Rhodococcus erythropolis: An Environmental Microbe Isolation Study
Microbes are an omnipresent living force found in every environment and habitat on the planet, from deep below the ocean in the crushing darkness at hydrothermal vents, to within human bodies and floating in the air around us. The diversity of microbes, especially bacteria, is staggering and constantly shifting through the tremendous evolutionary prowess and genetic flexibility of these organisms.
For the purposes of this research, I chose to investigate the microfauna of my aquarium biome. I have worked to culture a realistic and comfortable habitat for the organisms I keep, which has involved obtaining live plants, cultivating algae, using a heater to maintain constant temperature, and using a LED lamp to mimic sunlight. Nitrogen fixing and ammonia-oxidizing bacteria are necessary for a healthy aquarium environment (Urakawa et al., 2008)(Zehr et al., 2000), and bacteria considered pathogenic to fish and aquatic plants are perpetually present in the water (Baran et al., 2008)(Smith et al., 2012).
My goal was to isolate and identify a bacterial strain from my aquatic microbiome, then determine the likely role it plays within the system I have worked to cultivate. To do so, I sampled the aquarium water, allowed the bacteria to grow colonies from which to extract pure culture, and isolate a pure culture from which to run both genomic and physiological tests upon. I theorized the microbe I isolated is a form of nitrogen-fixing bacteria, as my tank is a healthy system with no diseased fish or plants, and has been thriving with minimal water changes, a sign of nitrogen cycling within the ecosystem.
However, the microbe I identified was Rhodococcus erythropolis, a versatile and occasionally pathogenic bacteria associated with aquatic and soil environments. This was somewhat surprising due to the predicted relative rarity of R. erythropolis in comparison to other microbial populations, such as nitrogen fixers in aquatic environments, but considering the methods I used, the bacteria I chose had already been through a selective process once I decided to isolate.
Methods
Sampling and Growth
The aquarium biome was sampled by submerging a sterile swab of cotton into the water and gently swiping it across a Betta splendens (Betta fish). The swab was then wiped across a plate of sterilized SA agar and left to incubate at room temperature for 6 days. Bacterial growth appeared after 72 hours in three small colonies. The SA plate had initially been intended to select for fungal growth, but I chose to isolate and study the acidophilic bacteria growing on the plate instead.
These bacterial colonies were isolated into pure culture by the quadrant streak method (Lab 2 Handout). Four successive streak plates were made over the course of 3 weeks, and were incubated at 37 °C during the growth phase of the bacterium.
Identification
When the isolate was sufficiently purified, a Gram stain was conducted to determine if the strain is Gram-positive or Gram-negative. This test was conducted according to the methods of Lab Handout 4.
The isolate was also tested for a variety of physiological abilities. A fluid thioglycollate test was used to determine the strain’s oxygen class, a catalase test establishes whether a bacterium contains the catalase enzyme, and a oxidase test to see if the isolate contained cytochrome c oxidase. An API 20E test strip was additionally used to test the isolate’s capacity for fermentation of various sugars, the presence of specific enzymes, and decarboxylations of amino acids, with 20 tests in total. All physiological tests were completed according to the methods of Lab Handout 6.
To obtain a more specific idea of the bacterium’s identification, DNA extraction and whole genome sequencing was conducted on the organism. Extraction was completed with a freshly streaked isolate and the PowerSoil Isolation kit according to the methods of Lab Handout 5. Genomic sequencing was completed in the Illumina MiSeq DNA sequencer at the University of Alaska-Fairbank’s DNA Core Lab, and analyzed in BaseSpace with SPAdes Genome Assembler and Kraken metagenomics according to the instructions of Lab Handout 6. These tests would determine the closest genetic match to the extracted DNA, and how many contigs, functional genes, and tRNA regions are identified, as well as what each section coded for, when relevant.
Unfortunately, after genomic extraction, the original isolate was lost. I attempted to reisolate the same bacteria again through similar sampling methods and selection of a colony of visually identical microbes, but was unsuccessful. Therefore, any antibiotic resistance by my original strain remains unknown, as well as its ability to ferment lactose or sugars, as would have been tested in Labs 8 and 9.
Results
This isolate was observed to grow most quickly at 25 °C. Colonies were matte white and circular with defined edges and a convex shape. This bacteria was able to grow under both refrigeration and at 37 °C, though slowly.
Gram staining of the isolate showed the microbe as a gram positive bacillus. The rods most commonly appeared clustered in pairs (diplobacilli), bent at an angle so as from a distance they appear to be one spiral bacteria. The individual bacilli are approximately 0.5μm long.
Figure 1. Isolate gram-stained and magnified at 1000X. This organism is clearly gram-positive and is clustered in pairs with few exceptions.
In physiological tests, the isolate was positive in the catalase and oxidate tests. (Acharya, 2015). Complete results for the API 20E test strip are listed in Table 1. The ONPG test (tests for β-galactosidase enzyme) was inconclusive due to an unfixable air bubble that hindered results, and is not included.
| Test | Result |
| ADH- determines decarboxylation of the amino acid arginine by arginine dihydrolase | Negative |
| LDC- determines decarboxylations of the amino acid lysine by lysine decarboxylase | Negative |
| ODC- determines decarboxylations of the amino acid ornithine by ornithine decarboxylase | Negative |
| CIT- detects utilization of citrate as only carbon source | Negative |
| H2S- detects production of hydrogen sulfide | Negative |
| URE- detection of enzyme urease | Positive |
| TDA- detection of the enzyme tryptophan deaminase | Positive |
| IND- detection of production of indole by the enzyme tryptophanase | Positive |
| VP- determines if fermentation of glucose by bacteria utilizing the butylene glycol pathway is being utilized | Positive |
| GEL- tests for the production of the enzyme gelatinase | Negative |
| GLU- detects fermentation of glucose (a hexose sugar) | Positive |
| MAN- detects fermentation of mannose (a hexose sugar) | Positive |
| INO- detects fermentation of inositol (a cyclic polyalcohol) | Positive |
| SOR- detects fermentation of sorbitol (a alcohol sugar) | Positive |
| RHA- detects fermentation of rhamnose (a methyl pentose sugar) | Positive |
| SAC- detects fermentation of sucrose (a disaccharide) | Negative |
| MEL- detects fermentation of melibiose (a disaccharide) | Negative |
| AMY- detects fermentation of amygdalin (a glycoside) | Negative |
| ARA- detects fermentation of arabinose (a pentose sugar) | Negative |
Table 1. A complete listing of the results from my API 20E test strip for my isolate (Acharya, 2015).
In the fluid thioglycollate test, the isolate proved able to survive in all areas of oxygenation, but preferred to grow at the surface, making it a facultative aerobe. Growth was thickly concentrated at the surface and directly below the surface of the agarose, but a thin line of microbial growth was visible extending from the surface to the base of the tube.
Genomic tests indicated only approximately 70% of genetic data was usable, as the other data was not even classified as bacteria in the analysis. However, within the usable information, the confidence level of the identified sequences were 95% or greater. Complete percentages of taxonomic rank are illustrated in Table 2, indicating a high level of precision in the identification of the isolate as Rhodococcus erythropolis (BaseSpace, 2017).
| Kingdom | Phylum | Class | Order | Family | Genus | Species |
| Bacteria | Actinobacteria | Actinobacteria | Actinomycetales | Nocardiceae | Rhodococcus | erythropolis |
| 99.11% | 98.41% | 98.41% | 98.34% | 95.99% | 96.05% | 95.91% |
Table 2. The confidence levels of identified sequences within the bacterial genome and the identified classifications. Note all of these values are derived from ∽70% of total data, but the remaining 30% of data was completely unidentifiable, even to the kingdom level, and as such has been disregarded in this interpretation.
Only one contig of 1420bp was identified. Additionally, only one tRNA was observed, and 169 coding regions.
Lastly, BLAST analysis was used as a secondary classification system to check the accuracy of results from Kraken metagenomics. In BLAST, the result for nucleotide analysis on contigs came back as 97-94% match for R. erythropolis, confirming the Kraken results (National Center for Biotechnology Information, 2017).
Discussion
The results of the genomic tests led me to believe the strain I isolated was a variety of R. erythropolis, despite having relatively small amounts of genetic data to work with. The lack of contigs, functional genes, and tRNA leaves room for there to be a considerable amount of error in the identification, but the certainty of identification for what is present from the sample is considerably confident.
Additionally, if we are to analyze the characteristics of R. erythropolis, it is a Gram-positive, mesophilic, aerobic rod bacteria. Interestingly, it was recently observed to be a human pathogen under specific circumstances, causing septicemia in immunocompromised individuals (Park et al., 2011). Ironically, a fish in the tank I sampled died of septicemia earlier this year, though there is no means to determine if R. erythropolis was responsible for the death.
These organisms are commonly found in both soil and aquatic environments, as well as eukaryotic cells. In wild environments, they are known for utilizing a wide variety of organic compounds. Different strains have demonstrated the ability to undergo oxidations, dehydrogenations, epoxidations, hydrolysis, hydroxylations, dehalogenations and desulfurization in order to process the resources of their habitat (de Carvalho, 2005). Due to this diversity, Rhodococcus sp. are considered important industrial organisms, utilized in production of bioactive steroids and fossil fuel biodesulfurization, and is also regarded as the most commercially successful microbial biocatalyst (McLeod, 2006).
Comparatively, my isolate demonstrated several traits associated with Rhodococcus species. My isolate was gram-positive and grew in rods. The diplobacilli arrangement noted in my isolate was never mentioned as a distinguishing factor of R. erythropolis or other Rhodococcus species. I collected my sample from an aquatic environment, and though the environment I collected it from was warm, I found the bacterium grew much more successfully at room temperature. Additionally, my bacteria was established as tolerant of acidic environments from the initiation of the experiment. R. erythropolis is not characterized as an acidophile, so I believe it is an ability it possesses, but as demonstrated by growth on TSA plates and TSB, not the preferred environment of the organism.
Physiological tests revealed my bacteria sample can process urea, produces the enzymes tryptophan deaminase and tryptophanase, and can ferment a variety of simple sugars, but does not appear to have the dramatic abilities some R. erythropolis strains, though some were never tested. Although my isolate thrived in an aerobic environment, a characteristic of Rhodococcus sp., it also showed the capacity to persist in all oxygen zones, something not all Rhodococcus sp. can do.
Due to the genomic and physical evidence presented, I believe my isolate is a variety of Rhodococcus, though it may be debatable whether it is R. erythropolis, considering the lack of additional genomic data, and the diversity of the Rhodococcus genus. It is commonly acknowledged that Rhodococcus is environmentally widespread, and many more species may be present than currently documented in journals or databases (Park, 2011).
Several tests were conducted in lab research on other isolates which were not possible to do on my bacterium, due to its unfortunate and premature loss from the lab samples. As such, there can be no further research on this specific organism, though if there were, would like to investigate its abilities in several other ways.
I especially would have liked to analyze antibiotic resistance of my isolate. As it is an environmental microbe, especially one which can compete in the same pH zone as fungi, I would imagine it would have resistance to fungal antibacterials so as to better contend with fungi living in the same niche. Additionally, since it has potential to be pathogenic, it would have been beneficial to see what resistance or susceptibility it may have.
Though it would have been interesting and insightful to test my isolate’s ability to process petroleum, considering the use of R. erythropolis as a biodegradation, this was an opportunity not presented to me in this study. Another experiment I would have liked to complete in future research is analysis of the pathogenic potential of my specific strain. As there are multiple accounts of R. erythropolis causing septicemia in patients, perhaps analyzing growth on blood agar would show how quickly an infection of my isolate could become threatening.
In conclusion, my bacterium sampled was not a nitrifying organism, as I had predicted. Rather, it is a decomposer with the capacity to degrade a variety of environmental compounds, likely taking full advantage of the waste products and other organic matter present in the aquarium biome I took it from. There is a wide range of further experiments I would have liked to have conducted on this organism, but due to the follies of lab, I have, for the moment, lost the ability to further study this opportunistic organism. However, this entire study was a reinforcement of the staggering amount of diversity within the microbial world and the startlingly prolific world just out of our sight at all times.
References
Archarya, T. (2015). API 20E Test System: Introduction, Procedure Results and Interpretations. Retrieved from https://microbeonline.com/api-20e-test-system-introduction-procedure-results-interpretations/ on April 2, 2017.
Baran V., Dvorska L., Matlova L., Pavlik I., Svastova P. (2008). Distribution of mycobacteria in clinically healthy ornamental fish and their aquarium environment. Journal of Fish Diseases 29: 383—393.
de Carvalho, C.C.C.R., da Fonseca, M.M.R. (2005). The remarkable Rhodococcus erythropolis. Applied Microbiology and Biotechnology 67: 715—726.
Illumina BaseSpace. (2017). SequenceHub. Retrieved from https://basespace.illumina.com/dashboard on April 2, 2017.
McLeod, M.P., et al. (2006) The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proceedings of the National Academy of Sciences of the United States of America 103: 15582—15587.
National Center for Biotechnology Information. (2017). BLAST Nucleotide Sequence. Retrieved from https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome on April 2, 2017.
Park, S.D., Uh, Y., Jang, I.H., Yoon, K.J., Kim, H.H., Bae, Y.J. (2011). Rhodococcus erythropolis septicaemia in a patient with acute lymphocytic leukaemia. Journal of Medical Microbiology 60: 252—255.
Smith K., Schmidt V., Rosen G.E., Amaral-Zettler L. (2012). Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water. PLOS One: https://dx.doi.org/10.1371/journal.pone.0039971.
Urakawa H., Tajima Y., Numata Y., Tsuneda S. (2008). Low Temperature Decreases the Phylogenetic Diversity of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofiltration Systems. Applied and Environmental Microbiology 74: 894-900.
Zehr J.P., Carpenter E.J., Villareal T.A. (2000). New perspectives on nitrogen-fixing microorganisms in tropical and subtropical oceans. Trends in Microbiology 8: 68-73.
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