White, A.

Alexis White Organismic Biology 121 4/16/2013 Foodborne Illness: Listeria Monocytogenes

White, A.

Abstract:
1 out of 6 Americans get sick each year from foodborne illnesses, a condition that causes infection in the gastrointestinal tract results from contaminated foods. One important foodborne illness is listeria which presents 1,600 cases yearly and 250 deaths annually. Our research team designed an experiment to test the effects of sodium lauryl sulfate, liquid smoke, a combination of SLS and LQS, and water as a control against microbial populations in ready- to -eat meats, specifically, hot dogs, over the course of 21 days. Results proved that a combination of SQL and LQS is most effective for decreasing microbial populations in contaminated ready to-eat meats.

Introduction:
Foodborne illness, also christened food poisoning, is any illness arising from a food contagion that harvests an infection in the gastrointestinal tract. Foodborne illness can be parasitic, viral, or caused by bacterial contamination, which results in 39% of food poisoning cases yearly (Scallan, E., Hoekstra, Angulo, F.J., Tauxe, R.V., Widdowson, R., Roy, S.L., Jones, J.L., and Griffin, P.M. 2011.). Bacteria that cause foodborne illness are normally present in most meats and produce, and are spread due to poor food preparation. Most common of these are Salmonella, Campylobacter, and Escherichia Coli. According to the Centers of Disease Control & Prevention, there are

White, A.

over 1,000,000 cases of Salmonella poisoning and about 400 deaths reported each year in the United States. These odds are favorable in comparison to a bacterial infection called Listeria, resulting in roughly 1,600 cases and 250 deaths annually. Listeria, one of the food industrys primary threats, is a ubiquitous disease, caused by Listeria monocytogenes, a gram positive, rod-shaped bacterium (CDC). Individuals with serious medical illnesses, weakened immune systems such as individuals carrying the HIV virus, the elderly, and women who are pregnant, and their fetuses are most vulnerable to Listeriosis (CDC).Listeria is killed during the heating of food, but it can be found in ready- to- eat products such as hot dogs and deli meats (CDC). These foods run a greater risk of infection when they are later repackaged. Two major agents used in combating bacterial infection in the meat packaging industry are sodium lauryl sulfate, and liquid smoke. A 2004 study from Colorado State University titled LISTERIA MONOCYTOGENES IN READY-TO-EAT MEAT PRODUCTS: RISKS, CONTROLS AND EDUCATION FOR PREVENTION, tested for the effects of sodium lauryl sulfate on ready-to-eat meats, specifically, frankfurters. According to the study, bathing frankfurters in a mixture of Sodium lauryl sulfate and Lactic acid greatly reduced the count of bacteria. Sodium lauryl sulfate is effective at killing bacteria because of its protein denaturing properties (Final Report on the Safety Assessment of Sodium Lauryl Sulfate, 1983.). Once the cell membrane is compromised by sodium lauryl sulfate, the cell is susceptible to other foreign agents which will culminate in the death of the cell. Another plausible method for controlling the spreading of Listeria in
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White, A.

the food packaging industry is the use of liquid smoke. Because of liquid smokes acidic nature, pH of about 2-3, it can effectively kill bacteria which usually grow in a pH of about 4-9 (Patent 5043174). However, because listeria monocytogenes is a facultative anaerobe, and is psychotropic, it can survive extremely harsh conditions. The previous methods can kill most but not all bacteria. Since Listeria thrives in conditions suitable for ready to eat meats (Grows at refrigeration temperatures of (4C)), can survive freezing, and because of the speedy generation time of bacteria, probability for re-inoculation and recontamination is high. Monocytogenes can grow at wide range of temperature (1C to 45C). Saritha Gedela states that some monocytogenes have developed heat resistance due to its environment (2005). Because listeria is consistently exposed to an environment that is toxic and lethal to the organism, it develops a thermostable membrane equipped with heat shock proteins that allow the organism to thrive at even higher temperatures. The purpose of this experiment is to identify the most effective treatment for listeria decontamination, testing both sodium-lauryl sulfate and liquid smoke. I predict the coupling of both liquid smoke and sodium lauryl sulfate will kill the most bacteria. Sodium lauryl sulfate will attack the cell walls of bacteria by denaturing its proteins, and liquid smoke, because of its acidic nature, will cause normal cell function to stop, and the bacteria will die.

White, A.

Methods and Procedure:

Bacterial strains and preparation of inoculum. Three cultures Listeria monocytogenes was used to inoculate frankfurters used for the study. From each L. monocytogenes working culture, 0.1 ml was aseptically transferred to10 ml of Tryptic Soy Broth and incubated at 35C for 18 h. A 3-strain mixture of L. monocytogenes was prepared by combining 10 ml of each culture to obtain a 30 ml mixture in a sterile 50-ml centrifuge tube. The cells were harvested by centrifugation (10,000 x g for 10 min at 4C) The supernatant was discarded and the pelleted cells were resuspended in 0.1% peptone water and washed once by centrifugation (10,000 x g, 10 min, 4C). Pellets of washed cells were resuspened in 30 ml of fresh 0.1% peptone water to give a final cell concentration of approximately 109 colony forming units (CFU) per ml. These washed cells were used to inoculate the frankfurters.

Preparation and application of surface treatments 4 surface treatments were prepared for application: A 40% (v/v) solution liquid smoke, a 2% (w/v) solution sodium lauryl sulfate (SLS) solution, a combination of the two (LQS+SLS) and a treatment of distilled water for dipping frankfurters as a control. Frankfurters are to be dipped for two minutes in the assigned solutions, drained for 30 s, and placed in vacuum-package bags.

Inoculation of samples.

White, A.

Once frankfurters are placed in vacuum-packaging bags, each link is to be inoculated with 0.1 ml of washed cells to give a final concentration of 108 cells/link. The packaged frankfurters were manually massaged for 10 s to evenly spread the inoculum over their surfaces. Packages containing inoculated and non-inoculated frankfurters were packaged under vacuum in a Multivac A 300/51 vacuum packaging machine (Multivac Sepp Haggenmuller, Gmblt & Co., Wolfertschwenden, Germany). All vacuum-packaged samples were held overnight (18-20 h) in a walk-in refrigerator (2-4C)

Microbial analysis. The samples were stored at 4C and were analyzed for L. monocytogenes survivors on days 0, 7, and 21. Vacuum bags containing inoculated franks were opened and 20ml of sterile peptone water was added to each package. Massaging of the packages folled for 30s. .1ml aliquots were plated using the appropriate dulutions. For day 0, 7, and 21, plates were diluted to a factor of 10^2, 10^3, and 10^4. Serial dilutions were too low on day 0 for the control group. Only the serial dilution factor 10^2 yielded colonies which was obtained in a second trial. All inoculated agar plates were then incubated aerobically at 35C for 48 h. plates yielding colonies were then counted.

White, A.

Results:
# Log CFU/Link Days 0 7 Control 4.32 3.176 LQS 3.64 2.845 SLS 3.27 2.49 Combo 4.09 4.14

21

3.54

--

2.53

3.21

# Log Cfu/Link values indicate that, by day 7, a declination in the microbial population occurred in each group, except the combo group. However, by day 21, each group saw an increase in the microbial population, suggesting that each method had little long term effect on bacteria or bacteria developed resistance, allowing their proliferation in these environments. Moreover, if we look at microbial populations in regards to overall change in percentage, results would suggest that each method had little effect when compared to the control. Days 0 7 21 Control 4.32 73.50% 81.90% LQS 3.64 78.10% SLS 3.27 76% 77.40% Combo 4.09 101.20% 78.50%

White, A.

Looking at day 7 for the combo method, there is a sharp jump in microbial population followed by a large declination by day 21 that it not consistent with the other results. -

Conclusion:
After 21 days of microbial analysis, results suggest that the most effective method is using a combination of sodium lauryl sulfate as well as liquid smoke to combat microbial populations. However, it is still unexplainable why the population increased by day 7. Liquid smoke and sodium lauryl sulfate caused no significant changes in microbial population numbers compared to the control group.

White, A.

References

CDC (People at risk). Retrieved from http://www.cdc.gov/listeria/risk.html CDC(Listeria). Retrieved from http://www.cdc.gov/listeria/ CDC (Causes). Retrieved from http://www.cdc.gov/listeria/causes.html Colorado State University. (2004). LISTERIA MONOCYTOGENES IN READY-TO-EAT MEAT PRODUCTS: RISKS, CONTROLS AND EDUCATION FOR PREVENTION Retrieved from http://www.reeis.usda.gov/web/crisprojectpages/0200146listeria-monocytogenes-in-ready-to-eat-meat-products-risks-controls-andeducation-for-prevention.html Gedela, Saritha. (2005). APPLICATION OF LIQUID SMOKE ALONE AND IN COMBINATION WITH PRE-AND POSTPACKAGE PASTEURIZATION AGAINST LISTERIA MONOCYTOGENES ON READY-TO-EAT MEATS. Retrieved from http://digital.library.okstate.edu/etd/umi-okstate-1614.pdf Final Report on the Safey Assessment of Sodium Lauryl Sulfate. (1983). Journal of the American College of Toxicolog, 2.7. http://www.docstoc.com/docs/2423844/JOURNAL-OF-THE-AMERICANCOLLEGE-OF-TOXICOLOGY Patent. Retrieved from http://www.patentstorm.us/patents/5043174/fulltext.html Scallan, E., Hoekstra, Angulo, F.J., Tauxe, R.V., Widdowson, R., Roy, S.L., Jones, J.L., and Griffin, P.M. (2011). Foodborne Illness Acquired in the United StatesMajor Pathogens. Retrieved From http://wwwnc.cdc.gov/eid/article/17/1/P1-1101_article.htm

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