Are You Sure You Want to Share That?

1. Are You Sure You Want to Share That? By: Sarah Akkoush and Sao-Mai Nguyen-Mau BSCI223H 0101 May 10, 2006

2. Background: Food and water as vehicles for transmission. • Microbes can live in food and water • Normal flora from one person can contaminate a food item that is consumed by another person • Food can be contaminated with disease causing microbes that when consumed, can cause illness • Ingestion of pathogenic microbes can cause illness. • Food poisoning – ingestion of an exotoxin produced by a microbe and symptoms are usually quick • Food infection – ingestion of microbes that colonize inside the body

3. Our project: • Consumption of different liquids • Examination of plated liquid samples for diversity of bacteria • Measurement of increasing amount of bacteria as volume decreases

4. Hypothesis: • The consumption of liquids deposits bacteria from the mouth into the liquids and as the volume decreases, the amount of bacteria increases. • Based on the following information: • Transmission of bacteria is facilitated through close living spaces and shared oropharyngeal secretions (saliva) in drinks, cigarettes, and utensils (Imrey et al, 1995). • Meningitis prevention has advocated the discretion of sharing beverages (“Meningococcal Disease”, 2005). • Based on the fact that dental personnel are required to wear protective gear to protect the personnel from “splatter” and the patient from “droplets” (Kohn, et al., 2003).

5. Method/Protocol:  Approach: Consume different types of beverages in different volume intervals, taking a sample from each volume to be plated and analyzed.  Rationale: By taking this approach, it can be determined whether or not the amount of bacteria increased as the volume of liquid consumed increased. Also, the approach is conducive to determining whether or not the diversity of organisms present increased as the volume of liquid consumed increased.

6. Methods • Obtain beverages to be tested: water, orange juice, milk, diet soda (all in sealed bottles) • Divide bottles into fourths by volume, with the last fourth being subdivided once more • Drink each beverage by taking multiple sips until each volume interval is reached (0, ¼, ½, ¾, and 7/8) • Plate 250l of the liquid on TSA for each of these interval • For the 7/8 sample, plate an additional 250l on each of the following: Mannitol Salt, MacConkey’s medium and blood agar. • Incubate all plates at 37 for 48 hours • Observe plates and select colonies to be gram stained for microscope viewing

7. TSA  0 1/4 1/2 3/4 7/8  Blood Agar Mannitol MacConkey’s

8. Expected Results • -  - For all beverages, the number of bacteria will increase as the amount of liquid consumed increases. The highest number of bacteria will be observed from the 7/8 sample • -   - Diversity of bacteria present will be greatest in the samples taken from the liquid at the bottom of the bottles (7/8 sample) • -         - Normal flora from the human mouth, specifically Streptococcus and Corynebacterium, will indicate that bacteria from the mouth entered the liquid

9. Results:  For water and soda, a clear trend in number of bacteria is apparent. As volume of liquid consumed increased, number of bacteria on TSA plates increased. This is consistent with our expectation.  A clear trend in the number of organisms is not apparent for orange juice and milk. For orange juice, the cell count decreased as volume consumed increased. For milk, the number of organisms was hard to judge due to the expansive nature of the growth on the plates.

10. Prior to consumption. Sample at ¼. Sample at ½. Sample at ¾. Sample at 7/8.

11. Results (cont’d): • As expected, the greatest diversity of organisms was present on platings from the final volume. • Bacteria consistent with Streptococcus was identified • isolation of Gram positive cocci • presence of beta and alpha hemolytic colonies consistent with S. pyogenes and S. pneumoniae

12. 7/8 water sample on blood agar. Green circle represents beta-hemolysis. Black circles represent alpha-hemolysis.

13. Soda on blood agar.

14. Gram negative cocci 1000x Gram positive cocci 1000x

15. Milk on blood. Majority of plate showed beta-hemolysis.

16. Milk before consuming 1000x. Milk from sample at end of consumption 1000x.

17. Orange juice on blood agar. White circles are gamma hemolysis. Green circles denote beta hemolysis.

18. Colony that only appeared on ¾ sample of orange juice.

19. Gram stain of unknown colony on ¾ orange juice. 1000x

20. Capsule stain of orange juice colony. 1000x

21. Results (cont’d): • Bacteria consistent with Corynebacterium was identified • Gram positive, curved slender rods in “V” shape • colonies appear convex and semiopaque on enriched media

22. Alpha-hemolytic colony on blood agar of water 1000x Gamma-hemolytic colony on blood agar of orange juice 1000x Beta-hemolytic colony 1000x.

23. Gram stain of brown colony on soda agar plate 1000x. Gram stain of white colony on soda agar plate 1000x

24. Discussion: • Indicator organisms • Streptococcus • Gram positive cocci in pairs or long chains • Facultative anaerobes • Exhibits alpha and beta hemolysis, depending on the species • Found in the normal flora of the mouth and upper respiratory tract • S. pyogenes and S. pneumoniae are the species that commonly cause disease in humans

25. Corynebacterium • Gram positive straight to slightly curved rods forming V- or L-shapes • Colonies are convex and semiopaque with a matted surface. • Found on the mucous membranes • Corynebacterium diphtheriae causes the disease diphtheria

26. Pneumonia: Inflammation of the lungs do to microbial infection. Can be acquired through contact with an infected person and airborne microbes. Symptoms include: shaking chills, a high fever, sweating, chest pain (pleurisy) and a cough that produces thick, greenish or yellow phlegm Medicinenet.com 2006 Treatment: antibiotics Prevention: take care of yourself, wash your hands, don’t smoke, don’t infect others

27. Strep throat: An infection of the upper respiratory tract, specifically the pharynx. Symptoms: Throat pain, difficulty swallowing, red and swollen tonsils, sometimes with white patches or streaks, swollen, tender lymph glands in the neck, fever, headache Treatment: antibiotics, such as penicillin, amoxicillin, and azithromycin. Unfinished prescriptions can lead to rheumatic fever. A.D.A.M. Medical Illustration Team 2004 Prevention: wash your hands and do not share drinks with infected person

28. Diphtheria Contagious infection of the throat and nose. Symptoms are caused by toxins secreted by the microbe. Transmission is through contact with infected individuals. Symptoms: sore throat and mild fever, membrane forms around throat and tonsils, lymph glands and tissue on both sides of the neck to swell a large size. Treatment: antibiotics (erythromycin) and antitoxins Virtual Pediatric Hospital Prevention: diphtheria vaccine

29. Significance of ‘backwash’: possible transmittance of pathogenic bacteria, such as S. pneumoniae, S. pyogenes, and C. diphtheriae. growth of bacteria from samples in experiment indicates that oral bacteria can be transferred to a beverage if that beverage is shared, the bacteria can enter another person and cause disease

30. Bibliography Boone, David and Garrity, George (ed.) Bergey's Manual of Systematic Bacteriology. Springer. 2005. Imrey, PB, Jackson, LA, Ludwinski, PH, England 3rd, AC, Fella, GA, Fox, BC, Isdale, LB, Reeves, MW and Wenger, JD. “Meningococcal carriage, alcohol consumption, and campus bar patronage in a serogroup C meningococcal disease outbreak.” Journal of Clinical Microbiology 33(12). 1995. Kohn, W., Collins, A., Cleveland, J., Harte, J., Eklund, K., and Malvitz, D. “Guidelines for Infection Control in Dental Health-Care Settins – 2003.” Morbidity and Mortality Weekly Report 52(RR17). 2003. Center for Disease Control and Prevention. 25 April 2006. http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5217al.htm. Madigan, M. and Martinko, J. Brock Biology of Microorganisms. Upper Saddle River: Pearson Education, Inc. 702.

31. “Meningococcal Disease.” Division for Bacterial and Mycotic Diseases. 2005. Centers for Disease Control and Prevention. May 2006 http://www.cdc.gov/ncidod/dbmd/diseaseinfo/meningococca l_g.htm #Is%20meningitis%20contagious. Prokaryotes. Springer New York, 2004. http://141.150.157.117:8080/prokPUB/index.htm.