens has been shown to produce the antibiotics  pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol (5).  It is encouraging to know that this genus has shown antibiotic production since my isolate appeared to show antibiotic production.

References
1) Liu, Y., et. al, Proposal of nine novel species of the Bacillus cereus group, International Journal of Systematic and Evolutionary Microbiology, 2017
2) Palleroni, N. J. (2015). Pseudomonas. In Bergey’s Manual of Systematics of Archaea and Bacteria (eds W. B. Whitman, F. Rainey, P. Kämpfer, M. Trujillo, J. Chun, P. DeVos, B. Hedlund and S. Dedysh).
3) Pantucek, R., et. al, Staphylococcus edaphicus sp. nov., isolated in Antarctica, harbours mecC gene and genomic islands with suspected role in adaptation to extreme environment., Applied and Environmental Microbiology, 2017
4) Javier Pascual, Marina García-López, Cristina Carmona, Thiciana da S. Sousa, Nuria de Pedro, Bastien Cautain, Jesús Martín, Francisca Vicente, Fernando Reyes, Gerald F. Bills, Olga Genilloud,
Pseudomonas soli sp. nov., a novel producer of xantholysin congeners, Systematic and Applied Microbiology, Volume 37, Issue 6, 2014, Pages 412-416
5) Sarniguet, A., et. al, The sigma factor sigma s affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5, Proceedings of the National Academy of Sciences, 1995

What were the results of your 16S analysis?
Although I originally only got one PCR product for my L.5 isolate, I obtained two 16S RNA gene sequences from Prof. Salvo. Using BLAST, I was able to determine the identity of my two producers, P.1 and L.5. Both isolates were identified with very high confidence to belong to Pseudomonas genus (P.1: 980 base pairs, 100% query cover, 99% identity; L.5: 885 base pairs, 100% query cover, 99% identity). 

Does your gram stain agree?
Unfortunately, because I only obtained a single PCR product, I just performed a Gram-stain on L.5 isolate but not on P.1 isolate. The Gram stain of L.5 producer allowed me to identify it as a Gram-negative rod-shaped bacterium, which is consistent for Pseudomonas as they are Gram-negative bacilli (1) (picture of my L.5 isolate’s Gram-stain can be found in my “Meet my Microbes!” blog post).

a) General cellular and morphological characteristics of the genus (taxonomic classification, nutrition, cell shape, habitat).
Pseudomonas are Gram-negative rod-shaped bacteria (1). They belong to Bacteria kingdom, Proteobacteria phylum, Gammaproteobacteria class, Pseudomonadales order, and Pseudomonadaceae family (2). This genus is found in soil, water, plants, and animals but is also known to inhabit in hospitals (1). Pseudomonas aeruginosa, a species of Pseudomonas, is known to have simple nutritional requirements as it can grow in just “distilled water” but can also withstand extreme physical conditions as it can grow in jet fuel or diesel (3). Additional information on the cellular and morphological characteristics of P. aeruginosa that belongs to this genus can be found in my “Meet the ESKAPE Pathogen” blog post. 

b) Information regarding antibiotic production in this genus.
There are few cases of antibiotic-producing bacteria from the Pseudomonas genus:

One of them is mupirocin. It is an antibiotic that is used topically to treat skin infections. It is isolated from Pseudomonas fluorescens and has a broad spectrum activity against Gram-negative and Gram-positive bacteria (4). It works by inhibiting the bacterial isoleucyl-tRNA synthetase (5). 

Another example of an antibiotic production is a bioactive organometallic compound that has shown to have an antibiotic ability against microorganisms; it can be isolated from Pseudomonas aeruginosa LV strain in the presence of copper chloride (6). 

Lastly, isolated Pseudomonas viscosa shows broad antibiotic spectrum against a wide range of Gram-negative and Gram-positive bacteria that seems to have greater antibiotic ability relative to P. aeruginosa and P. fluorescens (7). 

Works Cited
1. Iglewski, Barbara H. “Pseudomonas.” Current Neurology and Neuroscience Reports., U.S. National Library of Medicine, 1 Jan. 1996.
2.“Pseudomonas.” Encyclopedia of Life, eol.org/pages/83175/overview.
3. Putty, Murali. “Pseudomonas aeruginosa.” EMLab P&K, Mar. 2007
4. Matthijs, Sandra., et al. “Antimicrobial Properties of Pseudomonas Strains Producing the Antibiotic Mupirocin.” Research in Microbiology, 7 Oct. 2014.
5. Hughes, J, and G Mellows. “Inhibition of Isoleucyl-Transfer Ribonucleic Acid Synthetase in Escherichia coli by Pseudomonic Acid.” Current Neurology and Neuroscience Reports., U.S. National Library of Medicine, 15 Oct. 1978. Biochem J, 179 (1978), pp. 305-318.
6. Gionco Barbara., et al. “New Insights about Antibiotic Production by Pseudomonas aeruginosa: A Gene Expression Analysis.” Front Chem., 15 Sep. 2017.
7.Chinn, S. H. F. “An Antibiotic-Producing Bacterium of the Genus Pseudomonas.” Canadian Journal of Microbiology, 1973. 

What were the results of your 16S analysis?

The PCR results were successful in amplifying the DNA in all three strains that were tested, and their sequences were used to run individual BLAST procedures.  Strain 1A (seen in figure 1) yielded BLAST results (figure 2)that revealed that it was a Gm- bacteria belonging to the Klebsiella genus.

Figure 1: Strain 1A isolated streak plate

Figure 2: BLAST sequencing results for unknown strains show relatedness of different strains of bacteria to the proposed DNA sequence via the 16S analysis tool.

Successful results were also observed with strain 3A and 2B, while the BLAST sequences for these two retrieved 99% similarity between the tester strain’s DNA sequences and the BLAST 16S database [1].  It is important to note that not 100% similarity was observed with any of the BLAST results – which may be a cause of the PCR DNA sequences were not completely accurate.  Strain 3A’s 16S analysis was in line with strain 1A’s, as they were both of the Klebsiella genus.  2B’s 16S analysis, however, indicated a different genus than strains 1 and 3A.  2B’s results suggested that it was in the genus Massilia, another common soil bacteria [2].

Does your gram stain agree?

My gram staining agreed for the Gm+/-, but I found a more difficult time in identifying the shape of the individual bacterial cells. The gram staining that I produced suggested that samples 1A and 2B were coccus-shaped Gm- bacteria, while sample 3A was a rod-shaped Gm- bacteria (Figures 3, 4, and 5).  1A and 2B analysis contradicted the 16S analysis, however, after analysis of the strains with BLAST [1].  It was observed that all three bacteria strains were rod-shaped Gm- bacteria.  A reason for this mislabeling may have been that the bacteria cells were simply too small to accurately identify their shapes.

Figure 3: Gram staining from bacteria colony sample 1A showing Gm- rod-shaped Klebsiella.

Figure 4: Gram staining from bacteria colony sample 2B showing Gm- rod-shaped Massilia.

Figure 5: Gram staining from bacteria colony sample 3A showing Gm- rod-shaped Klebsiella.

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

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

Klebsiella bacteria are an aerobic bacteria that are part of the phylum proteobacteria.  They are of the class gamma proteobacteria, order Enterobacteriales, family Enterobacteriaceae, and genus Klebsiella [3].  They are gram negative rod-shaped bacteria that are often found in soil.  They do not require any special nutrients to grow, as they can survive off of citrate, glucose, and ammonia; this makes them effective at growing in low-nutrient environments.  They are normally found in the nose, mouth, and gastrointestinal tract in humans, but they can also be spread to other parts of the body to cause diseases like pneumonia and urinary tract infections.

b) Information regarding antibiotic production in this genus.

Not much information is known about the production of antibiotics in the genus Klebsiella, but they are the subject of antibiotic research as they are growing more and more resistant to normal antibiotics.  Their ability to survive in low-nutrient environments allows them to stay alive in dire environments, and they have recently evolved an ability to cleave carbapenem antibiotics with an enzyme carbapenamase.  This enzyme has rendered the entire line of carbapenem antibiotics useless against Klebsiella bacteria[4].  It is mutations like this that has researchers working toward discovering new antibiotics, because bacteria are constantly evolving to survive – posing a greater threat to humanity every day.

Works Cited

1. “BLAST: Basic Local Alignment Search Tool.” National Center for Biotechnology Information, U.S. National Library of Medicine.
2. Shen, Liang, et al. “Massilia Eurypsychrophila Sp. Nov. a Facultatively Psychrophilic Bacteria Isolated from Ice Core.” International Journal of Systematic and Evolutionary Microbiology, Microbiology Society, 1 July 2015.
3. Thomas, Gavin. “Klebsiella Pneumoniae.” What Is Electron Microscopy?.
4. Daikos, George L., et al. “Carbapenemase-Producing Klebsiella Pneumoniae Bloodstream Infections: Lowering Mortality by Antibiotic Combination Schemes and the Role of Carbapenems.” Antimicrobial Agents and Chemotherapy, American Society for Microbiology Journals, 10 Feb. 2014.

PCR resulted in successful amplification of two of my three isolates of interest (#5 and #11). Although #18 inhibited both Gram positive and Gram negative tester strains, BLAST sequence analysis could not be done to determine the genus, because no DNA was amplified to sequence! Additionally, neither Gram staining nor extraction were performed for isolate #5. Thus, here I identify and discuss the genus of isolate number 11 for which Gram stains and BLAST were both successfully performed.

Streak plate of isolate 11.

What were the results of your 16S analysis? 
BLAST retrieved a list of organisms (genus and species) that contain a high level of sequence similarity in the DNA region coding for the 16S rRNA subunit (693 base pairs analyzed). The results are seen below… and my microbe is…Bacillus! The genus Bacillus dominated the sequence similarity results in both BLAST and the Ribosomal Database Project (RDP) (results seen below). Both proposed a list of species and strains within the Bacillus genus that demonstrated similarity in 16S sequence. Bacillus subtilis was the most common species to appear in these lists, in a variety of strains. This is interesting because Bacillus subtilis was one of our safe-analog ESKAPE tester strains! A few such proposed strains were Bacillus subtilis NBRC 13719, Bacillus subtilis DMS 10, and Bacillus subtilis IAM 12118. However, Bacillus strain identification is limited to the species level in 16S rRNA analysis (1).

Another species that was listed as similar was Bacillus licheniformis, which has strains known to produce antibiotics such as bacitracin and bacteriocin (1). Since isolate 11 inhibited tester strains, it is possible that it was Bacillus licheniformis. Nevertheless, 16S results definitely point to Bacillus.

BLAST results listing organisms with highly similar sequences of DNA coding for 16S rRNA. Genus and species of similar organisms are included.

RDP results, affirming the genus as Bacillus in accordance with BLAST. Bacillus subtilis appears often in the list of similar strains, as it did in BLAST. This suggests isolate 11 may be a strain of Bacillus subtilis.

Does your gram stain agree? 
YES! My Gram stain showed that isolate 11 was Gram positive rods and upon close examination it appeared that they formed spores. We also know that this isolate must have been aerobic since it grew in our aerobic conditions.

The genus of Bacillus contains Gram-positive, spore-forming, rod-shaped, aerobic bacteria (1). All of this aligns with what we know to be true about my isolate 11! Thus, I feel comfortable claiming that isolate 11 is some species of Bacillus.

Isolate number 11. Gram positive rods. Looks like they might be spore formers.

Gram positive rods, just like isolate 11. Form endospores which resemble the spore-looking characteristic of isolate 11. https://www.medschool.lsuhsc.edu/Microbiology/DMIP/dmex17.htm

This streak plate of Bacillus subtilis found online resembles closely the morphology of my isolate 11 streaks in particular the color and unique pattern of growth on the edges of colonies. https://www.scienceprofonline.com/science-image-libr/sci-image-libr-bacterial-colonies.html

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

Taxonomic classification. Domain: Bacteria; Phylum: Firmicutes; Class: Bacilli; Order: Bacillales; Family: Bacillaceae 1; Genus: Bacillus

These are Gram-positive, spore-forming, rod-shaped, aerobic bacteria (1). They are widely distributed in the soil as well as the aquatic environment (2). Their ability to inhabit diverse habitats results in part due to their spore forming ability (2). Some Bacillus species inhabit extreme environments such as high temperature or extreme pH since spores can survive dormant in extreme environmental conditions (3). Bacillus can also survive in low-nutrient environments, including a lack of elements such as phosphorus, nitrogen or oxygen, provided that the organism has a sufficient supply of carbon sources (1). Bacillus represent heterogeneous species that also form bioactive polymers useful in industry including medicine, biodefense, biofuels, and bio-pesticides. For example, Bacillus thuringiensis is used for biological control of insects to protect crops (1). Spores of Bacillus subtilis have also been used as probiotics in humans (1).

b) Information regarding antibiotic production in this genus. 

The number of antibiotics produced by the genus Bacillus is in the hundreds (4). Many of these antibiotics are peptide antibiotics, which tend to be smaller than proteins but range in molecular weights from 270 (bacilysin) to 4500 (licheniformin) (4). 66 different peptide antibiotics are elaborated by strains of Bacillus subtilis (4) and 23 by Bacillus brevis (4). Most of these peptide antibiotics are made exclusively from amino acids, but some contain additional substituents (4). As mentioned above, the species Bacillus licheniformis is known to contain strains that produce antibiotics including bacitracin and bacteriocin (1). Additionally, polymyxin and gramicidin S have been isolated from species of Bacillus. Antibiotics of Bacillus have been seen to be more effective against Gram positive bacteria (4), which is consistent with my observations of isolate 11.

References

(1) Porwal S., Lal S., Cheema S., Kalia V.C. (2009). Phylogeny in Aid of the Present and Novel Microbial Lineages: Diversity in Bacillus. Plos One.

(2) Parvathi A., Krishna K., Jose J., Joseph N., Nair S. (2009). Biochemical and molecular characterization of Bacillus pumilus from coastal environment in Cochin, India. Brazilian Journal of Microbiology. 40(2):269-275.

(3) Nicholson W.L., Munakata N., Horneck G., Melosh H.J., Setlow P. (2000). Resistance of Bacillus endospores to extreme terrestreial and extraterrestrial environments. Microbiology and Molecular Biology Reviews. 64(3):548-572.

(4) Katz E. and Demain A.L. (1977). The peptide antibiotics of Bacillus: Chemistry, Biogenesis, and Possible Functions. Bacteriological Reviews. 41(2):449-474.

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

What were the results of your 16S analysis?

Unfortunately, I was only able to obtain 16S results for one of my isolates, #20, as the other two, #15 and #23 did not work. However, for #20, I was able to run 677 bp of reliable sequence in my Blast and Ribosome Database Project (RDP) searches. Both searches agreed on Streptomyces genus as the identify of my isolate with 99% identity match and 99% query coverage. Further, my searches determined the closest relative to be Streptomyces of the vinaceus or cirratus strains, but more informations and biochemical tests would be needed to distinguish any further.

Does your gram stain agree?

This is a photo of my gram stain for isolate #20 (photos of controls and other info in my “Meet My Microbes!” post). From my gram stain, I determined my isolate to be Gram + with a rod-shape/bacillus and filamentous microscopic morphology. Therefore, my gram stain agrees with my 16S data because Streptomyces is a genus of Gram positive bacteria with a filamentous shape [1].

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

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

b) Information regarding antibiotic production in this genus.

In terms of taxonomy, Streptomyces is the genus of the Streptomycetaceae family, which is further broken down into over 575 species, with more and more still being discovered. Additionally, Streptomyces belongs to the Bacteria Kingdom, Actinobacteria Phylum and Class, and Actinomycetales Order [2]. Streptomyces is known to be Gram positive bacteria that can grow in various environments, and has a filamentous morphology [1]. In their simplest form, Streptomyces are unicellular spheres and rods and are filamentous. However, in more complex forms, they are described as “mycelium of branching hyphal filaments, and reproduce in a mould-like manner by sending up aerial branches that turn into chains of spores” [3]. Streptomyces are primarily found in soil and decaying vegetation, which further confirms that this could be my isolate #20 because I isolated it from my soil sample from a garden [2, 4]. Interestingly, the first Streptomyces are thought to date back 400 millions years ago to when Earth last was first colonized by plants and as such their job was to solubilize the cell walls or other other components of plants, fungi, and insects. This function is further thought to be retained as certain species have genes for proteins that are involved in the binding and degradation of certain carbohydrates[4]. In terms of nutrition, the genus is primarily found in soil and so upon studies for optimum growth of Streptomyces in media, it was determined that various trace elements proved to be important including copper, manganese, and zinc, as well as using sodium nitrate, potassium phosphate, and magnesium sulphate [5].

In today’s society, Streptomyces are of utmost importance as a major source of antibiotics in medical, veterinary, and agricultural use [3]. Further, Streptomyces best known for their ability to produce bioactive secondary metabolites including antifungals, immunosuppressants, and perhaps most importantly in our case, antibiotics. The production of antibiotics is species specific and are important for them to compete with other microorganisms they meet [1]. Finally, the importance of this genus is expressed in the statistic that 80% of antibiotics commonly used today come from the Streptomyces genus [1].

[1] De Lima Procópio, Rudi Emerson, et al. “Antibiotics produced by Streptomyces.” Antibiotics produced by Streptomyces. https://www.sciencedirect.com/science/article/pii/S1413867012001341

[2] Streptomyces, Wikipedia. https://en.wikipedia.org/wiki/Streptomyces

[3] “Streptomyces inside-out: a new perspective on the bacteria that provide us with antibiotics” Philosophical transactions of the Royal Society of London. Series B, Biological sciences vol. 361,1469 (2006)

[4]. “Recent advances in understanding Streptomyces” F1000Research vol. 5 2795. 30 Nov. 2016, doi:10.12688/f1000research.9534.1

[5] Spilsbury, J. F. “Observations on the nutritional requirements of Streptomyces griseus (Krainsky) Waksman & Schatz.” Transactions of the British Mycological Society.

Two out of the three samples I sent out for sequencing came back with results.  My first unknown sample sample was a bit noisy, so I only blasted 687 nucleotides.  The closest genetic match was Acinectobacter baylyi strain B2 (Query 100%, Identity 96%). The majority of the bases that did not match were “n” so they could have been any nucleotide. Ironically, this is one of the tester strains used in lab. Colonies are described as “circular, convex, smooth and slightly opaque,” and the genus has general characteristics such as being aerobic, gram-negative bacilli (Carr et. al.).  This corresponds with my data. I put the picture of my gram stain below, because initially I thought that I had spore formation or a mix of 2 colonies, but with the BLAST data I can probably say this is not spore formation and is indeed gram-negative.

For my second sample, I was able to run 792 bases.  The BLAST results only had members of the genus streptomyces.  The closest match was Streptomyces spororaveus strain NBRC 15456 (query 99%, identity 99%).   Streptomyces spororaveus gram positive, forms dusty gray multicellular complexes, as well as spore chains, which I observed in lab (Podstawka). The spore chains made it hard to pick, spread, and gram stain this organism.  Below is an image of the spore chains from Streptomyces spororaveus, as well as spread plates on various media. My spread plates looked very similar.

Image citation: Joachim M. Wink, HZI – Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany

Streptomyces is in the domain Bacteria, phylum Actinobacteria, class Actinobacteria, order Actinomycetales, and family Streptomycetaceae (Podstawka).  Optimal growth occurs at 28 degrees celsius, and streptomyces are commonly found in soil. (Podstawka). Streptomyces can survive in vastly diverse environmental / nutritional conditions due to their ability to form spores, and commonly form symbiotic relationships with plant roots (de Lima Procópio). “The most interesting property of Streptomyces is the ability to produce bioactive secondary metabolites, such as antifungals, antivirals, antitumorals, anti-hypertensives, immunosuppressants, and especially anitbiotics” (de Lima Procópio).  Over 60% of naturally derived antibiotics were isolated from members of the genus streptomyces.  The first, streptothricin, was discovered in 1942 (Wavte).  It is predicted that members of the genus streptomyces are capable of producing over 100,000 secondary metabolites that have antibiotic properties (Wavte). The antibiotic Streptomicin is derived from S. griseus, and Avermictin is derived from S. avermitilis (de Lima Procópio). Notice below how many antibiotics are from S. ___ indicating they are derived from streptomyces.

Image source: de Lima Procópio, Rudi Emerson. “Antibiotics produced by Streptomyces.” Braz journal of infect dis. 2012;16(5):466–471

Carr, E. L. “Seven Novel Species of Acinetobacter Isolated from Activated Sludge.” International Journal Of Systematic And Evolutionary Microbiology, vol. 53, no. 4, 2003, pp. 953–963., doi:10.1099/ijs.0.02486-0.

de Lima Procópio, Rudi Emerson. “Antibiotics produced by Streptomyces.” Braz journal of infect dis. 2012;16(5):466–471

Podstawka, Adam. “Streptomyces Spororaveus | Type Strain | DSM 41462, ATCC 43694, INMI 101, VKM Ac-318 | BacDiveID:16196.” BacDive | The Bacterial Diversity Metadatabase, bacdive.dsmz.de/strain/16196.

Watve, Milind, et al. “How Many Antibiotics Are Produced by the Genus Streptomyces ?” Archives of Microbiology, vol. 176, no. 5, 2001, pp. 386–390.,

I only got sequencing results for one of my three producers, so the identity of the other two will have to remain a mystery. The one that did work yielded about 725 base pairs of reliable sequence which matched 100% to the 16S ribosomal sequence for the Streptomyces genus. This genus is made of gram positive species and my gram stain results do agree: my gram stain yielded a purple cluster of rod-shaped bacteria set up in chains (see below; image taken from the “Meet my microbes” post – please see that post for further explanation).

As far as taxonomy goes, the organism belongs to the Bacteria kingdom, Actinobacteria phylum, Actinobacteria class, Actinomycetales order, and Streptomycetaceae family (3). Streptomycesis typically live in soil, decaying plant material, and/or water (3,4). They are also known for their quite wonderful smell, which has been called “earthy” and definitely contributed to the smell of the lab that we all became so fond of (3). This genus is known for forming branching chains, as was seen during the gram stain process (4). About 80% of antibiotics used today come from species in the Streptomyces genus, including chloramphenicol, daptomycin, neomycin, streptomycin, and so many more, so production of an antibiotic from my organism is more than plausible, suggesting the experimental method used here was sufficient for our purposes (2,3). Some species also produce antifungals and antiparasitic compounds (3). One study that was done to evaluate the specific nutrient requirements to produce optimal antibiotic production from one species found that they rely on copper, magnesium, zinc, and tryptophan heavily, but excel when provided sodium nitrate, potassium phosphate, and magnesium sulfate (3).

Sources:

What were the results of your 16S analysis?

The 16S analysis results showed the genus of my species is Bacillus. The closest relative of my bacteria, determined by BLAST, is Bacillus subtilis. This relative had a percent identity of 99% and the most common sequence alignment. The phylogenetic tree produced by BLAST explains that other relatives with high percent identity values are close together in the tree. For example, Bacillus glycinifermentans, Bacillus haynesii, and Bacillus sonorensis are all of 99% identity values, but had less similar “hits” or or sequence comparisons than B. subtilis.

Figure 1. Phylogenetic tree, produced by BLAST, showing the relationship between bacteria that produce 99% identity matches. Although they produce similar % identities to B. subtilis, they have less overall sequence similarities to my unknown sample.

Does your gram stain agree?

My gram stain agrees! Bacillus is Gram positive, rod-shaped, spore forming bacterium. My Gram stain shows all of these features. The image below shows the Gram stain of my bacteria. The rods are purple in color with a pink center. The purple color signifies a Gram positive bacteria. The pink center shows that the bacteria is spore-forming.

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

I only have one isolate, so that is the one that I have picked (KM5). The genus is Bacillus. 

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

There are over 200 known species of Bacillus, but only two are known to impact humans. Bacillus can be classified as follows: Kingdom- Bacteria, Phylum- Firmicutes, Class- Bacilli, Order- Bacillales, Family- Bacillaceae. Bacillus are rod-shaped, Gram positive, endospore-forming bacteria. The bacteria that has been isolated in lab, as well as most of the Bacillus genus is aerobic. There are some species that act as facultative anaerobes. Due to most species being aerobic, their nutritional well-being depends on having oxygen. To achieve optimal growth in a lab setting, Bacillus prefers a rich media. With that being said, the genus would grow best in its natural environmental conditions and it is hard to replicate those. Their optimal temperature range for growth and life is between 25-35 degrees Celsius. Finally, the genus are known to inhabit soil. However, they can also be found in aquatic environments. Some species, such as B. subtilis can be found naturally in the human gut.

b) Information regarding antibiotic production in this genus.

The Bacillus species produces antibiotics. Antibiotics act as the bacteria’s “fight” mechanism. Some examples of common antibiotics that the Bacillus genus excretes are polymyxin, bacitracin, and gramicidin. The antibiotics that the Bacillus genus produces are effective against both Gram positive and Gram negative bacteria. From my experiments, I determined that my isolate was only effective against Gram positive bacteria. This is not unusual for a variety of species of Bacillus. The genus can also have other antimicrobial uses, such as being an anti-fungal.

Sources:

https://www.sciencedirect.com/science/article/pii/S0944501305000704

https://www.ncbi.nlm.nih.gov/books/NBK7699/

https://www.flandershealth.us/microbiology/i-characteristics-of-bacillus-subtilis.html

http://www.heathermaughan.ca/resources/Maughan%26vanderAuwera11.pdf

https://microbewiki.kenyon.edu/index.php/Bacillus_subtilis

https://www.ncbi.nlm.nih.gov/blast/treeview/treeView.cgi?request=page&blastRID=Y38Z9EVS014&queryID=lcl|Query_28503&entrezLim=&ex=&exl=&exh=&ns=100&screenWidth=1280&screenHeight=800