Escherichia coli O157:H7

„E.coli
E.coli belongs to the family enterobacteriaceae.
enterobacteriaceae.

Pathogens „Bacillus gram negative.

„Anaerobic facultative
„E.coli can ferment lactose and glucose.

Determination by PCR „E. coli comprises about 1% of the total fecal bacterial flora of humans and
most warm-
warm-blooded animals
„Mobile with Peritrichous flagella
„Some serotypes of Escherichia coli cause soft intestinal diseases and urinary
tract infection
„Escherichia coli O157:H7can
O157:H7can develop diarrhea with blood, and abdominal
B.K.Kolita Kamal Jinadasa,
Jinadasa, cramps.
„E,coli is an indicator of fecal contamination
Post Harvest Technology Division,
NARA,
Colombo-
Colombo-15,
Sri Lanka.

Salmonella Shigella spp

„ Belongs to the family Enterobacteriaceae

„ Bacillus gram negative
„ Bacillus gram negativo.
negativo.
„ Anaerobic facultative
„ Anaerobic facultative.
„ Mobile with Peritrichous flagella..
„ Immobile.
„ There are different serovars that cause fooborne illness.
„ Shigella can’
can’t ferment lactose.
„ The most common illness is caused by Salmonella enterica.
enterica. This illness is named
salmonellosis. „ Different types of Shigella causes foodborne illness.
salmonellosis. Salmonella cause foodborne illness especially from poultry and
raw eggs and more generally from food that has been cooked or frozen, and and „ Shigellosis is an infectious disease caused by Shigella.
higella.
not eaten straight away. „ Shigellosis develop diarrhea, fever, and stomach cramps. The diarrhea is often
often
„ Salmonellosis develop diarrhea, stomach cramps, headache, fever, vomiting, bloody.
dehydration (fluid loss), especially among infants and the elderly.
elderly. „ Shigella dysenteriae causes deadly epidemics.
„ Salmonella is an indicator of fecal contamination. „ It’
It’s very important the control of Shigella in freshwater products.
„ The presence of salmonella is not allowed in food.

Vibrio parahaemolyticus Vibrio cholerae

„ Gram negative
„ Halofilic
„ V. cholerae occurs naturally in the plankton of fresh,fresh, brackish,
brackish, and salt water,
water, attached
primarily to copepods in the zooplankton.
zooplankton.
„ Illness named cholera is caused by the ingestion of viable bacteria
bacteria
„ Person may get cholera by drinking water or eating food contaminated
contaminated with the cholera
bacterium. In an epidemic, the source of the contamination is usually usually the feces of an
„ Gram negative. infected person
„ Halophilic.
Halophilic. „ V. cholerae colonizes the gastrointestinal tract, where it adheres to villous absorptive cells
It’ via pili,
pili, and secretes a Binary toxin,
toxin, called cholera toxin
„ It’s found in saltwater.
„ V. cholera develop abdominal cramps, nausea, vomiting, dehydration, and shock; shock; after
„ Outbreaks tend to be concentrated along coastal regions during the the summer severe fluid and electrolyte loss, death may occur
and early fall when higher water temperatures favor higher levels
levels of bacteria „ It’
It’s very important the control of Vibrio cholera in freshwater products.
„ Infection is via fecal-
fecal-oral contamination.
contamination. „ Some of the pathogenic serogroups of Vibrio cholera are O139 and O1
„ Seafood most often implicated includes squid, mackerel, tuna, sardines,
sardines, crab,
shrimp, and bivalves like oysters and clams.
„ The incubation period of ~24 hours is followed by explosive, watery diarrhea
accompanied by nausea,
nausea, vomiting,
vomiting, abdominal cramps,
cramps, and sometimes fever.
fever.
Vibrio parahaemolyticus symptoms typically resolve with-
with-in 72 hours.

1
 Staphylococcus aureus Sequences of Escherichia coli
„ Coccus gram positive
„ Anaerobic facultative ( better in aerobiosic conditions)
„ Catalase and coagulase positive „ Escherichia coli APEC O1, complete genome 2006/11/08.
„ S. aureus may occur as a commensal on human skin (particularly the scalp, armpits and groins); it also occurs in
the nose (in about 25% of the population) and throat and less commonly,
commonly, may be found in the colon and in
urine „ Escherichia coli UTI89, complete genome 2006/04/07.
„ Staphylococcal food poisoning (staphyloenterotoxicosis;
staphyloenterotoxicosis; staphyloenterotoxemia)
staphyloenterotoxemia) is the name of the condition
caused by the enterotoxins which some strains of S. aureus produce.
„ Human intoxication is caused by ingesting enterotoxins produced in food by some strains of S. aureus,aureus, usually „ Escherichia coli B, unfinished sequence,
sequence, whole genome shotgun sequencing
because the food has not been kept hot enough (60°(60°C, 140°
140°F, or above) or cold enough (7.2°
(7.2°C, 45°
45°F, or below project 2007/02/07.
„ The most common symptoms are nausea, vomiting, retching, abdominal abdominal cramping, and prostration. Some
individuals may not always demonstrate all the symptoms associated
associated with the illness
„ In more severe cases, headache, muscle cramping, and transient changes
changes in blood pressure and pulse rate may „ Escherichia coli O157:H7 EDL933, complete genome 2001/09/27.
occur
„ Foods that are frequently incriminated in staphylococcal food poisoning
poisoning include meat and meat products;
poultry and egg products; salads such as egg, tuna, chicken, potato,
potato, and macaroni; bakery products such as
cream-
cream-filled pastries, cream pies, and chocolate eclairs;
eclairs; sandwich fillings; and milk and dairy products „ Escherichia coli W3110 DNA, complete genome.
genome.
„ Emphasis on basic hand washing techniques are therefore effective in preventing the transmission
transmission of S. aureus 2006/03/01
„ Escherichia coli CFT073, complete genome 2002/12/09 .

Sequences of Salmonella Sequences of Shigella

„ Salmonella typhimurium plasmid R64, complete sequence 2003/07/08 „ Shigella sonnei Ss046, complete genome 2005/09/09.
„ S. enterica subsp.
subsp. enterica serovar Choleraesuis str.
str. SC-
SC-B67, complete genome
2005/04/04
„ S. flexneri virulence plasmid pWR501,
pWR501, complete
„ S. enteritidis plasmid pK,
pK, complete sequence 2002/03/17 sequence 2001/03/13.
„ S. enteritidis plasmid pP,
pP, complete sequence 2002/03/17
„ S. flexneri 2a str.
str. 301, complete genome 2002/10/16.
„ S. choleraesuis plasmid pSFD10,
pSFD10, complete sequence 2001/08/20

„ S. typhi plasmid R27, complete sequence 2000/05/14 „ S. boydii Sb227, complete genome 2005/11/18.
„ S. typhimurium LT2, complete genome 2001/11/07
„ S. flexneri 2a str.
str. 2457T, complete genome 2003/04/23.
„ S. enterica subsp.
subsp. enterica serovar Typhi Ty2, complete genome 2003/03/21

Sequences of Vibrio
Sequences of Staphylococcus aureus
parahaemolyticus
„ Staphylococcus aureus subsp.
subsp. aureus MSSA476, complete genome „ Vibrio parahaemolyticus RIMD 2210633
2001/11/06
„ S. aureus subsp.
subsp. aureus USA300, complete genome 2006/02/11 chromosome I, complete sequence 2003/03/10
„ S. aureus subsp.
subsp. aureus USA300 plasmid pUSA03,
pUSA03, complete „ Vibrio parahaemolyticus AQ3810, unfinished
sequence 2006/02/11
„ S. aureus subsp.
subsp. aureus USA300 plasmid pUSA02,
pUSA02, complete
sequence,
sequence, whole genome shotgun sequencing
sequence 2006/02/11 project 2007/01/11
„ S. aureus subsp.
subsp. aureus USA300 plasmid pUSA01,
pUSA01, complete
sequence 2006/02/11
„ Vibrio parahaemolyticus plasmid pO3K6,
pO3K6, complete
„ S. aureus subsp.
subsp. aureus Mu50, complete genome 2001/10/04 sequence

2
 Primers
Sequences of Vibrio cholerae Species Target gene Product size Reference
„ V. cholerae 2740-
2740-80, unfinished sequence,
sequence, whole genome
E.Coli hlyA gene 361 bp Wang et al
shotgun sequencing project 2007/01/10. O157:H7 (1997)
„ V. cholerae V52, unfinished sequence,
sequence, whole genome
shotgun sequencing project Shigella spp Ipah gene 610 bp Sethabutr et
al.(1992)
2005/09/17.
„ V. cholerae 623-
623-39, unfinished sequence,
sequence, whole genome V.cholera Toxin gene 563 bp Fields et
al.(1992)
shotgun sequencing project 2007/01/05.
„ V. cholerae MZO-
MZO-2, unfinished sequence,
sequence, whole genome V.parahaemol Genomic DNA 381 bp Lee et al
shotgun sequencing project yticus (1992)

2007/01/04. S.aureus Nuclease gen 276 bp Brakstad et al

(1992)

Detection of Escherichia coli O157:H7

Detection of Salmonella spp
„ Collect 25 g of food samples from different parts of the food samples,
samples, then
cut it into small pieces. „ 25 g of food samples were collected from different parts of the
„ Inoculate the small pieces into 225 ml of TSBY medium. food samples, then cut it into small pieces.
„ Incubate the samples at 37 ˚C overnight (16-
(16-20h) „ Inoculate the small pieces into 225 ml of TSBY medium.
„ Mix 0,5 ml of the samples with 1 ml of PBS and centrifugation the mixed at „ Incubate the samples at 37˚
37˚C overnight (16-
(16-20h)
9000g for three minutes. „ Mix 0,5 ml of the samples with 1 ml of PBS and centrifugation
„ Wash the pellet three times with PBS and once with water. the mixed at 9000g for three minutes.
„ Resuspend in 50 µl of water. „ Wash the pellet three times with PBS and once with water.
„ Dilute the samples (1:10) with 1% Triton X-X-100. „ Resuspend in 50 µl of water.
„ Add 2 µl of each DNA sample to 23 µl of PCR mixture.
mixture. „ Dilute the samples (1:10) with 1% Triton X- X-100.
„ The PCR products will be separated by electrophoresis in 2%agarose
2%agarose gels Add 2 µl of each DNA samples to 23 µl of PCR mixture.
containing ethidium bromide.
„ mixture.
„ The PCR products will be separeted by electroforesis in
2%agarose gels containig ethidium bromide.

Detection of Staphylococcus aureus Detection of Shigella spp

„ 25 g of food samples were collected from different parts of the
food samples, then cut it into small pieces.
„ Inoculate the small pieces into 225 ml of TSBY medium.
„ Incubate the samples at 37˚
37˚C overnight (16-
(16-20h)
„ Mix 0,5 ml of the samples with 1 ml of PBS and centrifugation
the mixed at 9000gor for three minutes.
„ Wash the pellet three times with PBS and once with water.
„ Resuspend in 50 µl of water.
„ Dilute the samples (1:10) with 1% Triton X- X-100.
„ Ad 2 µl of each DNA samples to 23 µl of PCR mixture.
mixture.
„ The PCR products will be separated by electrophoresis in
2%agarose gels containing ethidium bromide.
„ 25 g of food samples were collected from different parts of the
food samples, then cut it into small pieces.
„ Inoculate the small pieces into 225 ml of TSBY medium.
„ Incubate the samples at 37˚
37˚C overnight (16-
(16-20h)
„ Mix 0,5 ml of the samples with 1 ml of PBS and centrifugation
the mixed at 9000g for three minutes.
„ Wash the pellet three times with PBS and once with water.
„ Resuspend in 50 µl of water.
„ Dilute the samples (1:10) with 1% Triton X- X-100.
„ Ad 2 µl of each DNA samples to 23 µl of PCR mixture.
mixture.
„ The PCR products will be separeted by electroforesis in
2%agarose gels containig ethidium bromide.

3
 Detection of Vibrio cholerae
„ 25 g of food samples were collected from different parts of the
food samples, then cut it into small pieces.
„ Inoculate the small pieces into 225 ml of TSBY medium.
„ Incubate the samples at 37 ˚C overnight (16-
(16-20h)
„ Mix 0,5 ml of the samples with 1 ml of PBS and centrifugation
the mixed at 9000g for three minutes.
„ Wash the pellet three times with PBS and once with water.
„ Resuspend in 50 µl of water.
„ Dilute the samples (1:10) with 1% Triton X- X-100.
„ Ad 2 µl of each DNA samples to 23 µl of PCR mixture.
mixture.
„ The PCR products will be separeted by electroforesis in
2%agarose gels containig ethidium bromide.
Detection of Vibrio parahaemolyticus
„ 25 g of food samples were collected from different parts of the
food samples, then cut it into small pieces.
„ Inoculate the small pieces into 225 ml of TSBY medium.
„ Incubate the samples at 37 ˚C overnight (16-
(16-20h)
„ Mix 0,5 ml of the samples with 1 ml of PBS and centrifugation
the mixed at 9000g for three minutes.
„ Wash the pellet three times with PBS and once with water.
„ Resuspend in 50 µl of water.
„ Dilute the samples (1:10) with 1% Triton X- X-100.
„ Add 2 µl of each DNA samples to 23 µl of PCR mixture.
mixture.
„ The PCR products will be separeted by electroforesis in
2%agarose gels containing ethidium bromide.

GENERAL DIAGRAM
Detection Vibrio Cut 25gr of food samples
↓
Inoculate the small pieces into 225 ml of TSBY
↓
Detection of Vibrio spp with primers VC 16S F / VC 16S R Incubate the samples at 37˚
37˚ C
↓

↓
Mix 0,5 ml of the samples with 1ml of PBS
↓
Centrifugate at 9000 g or 10.000 rpm for three minutes
If Vibrio spp is detected, there are two couple of primers that can ↓
Wash the pellet three times with PBS and once with water
detect and distinguish Vibrio parahaemolyticus and Vibrio cholerae.
↓
Primers are ctxA94 F / ctxA614 R for Vibrio cholerae and tcpA72 Resuspend in 50 µl of water
F / tcpA647F for Vibrio parahaemolyticus. ↓
Dilute the samples 1:10 with Triton X-
X-100
↓ ↓
Incubated in boiling water for 5 minutes and cooled in ice water
↓
There are two important pathogenic types of Vibrio cholera, Vibrio Centrifugate 1 minute at 9000 g
cholerae O1 and Vibrio cholara O139. There are two couple of ↓
primers primers that distinguish the two types. VCO1 F2 / VCO1 Ad 2µ
2µl of each DNA sample to 23 µl of PCR mixture
R2 for Vibrio cholerae O1 and VCO139 F2 / VCO139 R2 for Vibrio ↓
Electroforesis in 2% agarose gel
cholera O139.

Specific primers of E.coli O157:H7

Example of PCR calibration 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 pGEM

„ Set up of the suitable temperature of melting. 361 bp

1 2 3 4 5 6 L-50

Lane 1. Negative control 7. L. monocytogenes

1. V.cholera 13. E. faecalis
610 bp Lane 2: 71˚C
2. V. parahaemolyticus 8. L. seeligeri 14. Bacillus cereus
Lane 3: 66.7 ˚C
3. S. aureus 9. L. ivanovi 15. S. enterica
Lane 4: 62.5 ˚C
Lane 5: 52.6 ˚C 4. E. coli 516 10. L. innocua 16. Negative control
Lane 6: 48 ˚C 5. E.coli 434 11. C perfringens 17. E.coli O147:H7
6. S. sonnei 12. C. biferment

4
 Pathogens mix. PCR
DNA 2 µl
„ PCR components are:
„ Detection of one pathogen in a mix of pathogens.
Taq polymerase 0,1 µl
L50 1 2 3 4 5 6 7 8 9 1. DNA template
2. Taq polymerase dNTPs 0,2 µl
Lane 1: E.coli O157:H7 3. Deoxynucleotide triphosphates
Lane 2: E.coli O157:H7+Shigella sonnei 4. Buffer solution Buffer 2,5 µl
5. Divalent cation,
cation, magnesium or
Lane 3: E.coli O157:H7+Salmonella enterica manganese ClMg 1 µl
276 bp Lane 4: E.coli O157:H7+S.aureus 6. Primers
276 bp
7. Water H 2 µl
Lane 5: Mix of the four pathogens
Lane 6: S.enterica+S.sonnei L 2 µl

Lane 7: S.sonnei+S.aureus Water 15,2 µl

Lane 8: S.enterica+S.auerus
Total 25 µl

PCR mix Vibrio detection

„ You have 230 µl of mix. You have 10 tubes, so you have to inoculate 23 µl of
mix on each tube. 1 2 3 2 4 2 pGEM

„ 23 µl(MIX)+
l(MIX)+ 2 µl (DNA)=25 µl.
Lane 1: results with primer VC 16S F
„ mixture is inserted into the thermocycler.
thermocycler. Choose the PCR program suitable / VC 16S R
to the type of samples.
Lane 2: negative control.
„ The correct PCR product can be identified by its size, using agarose gel
Lane 3: results with primers VCO1
electrophoresis.
electrophoresis. Agarose gel electrophoresis is a procedure that consists of
F2 / VCO1 R2
loading DNA into small wells of an agarose gel and then applying an electric
current to the gel Lane 2: negative control.
Lane 4: results with primers VCO139
F2 / VCO139 R2
Lane 2: negative control

Result confirm that the pathogen is Vibrio cholera O139

Limit of detection calibration

Aplication of PCR
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