Download Food Borne Illness

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8/15/2011
Name: ________________________
Food Borne Illness
(What Type of Infection Do You Have?
Learning Objectives:
1. Define PCR
2. Explain the process of PCR.
3. List various types of food borne illness.
4. Analyze results of DNA sequencing. (Blast search).
5. Perform PCR.
What is Escherichia coli (E. coli) 0157:H7 infection?
E. coli are bacteria that normally live in the intestines of humans and animals. Although
most strains of this bacteria are harmless, several are known to produce toxins that can
cause diarrhea. One particular E. coli strain called 0157:H7 can cause severe diarrhea and
kidney damage.
Who gets E. coli 0157:H7 infection?
Anyone of any age can become infected with E. coli 0157:H7, but children and the
elderly are more likely to develop serious complications.
How does one get infected with E. coli 0157:H7?
The bacteria are acquired by eating food containing the bacteria. The bacteria live in the
intestines of some healthy cattle, and contamination of the meat may occur in the
slaughtering process. Eating meat that is rare or inadequately cooked is the most common
way of getting the infection. Infection can also occur after consuming foods such as
lettuce, alfalfa sprouts, salami, and unpasteurized milk, juice or cider. Person-to-person
transmission can occur if infected people do not wash their hands after using the toilet.
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How can infection with E. coli 0157:H7 be prevented?
Do not eat undercooked hamburger or other ground beef products. Cook all ground beef and
hamburger thoroughly. Make sure the cooked meat is brown throughout (not pink), and the
juices run clear. Drink only pasteurized milk, juice or cider. Wash fruits and vegetables
thoroughly, especially those that will not be cooked. Children under five years,
immunocompromised persons, and the elderly should avoid eating alfalfa sprouts. Make sure
infected people, especially children, wash their hands carefully with soap after using the toilet
to reduce the risk of spreading the disease.
Salmonella enterica
Most cases of salmonellosis are caused by food infected with S. enterica, which often
infects cattle and poultry, though also other animals such as domestic cats and hamsters
have also been shown to be sources of infection to humans.
Salmonella enterica serovar Typhimurium (Also called Salmonella Typhimurium
or abbreviated to S. Typhimurium)
Until recently the most common cause of food poisoning by Salmonella species was due
to S. Typhimurium. As its name suggests, it causes a typhoid-like disease in mice. In
humans S. Typhimurium does not cause as severe disease as S. Typhi, and is not normally
fatal. The disease is characterized by diarrhea, abdominal cramps, vomiting and nausea,
and generally lasts up to 7 days. Unfortunately, in immunocompromized people, that is
the elderly, young, or people with depressed immune systems, Salmonella infections are
often fatal if they are not treated with antibiotics.
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Listeria monocytogenes
This is a Gram-positive bacterium2, motile by means of flagella. Some studies suggest
that 1-10% of humans may be intestinal carriers of L. monocytogenes. It has been found
in at least 37 mammalian species, both domestic and feral, as well as at least 17 species
of birds and possibly some species of fish and shellfish. It can be isolated from soil,
silage, and other environmental sources. L. monocytogenes is quite hardy and resists the
deleterious effects of freezing, drying, and heat remarkably well for a bacterium that does
not form spores. Most L. monocytogenes are pathogenic to some degree.
Listeria is the name of a bacteria found in soil and water and some animals, including
poultry and cattle. It can be present in raw milk and foods made from raw milk. It can
also live in food processing plants and contaminate a variety of processed meats.
Listeria is unlike many other germs because it can grow even in the cold temperature of
the refrigerator. Listeria is killed by cooking and pasteurization.
Symptoms
Fever, stiff neck, confusion, weakness, vomiting, sometimes preceded by diarrhea
Associated Foods:
L. monocytogenes has been associated with such foods as raw milk, supposedly
pasteurized fluid milk, cheeses (particularly soft-ripened varieties), ice cream, raw
vegetables, fermented raw-meat sausages, raw and cooked poultry, raw meats (all types),
and raw and smoked fish. Its ability to grow at temperatures as low as 3°C permits
multiplication in refrigerated foods.
Bacillus cereus and other Bacillus spp.
Bacillus cereus is a Gram-positive,1 facultatively aerobic sporeformer whose cells are
large rods and whose spores2 do not swell the sporangium. These and other
characteristics, including biochemical features, are used to differentiate and confirm the
presence B. cereus, although these characteristics are shared with B. cereus var.
mycoides, B. thuringiensis3 and B. anthracis4. Differentiation of these organisms depends
upon determination of motility (most B. cereus are motile), presence of toxin crystals (B.
thuringiensis), hemolytic activity (B. cereus and others are beta hemolytic whereas B.
anthracis is usually nonhemolytic), and rhizoid growth which is characteristic of B.
cereus var. mycoides.
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Nature of Acute Disease:
B. cereus food poisoning is the general description, although two recognized types of
illness are caused by two distinct metabolites. The diarrheal type of illness is caused by a
large molecular weight protein, while the vomiting (emetic) type of illness is believed to
be caused by a low molecular weight, heat-stable peptide.
Associated Foods:
A wide variety of foods including meats, milk, vegetables, and fish have been associated
with the diarrheal type food poisoning. The vomiting-type outbreaks have generally been
associated with rice products; however, other starchy foods such as potato, pasta and
cheese products have also been implicated. Food mixtures such as sauces, puddings,
soups, casseroles, pastries, and salads have frequently been incriminated in food
poisoning outbreaks.
Nature of Disease:
The symptoms of
B. cereus diarrheal type food poisoning mimic those of Clostridium
perfringens5 food poisoning. The onset of watery diarrhea, abdominal cramps, and pain
occurs 6-15 hours after consumption of contaminated food. Nausea may accompany diarrhea,
but vomiting (emesis) rarely occurs. Symptoms persist for 24 hours in most instances.
Experiment
Supplies:
Pico-fuge
PCR HiFi Mix
5’ & 3’ Primers: E.coli, Salmonella, Bacillus E or H, Listeria
Extracted DNA from food sources
Agarose or E-gels
PCR machine
Finnpipette (10, 100, 1000ul)
Pipette tips
Track Dye
UV light
Power supplies
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Signs : (Beef, Pork, Chicken, Spinach, Bean Sprouts, Turkey)
1. Students will work in groups of 5
2. Each student group will pick a food to test.
3. One student will pick to make one of the following master mixes :
a. E.coli 0157
b. Bacillus E
c. Bacillus H
d. Salmonella
e. Listeria
4. Master Mix
a. 23 ul of Hi FI PCR mix
b. 1 ul of 5’ primer
c. 1 ul of 3’primer
d. 1 ul of Food product extracted DNA
5. Gently mix and spin 2 seconds in pico-fuge
6. PCR
a. 94-950C for 4 minutes (1 cycle)
b. 94-950C for 30 seconds
c. 520C (E.coli & Listeria) 560C (Salmonella & Bacillus) 30 seconds
d. 680C for 30 seconds (30 cycles)
e. 680C 7 minutes (1 cycle)
f. 4 degree hold infinite
7. Take add 5 ul of track dye and load 20 ul on the gel.
8. Run agarose gel 150 volts 30 – 40 minutes
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9. If using E-gel run 30 minutes
10. View on UV light box and determine pathogen.
Food Poison Experiments PCR
1. E. coli 0157:H7
182 b.p.
2. Salmonella enterica
285 b.p.
3. Bacillus cereus enterotoxin
645 b.p.
4. Bacillus cereus hemolysin
873 b.p.
5. Listeria monocytogenes
90 b.p.
Links:
http://www.health.state.mn.us/
http://www.ncbi.nlm.nih.gov/
http://www.cdc.gov/