Identifying Bacteria in the Canine Oral Microbiome
A study conducted in 2015 (by Oh C, et al) found that there is little to no transmission from pets to their owners, but the findings of this study contradict that of another study conducted in 2009 (by Oehler RL, et al) which did find a transmission risk when bacteria were able to get past the skin, such as through bites or some licks. Does the environment in which they are kept play a role in the oral microbiome of dogs?
We intend to discover whether there is a difference in the oral microbiomes of dogs based on the environment in which they are kept. We hypothesize that the dogs found in the animal shelter will carry the most harmful types of bacteria. This hypothesis is based on the knowledge of the animal shelter environment and the conditions that the animals are kept in because of the surplus of dogs and the lack of employees and volunteers. The conditions that shelter dogs are kept in are quite subpar in comparison to dogs that are placed in normal, stable conditions such as in a household with a primary caretaker.
Introduction
Materials:
Culture swabs, conical tubes, sterile PBS, TSA plates, eppendorf tubes, PCR tubes, chem glass beads, solution 5, solution 6, solution 7, spin filter, spin column, micropipettes, micropipet tips, master mix, inoculating loops, sharpie, centrifuge, vortexer, heat block, PCR thermocycler, spectrophotometer, loading dye, 1 kb DNA ladder, nucleic acid gel stain, 0.8% agarose gel, gel electrophoresis tray, gel electrophoresis box, TAE buffer, gel electrophoresis comb, gel electrophoresis imager.
Method:
Methods and Materials
Results
We hypothesized that the conditions in which dogs are kept influences their oral microbiome, such that dogs raised in less hygienic environments have more dangerous and more commutable bacteria. Our findings supported our hypothesis. Of the samples collected from dog one, a shelter dog, three of them are zoonotic pathogens that are very serious and can even be fatal. One of them, Bacillus anthracis, is the bacteria responsible for the anthrax disease. The bacteria isolated from the third dog, a dog in a retail store, was generally safe. Two of the bacteria affect humans if they are immunocompromised or if they infect an injury. The second dog which was a house dog, contained mostly bacteria that is non transmissible and one that can be spread through a bite.
These findings generally do support or hypothesis, since the majority of the dangerous and zoonotic microbes were cultured from the dog kept in the poorest conditions. The bacteria that were isolated from the dogs’ saliva are clearly not all of the bacteria that can be found, but the bacteria that were found do seem reasonable and potentially representative of the respective proportions found in the oral microbiome. The Staphylococcus, Bacillus, and Corynebacterium genera are some of the the most commonly found bacteria in canines.
One way the experiment’s reliability could be improved is to take more saliva samples from each dog and have more dog participants. The saliva could also be cultured on different types of media to help demonstrate the full range of variety in the bacteria. A way to improve the validity of the experiment is to have more controls, such as the species, age, and gender of the dogs selected for saliva samples. If possible, it would also be beneficial to have all of the dogs on the same diet, since there is a correlation between diet and microbes found in the mouth, stomach, and digestive tract.
Discussion
Earl, A. M., Losick, R., & Kolter, R. (2008). Ecology and genomics of Bacillus subtilis. Trends in microbiology, 16(6), 269–275. doi:10.1016/j.tim.2008.03.004
Ghasemzadeh, I., & Namazi, S. H. (2015). Review of bacterial and viral zoonotic infections transmitted by dogs. Journal of medicine and life, 4 (1-5). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5319273/
Grandolfo E. (2018). Looking through Staphylococcus pseudintermedius infections: Could SpA be considered a possible vaccine target?. Virulence, 9(1), 703–706. doi:10.1080/21505594.2018.1426964
McDowell RH, Sands EM, Friedman H. Bacillus Cereus. In: StatPearls. Treasure Island (FL): StatPearls Publishing; Jan 2019. Retrieved from: https://www.ncbi.nlm.nih.gov/books/NBK459121/
Oehler, R.L., Velez A.P., Mizrachi, M., Lamarche, J., Gompf, S. (2009). Bite-related and septic syndromes caused by cats and dogs. The Lancet: Infectious Diseases, 9:7 (393-454). doi: 10.1016/S1473-3099(09)70110-0
Oh, C., Lee, K., Cheong, Y., Lee, S. W., Park, S. Y., Song, C. S., … Lee, J. B. (2015). Comparison of the Oral Microbiomes of Canines and Their Owners Using Next-Generation Sequencing. PloS one, 10(7), e0131468. doi:10.1371/journal.pone.0131468
Stoica C., Sorescu I. ABIS Online Encyclopedia - Advanced Bacterial Identification Software. Retrieved from: http://www.tgw1916.net/ABIS/encyclopedia.html
Swe, T., Naing, A. T., Baqui, A., & Khillan, R. (2016). Methicillin-Resistant Staphylococcus schleiferi Subspecies coagulans Infection in a Patient With Hepatocellular Carcinoma. Journal of investigative medicine high impact case reports, 4(3), 2324709616671148. doi:10.1177/2324709616671148
References
Justin Lumerman, Madison Ragazzone, Stephen Sedwick, Rayhanah Waldin
University of Florida, MCB3023L, Fall 2019
Dog 1 (Environment: Shelter) | ||
Sample | Organism | Description |
Sample 1.1 | Bacillus subtilis | Gram positive. Commonly found in gastrointestinal tracts of animals and soil, near plant roots. Can form biofilms. Produces antibiotics. Used as a probiotic. |
Sample 1.2 | Bacillus cereus | Gram positive. Toxin producing. Commonly found in soil and produce. Quickly multiplies at room temperature. Causes intestinal illness. |
Sample 1.3 | Bacillus thuringiensis | Gram positive. An endospore. Widespread and commonly found in soil. �An Insect larvae pathogen and used as a bio-pesticide. |
Sample 1.4 | Bacillus anthracis | Gram positive. Inhabits the soil in sporulated form. Isolated from blood of animals with anthrax. Pathogenicity factors include: capsules, enzymes, and toxins. |
Dog 2 (Environment: House) | ||
Sample | Organism | Description |
Sample 2.1 | Staphylococcus pseudintermedius | Gram positive and typically isolated from dogs, cats, horses, and parrots. It is the most prevalent inhabitant of the skin and mucosa of dogs and cats, as well as the major bacterial pathogen causing skin and ear infections. |
Sample 2.2 | Microbacterium maritypicum | Gram positive. Isolated from sea water and marine mud. Undetermined pathogenicity. |
Sample 2.3 | Neisseria canis | Gram negative. Isolated from oral mucosa of dogs, throats of cats, and cat-bite wounds in humans. Opportunistic pathogen. |
Sample 2.4 | Corynebacterium sputi | Gram positive. Is generally non-pathogenic, but it can cause canine otitis externa (ear infections) and can cause infection in human bites. In certain circumstances can lead to pneumonia. |
Dog 3 (Environment: Retail) | ||
Sample | Organism | Description |
Sample 3.1 | Corynebacterium auriscanis | Gram positive. Is generally non-pathogenic, but it can cause canine otitis externa (ear infections) and can cause infection in human bites. |
Sample 3.2 | Staphylococcus schleiferi | Gram positive. Causes skin and ear infections in dogs. Causes nosocomial infections mostly in immunocompromised humans. |
Sample 3.3 | Staphylococcus pseudintermedius | Gram positive and typically isolated from dogs, cats, horses, and parrots. It is the most prevalent inhabitant of the skin and mucosa of dogs and cats, as well as the major bacterial pathogen causing skin and ear infections. |
Sample 3.4 | Staphylococcus pseudintermedius | Gram positive and typically isolated from dogs, cats, horses, and parrots. It is the most prevalent inhabitant of the skin and mucosa of dogs and cats, as well as the major bacterial pathogen causing skin and ear infections. |
Figure 1: This figure depicts the pure culture isolations of 4 different types of bacteria from the swabs of Dog 1’s mouth. Dog 1 lives in an animal shelter.
Figure 2: This figure depicts the pure culture isolations of 4 different types of bacteria from the swabs of Dog 2’s mouth. Dog 2 lives in a house.
Figure 3: This figure depicts the pure culture isolations of 3 different types of bacteria from the swabs of Dog 3’s mouth. Dog 3 lives in a pet shop.
Figure 1:
Figure 2:
Figure 3:
Figure 4: A chart displaying the quality of the results of our DNA sequencing results indicated by the QC and length values. The sequencing was completed by Eton Bioscience Inc. on 22 November 2019.
Legend:
The QC scores only give an overall picture on data quality. QC Value: Average basecall quality value
QC20+: Total number
of bases in entire trace with basecaller quality Value of greater than
of 20.
Figure 5: A successfully imaged gel to confirm the isolation of the 16S rRNA permitting PCR to be run and DNA sequencing to be completed. Notice that the bands are consistent and fluoresce around 1200 base pairs.