alfrary Author

 

Alex Frary

28APR2017

342 F03

Micrococcus luteus and the Catahoula

Introduction

The 21^st century is an exciting time to be a microbiologist with all the new discoveries and advances in technology such as a portable, real-time DNA sequencer. In this new and exciting time, there has also been an increase in public notice and awareness of microbes like bacteria and viruses as well as an increase in access to advanced technology. The general population is now becoming curious as to what microorganisms surround them everyday such as those found in their fridge, on the floors, or even on their pets. As any owner knows, dogs are generally affectionate and have a tendency to express that affection through the transfer of their saliva when licking their owners. And so it leads one to wonder: what exactly is in dog’s saliva? After all, a dog’s mouth can be an ideal breeding ground for all kinds of microorganisms with its warm, moist climate and abundance of food being passed through.

In order to satisfy my curiosity as a dog owner and scientist, I swabbed the inside cheek and gums of my 21 month old Catahoula. One specimen of this sample was then isolated by streaking, characterized by several physiological tests and genome sequencing, and then identified. According to a similar study of a beagle, several genera of microorganisms were identified from the dental plaque of the animal to include Micrococcus, Pseudomonas, and Streptococcus (Wunder 1976). It is among these genera that the specimen taken from the Catahoula would more than likely fall under. My isolate was eventually identified as being Micrococcus luteus. This bacterium is commonly found not only in the human flora, but also in the microflora of skin, mucosa, and oropharynx of mammals (Engelkirk 2008). It is considered an opportunistic pathogen that is pretty much harmless and very common.

 

Methods

Collection and Isolation:

                      Using two sterile swabs, I lightly sampled the inner cheeks and gums of the Catahoula female canine and transferred the samples onto and a TSA plate. Over the next 48 hours, substantial growth of two different types of colonies could be seen on the Tryptic soy agar plate. Choosing the lighter colored bacteria, I used the Streak Plate Method to transfer to a new TSA plate, and over the course of the week, was able to isolate the strain. In between streaks, the plates were kept at 37 degree Celsius in an incubator.

Identification and Characterization:

                      After having isolated the strain, I utilized the Gram-staining process to determine whether the strain was Gram-positive or —negative (as per Lab 4 guidelines). Following this, I went on to use several physiological tests to further characterize the bacteria. The first of which was the fluid thioglycollate test, which I used to determine oxygen class. The next test was the Oxidase Test, which I used to determine if the strain has cytochrome c oxidase (which is helpful in differentiating between pseudomonas species and enteric species). The following test was then the catalase test, which I used to determine if the bacteria has the enzyme catalase. Lastly, the API test strip, which is used to identify enteric Gram negative rods, was then used. The exact methods used for the physiological tests can be found in the Lab 6 Manual.

In order to taxonomically identify the bacteria, I extracted DNA from my isolate using the PowerSoil DNA kit and using techniques described in Lab 5. The genome was then sequenced by the UAF DNA Core Lab Technician using Illumina MiSeq technology. Following the return of the data (bioinformatics) on BaseSpace, I was able to analyze genomic sequencing of my isolate following Lab 7 instructions. The applications used in BaseSpace were Genomic Assembly (to determine what genes are present and what their functions might be), Taxonomic Assignment (to identify isolate to the species/genus level), and the Functional Gene Annotation (to annotate genes and determine the function of those genes). One of the last tests I administered to the isolate was the antibiotic susceptibility test. In this test, I used the Amikacin, Cefazolin, Cefoperazone, Gentamicin, Oxacillin, Tobramycin, Trimethoprim, and Vancomycin disks to observe the zone of inhibition around each disk after being incubated at 37 degrees Celsius for 48 hours (Lab 9).

 

Results

After staining the isolate, the resulting images underneath the microscope showed purple, cocci bundles of bacteria, this lead to the conclusion that the isolate was Gram-Positive (Figure 1). During the physiological testing, the growth in the fluid thioglycollate tube revealed that the bacteria only grew on the top most layer of the tube, suggesting that the isolate is strictly aerobic. After performing the oxidase test, it was conclusive that there was color change on the oxidase test strip, which indicates the isolate contains cytochrome c oxidase. Next was the catalase test in which when hydrogen peroxide was added to the isolate, bubbles appeared. This reaction indicates that the isolate was a reactive oxygen species and contains the catalase enzyme. Following the catalase test, an API 20E test strip was utilized to further characterize the isolate. The results of the strip, however, came out to be negative for each test (Figure 2). It is because of the lack of results that an additional Staph API test strip was then inoculated. The results of this API test strip, however, came out to be negative for all sections just as the previous (Figure 3).

After using the PowerSoil DNA isolation kit to extract DNA from the isolate, the sample was sent off to the UAF DNA Core Lab for sequencing. The results on the BaseSpace application, Kraken Metagenomics, outlined the “Percent Reads Classified’ for “-species level identification-‘ were approximately 25.12%, suggesting that the classification revealed in the program has lower support and can be less certain of the result possibly due to contamination. Based on this data, however, the isolate was identified as (probably) being Micrococcus luteus.

Further testing of the isolate showed the strains antibiotic resistance (Figure 4). Among the antibiotics tested, Amikacin, Cefazolin, Cefoperazone, Gentamicin, and Vancomycin were the ones that the isolate was susceptible to. The isolate showed only intermediate susceptibility to Trimethoprim, and was resistant to Oxacillin and Tobramycin (Table 1).

Figure 1: Microscope view of Isolate

Figure 2: API Test Strip (negative results)

Figure 3: Staph API Test Strip (negative results)

Figure 4: Antibiotic Resistance Test

Antibiotic	Diameter	Result
Amikacin	38mm	Susceptible
Cefazolin	30mm	Susceptible
Cefoperazone	34mm	Susceptible
Gentamicin	36mm	Susceptible
Oxacillin	0mm	Resistant
Tobramycin	8mm	Resistant
Trimethoprim	12mm	Intermediate
Vancomycin	20mm	Susceptible

Table 1: Antibiotic Resistance Results

 

 

Discussion

Although the isolate sample sent in for genetic sequencing did not yield ideal results, due most likely to contamination , the physiological testing results were able to provide additional support that the isolate was indeed Micrococcus luteus. Micrococcus luteus is a Gram-positive, aerobic chemoorganotroph in the Genus: Micrococcus, Family: Micrococcaceae, Order: Antinomycetales, and Phylum: Actinobacteria (Slonczewski 2014).

Micrococcus luteus is also catalase positive, forms yellow colonies, oxidase positive, and also can occur in pairs, tetrads or clusters (Engelkirk 2008). The bacterium can be found in the microflora of skin, mucosa, and oropharynx of mammals, such as humans or Catahoulas as this study found (Engelkirk 2008). It is considered an opportunistic pathogen, meaning that certain strains of the bacteria have been linked to cases involving meningitis, pneumonia, and endocarditis (Whitman 2012).

Alexander Fleming discovered Micrococcus luteus in 1922 while studying an individual with a cold. He tried to find the specific cause of the individual’s runny nose. It was during this that he isolated Micrococcus lysodeikticus (now luteus), and it is now recognized as part of the normal flora of humans and that it is susceptible to lysozyme as Fleming discovered (Moticka 2015). I found the origin story particularly interesting as I found my sample in the saliva of my Catahoula.

In conclusion, I’ve found that the physiological and morphological results during the duration of the study provide strong evidence that my isolate is indeed Micrococcus luteus that I was able to isolate. My isolate was sampled from a yellow colony on the agar plate and is Gram-positive, strictly aerobic, catalase and oxidase positive, cocci clustered, and found in the saliva of a mammal. Based on the corresponding data available on the bacteria, my findings were supported. The end result, however, would have been more substantiated had I not possibly contaminated my DNA extract sample. Overall, proper laboratory technique was learned, and I believe that further studies into the possible pathogenic hazard of Micrococcus luteus should be considered given that it can be found on human skin and in the mouth of a pet and know to have caused illnesses such as meningitis.

 

 

 

REFERENCES

Engelkirk, P., Duben-Engelkirk J. 2008. Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology: p. 216.

Moticka, E. 2015. A Historical Perspective on Evidence-Based Immunology: p. 3.

Slonczewski, J., Foster, J. 2014. Microbiology: An Evolving Science, 3^rd Edition.

Whitman, W., Goodfellow, M. 2012. Bergey’s Manual of Systematic Bacteriology, Vol 5: p. 572.

Wunder, J., Brinner, W. 1976. Identification of the cultivable bacteria in dental plaque from a Beagle dog. Journal of Dental Research.