Transcript lab-9.-E.-coli-O157

University of Gaza
Faculty of Health Science
Medical laboratory Sciences Department
Lab 7 :
Isolation of E. coli 0157
in foods
Isolation of E. coli 0157 in foods
Objectives:
1. Detect E. coli O157:H7 in food.
2. Identify the pathogenic strain
based on its biochemical and
serological characteristics.
3. practice a rapid test using an
immunochromatographic
technique.
E. coli 0157
 Escherichia coli 0157:H7 is an important foodborne
pathogen causing symptoms ranging from mild
diarrhea to hemorrhagic colitis and haemolytic
uraemic syndrome.
 Most outbreaks and sporadic cases have been
associated with consumption of undercooked beef or
dairy products such as untreated milk.
infections due to E. coli 0157 are increasing and wide
ranges of foods have been associated with outbreaks.
These include cooked meat products, vegetables,
salad vegetables, coleslaw, and acid based foods such
as apple cider, yoghurt and fermented sausages.
Microbiological guidelines for ready to eat foods
recommend the absence of E. coli 0157 in 25 grams.
 Most strains of E. coli 0157 do not ferment sorbitol
within 24hrs and are B-glucuronidase negative
whereas most other serogroups of E. coli give
positive reactions.
 The method described has successfully isolated the
organism from naturally contaminated and
artificially inoculated foods.
 E. coli 0157 is pathogenic to man, has a low
infective dose and laboratory acquired infections
have been reported.
 Therefore, the isolation and identification of this
organism must be carried out by trained personnel
in a properly equipped laboratory, under the
control of a qualified microbiologist.
 Application
The method described is applicable to the detection of Escherichia
coli 0157 in all food types.

Description
• The method has been shown to produce satisfactory results with
artificially-contaminated meats (including beef, veal and pork),
vegetables, dairy products, spices and environmental samples.
• This method can be used successfully for the detection of E. coli
O157 in other foods, food ingredients and environmental
samples.
PRINCIPLE:
 The detection of E. coli 0157 involves the
selective enrichment with isolation from
enrichment culture by immunomagnetic
separation (IMS) followed by subculture to a
selective agar.
 The selective agar cultures are examined for
characteristic colonies.
 Confirmation of presumptive E. coli 0157 is
by serological and biochemical tests.
Materials and special equipment
 Broths and agars (base media and
supplements are commercially available)
1. Modified Tryptic Soy Broth with Novobiocin (mTSB-n)
2. Enterohemorrhagic E. coli (EHEC) Enrichment Broth (EEB)
3. Modified Hemorrhagic Coli Agar (mHC) with Tellurite and
Cefsulodin
4. Modified Sorbitol MacConkey agar (SMAC) with
Tellurite, Cefixime, and Cefsulodin
5. Purple broth base with cellobiose.
6.
7.
8.
0.5% K2SO4 (needed for some spices and foods containing large amounts of spices).
1N HCl and 1N NaOH.
pH meter or paper capable of distinguishing 0.3 to 0.5 pH units within a range of 6.0 to
7.5.
9.
Stomacher, blender, or equivalent.
10. Control cultures (use ATTC cultures or equivalent) positive control:
E. coli O157 (H7 or other serovars) negative control: E. coli (NOT an O157)
11. Incubators capable of maintaining 35 and 42°C Confirmation media and
reagents (commercially available).
12.
Trypticase Soy Agar with Yeast Extract (TSA-YE)
13.
Rapid Identifcation kits
14.
Latex Agglutination kits
15.
IMVIC Reagents (see MFHPB-19)
16.
urea agar slants
17.
MUG (4-methylumbelliferyl-$-D-glucuronide)
18.
BCIG (5-bromo-4-chloro-3-indolyl-$-D-glucuronide) either –Na or-CHX
(cyclohexylammonium) salt.
19.
O157 and H7 antisera
Procedure
Handling of Sample Units
1. In the laboratory prior to analysis, except for shelf-stable
foods, keep sample units refrigerated (0-5oC) or frozen,
depending on the nature of the product. Thaw frozen
samples in a refrigerator, or under time and temperature
conditions which prevent microbial growth or death.
2. Analyze sample units as soon as possible after their
receipt in the laboratory.

 Preparation for Analysis
1.
2.
Have ready sterile mTSB-n (and/or EHEC enrichment broth (EEB)).
Clean the surface of the working area with a suitable disinfectant.
 Preparation of Sample
1. To ensure a truly representative analytical unit agitate liquids or
free flowing materials until the contents are homogeneous.
• If the sample unit is a solid, obtain the analytical unit by taking a
portion from several locations within the sample unit.
• To reduce the workload, the analytical units may be combined for
analysis.
• It is recommended that a composite contain not more than five
analytical units.

Note: When analyzing larger volumes,
the enrichment broth should be prewarmed
to 35oC
.
2. Prepare a 1:10 dilution of the food by aseptically adding 25 g or
mL (the analytical unit) into 225 mL of the enrichment broth
mTSB-n (and EEB if applicable). Stomach or blend.
•
A second primary enrichment broth started directly in EEB
should be done when there is a high bacterial load of competing
organisms in the sample.
Note : Some spices, such as onion and garlic powder are
antimicrobial in nature. When analyzing spices or
products containing large amounts of spices, add 0.5%
K2SO4 to mTSB-n before autoclaving. Garlic
especially affects E. coli O157, therefore garlic or garlic
containing products need to be diluted 1:100. Other
spices also may need to be analyzed using larger
dilutions.
3. After the addition of the sample to the broth adjust the
pH of the mixture, if necessary, to 6.0 to 7.0 with 1N
NaOH or 1N HCl.
4. A positive and a negative control should be set up at the
same time.
5. Incubate the enrichment mixture and controls for 22-24
h at 42oC.
6. Either screen the enrichment broth for E. coli O157 by
using rapid kits.
Secondary Enrichment in EEB
Note : This step must be used when a rapid kit has
identified the presence of E. coli O157 but it was not isolated
when the primary enrichment broths, mTSB-n and/or EEB,
were plated onto the selective agars.
• E. coli O157 may be difficult to isolate from some samples
with high ACC levels. After agitation of the enrichment
broth, transfer 1 mL of the mTSB-n and/or EEB to 9 mL
of EEB. Incubate 18-24 h at 35oC. Plate 0.1 mL of 10-4 to
10-6 dilutions (made in 0.1% peptone water)
from the enrichment broths onto the selective
agars, as below.
Follow confirmation steps for typical colonies.
Selective Isolation
1. Plate dilutions of 10-4 to 10-6 from each enrichment broth onto mHC and
SMAC agar plates. Incubate for 18-24 h at 42oC. Due to the increased
selectivity of SMAC, the counts may be one log less than on mHC.
2. On the mHC agar, typical E. coli O157:H7 colonies appear blue.
On SMAC, typical E. coli O157:H7 colonies appear colorless, bear the
tint of the medium or are gray to pink with Smokey centers. Other
serovars of E. coli, including O157 (not H7) will be yellow on mHC
and red on SMAC. Some of these sorbitol positive colonies may be
pathogenic also.
4.
5.
6.
7.
8.
9.
10.
11.
Use the purified colonies (step 1) and continue confirmation steps. Unless stipulated,
incubate all tests at 35oC for 22-24 h.
Do a Gram stain.
Use rapid identification kits following manufacturers’ instructions.
Confirm isolates as an O157 using latex agglutination kits.
Streak suspect colonies onto Phenol red sorbitol agar with MUG (PSRA), and/or
Sorbitol MacConkey agar with BCIG. Incubate plates at 35oC for 22-24 h.
Inoculate IMViC tests and Urea slants). Incubate at 35oC.
Complete serological testing using O157 antisera and H7 antisera.
Follow manufacturer’s instructions. The isolate must be "resuscitated“
in M broth or on motility agar several times (at least three times).
Sorbitol-MacConkey agar (SMAC)
majority of E. coli O157 do not ferment
sorbitol in contrast to most faecal E. coli
 modification of SMAC :
• supplemented with cefixime and
tellurite (CT-SMAC)
 suppress other sorbitol non-fermenting
organisms
 most non-O157 STEC ferment
sorbitol
ISO Standard for testing of E. coli O157 in Food
CHROM agar for E.coli O157
• Rapid and reliable detection of the
enterohaemorrhagic E.coli O157.
• Easily distinguishable colonies due to the
purple colouring they have acquired.
• Most other bacterial species are inhibited,
giving blue or colorless colonies.
• Despite specifity is improved in
comparison to Sorbitol MacConkey Agar,
when direct isolation method is used, the
false positive must be screened out by
additional tests and candidates must be
further studied for confirmation.
• E. coli O157 - purple
• other bacterial colonies - inhibited,
blue or colorless
Colonies appear as pink on CHROM
agar
 After incubation for 16–24 hours at 37°C, the plate
should be examined for possible O157 colonies, which
are colorless on SMAC or CT-SMAC and are mauve or
pink on CHROM agar O157
latex agglutination test
• To identify E. coli O157, a
portion of a well-isolated colony
(i.e., a distinct, single colony)
should be selected from the
culture plate and tested in
O157-specific antiserum or
O157 latex reagent as
recommended by the
manufacturer .
• Colonies that agglutinate with one of
the O157-specific reagents and do not
agglutinate with normal serum or
control latex reagent are presumed to
be E. coli O157.
latex agglutination test
anti-O157
anti-H7
Identification with biochemical methods
• The colony in which O157
STEC are detected should
be streaked onto SMAC or
a nonselective agar
medium such as tryptic
soy agar (TSA), heart
infusion agar (HIA), or
blood agar and
biochemically confirmed
to be E. coli
Singlepath® E. coli 0157 Test
 For the rapid detection of E. coli O157 in food.
 Sensitivity 97%, Specificity 98.4% ,Results In
Just 20 Minutes!
 Our new Singlepath® E. coli 0157 immunochromatographical test
offers all the benefits of traditional test methods with the added
benefits of speed, reliability and convenience. When used in
conjunction with our EC Broth Modified (mEC Broth) or Tryptic
Soy Broth Modified (mTSB) for enrichment, you can be sure that
after a 24 hour incubation, the count of the target E. coli 0157 will
be at levels that assure either a true positive or negative result
from the rapid test.
 The media already contain Novobiocin for convenience which
suppresses the growth of Gram-positive microbial flora.
Singlepath® E. coli 0157 Test Procedure
Selective Enrichment
• Add 25 g or 25 ml of the sample to 225 ml EC Broth
Modified (mEC) or Tryptic Soy Broth Modified (mTSB)
• Incubate at 35-37 °C for 18-24h
Detection
• Transfer 160 μl from the enrichment to
the circular sample port on the test
• Read after: 20 min
Confirmation
If result is positive:
Streak onto CT-SMAC Agar
for confirmation.
Negative
E.coli 0157
not present
Positive
E.coli 0157
present
Dynabeads® anti-E. coli O157
 Dynabeads® anti-E. coli O157 are supplied in a
suspension of phosphate buffered saline (PBS) pH 7.4
with 0.1% bovine serum albumin (BSA) and 0.02%
sodium azide.
Caution: Sodium azide may react with lead and copper
plumbing to form highly explosive metal azides.
 Dynabeads® anti-E. coli O157 is designed for rapid
selective separation of E. coli O157:H7 from food,
water, or environmental samples.
 Dynabeads® anti-E. coli O157 are designed for rapid, selective
concentration of E. coli O157 directly from a pre-enriched
sample aliquot using immunomagnetic separation (IMS).
 Dynabeads® anti-E. coli O157 reacts with all E. coli O157 strains
including pathogenic and non-pathogenic, sorbitol fermenting
and non-sorbitol fermenting isolates.
Dynabeads® anti-E. coli O157
 Dynabeads® anti-E. coli O157 are simply incubated with an
aliquot of the pre-enriched sample, and the antibodies coated
onto the beads will specifically bind the target bacteria. The
bead-bacteria complexes are subsequently separated by
applying a magnetic field.
 The whole IMS process can be automated using a
BeadRetriever™ instrument or performed manually.
 Any food, water, feed, or environmental sample that
has been pre-enriched for 6–18 hours in Buffered
Peptone Water (BPW), Tryptone Soya Broth (TSB), or
Brilliant-Green Bile Broth (BGBB) is suitable for IMS
with Dynabeads® anti-E. coli O157.
An electron micrograph showing E. coli O 157
bound to Dynabeads
END OF LECTURE