Microbial Detection of Enterobacter sakazakii: Food and Clinical

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Microbial Detection of Enterobacter sakazakii
Food and Clinical

• Donald H. Burr
• CFSAN/FDA

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Objective

• Provide a summary of the methods for isolating
  and quantifying levels of E. sakazakii in food
  and clinical samples.
• Not all of the material included in your White
  Paper will be discussed.

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Initial Isolation Reports of E. sakazakii

• 1980 - Farmer et. al.
• 1983 - Muytjens et. al.
• 1984 Postupa and Aldova
• All non-quantitative

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1980 - Farmer et. al.

• In designating E. sakazakii as a new species a
  can of dried milk listed as the source of one
  isolate.
• No isolation details were provided.

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1983 - Muytjens et. al.

• Studied 8 cases of neonatal meningitis associated
  with E. sakazakii.
• Isolated E. sakazakii several times from prepared
  formula but never from either the powdered
  formula itself or the water used in preparing the
  formula.
• No information was reported on the quantity of
  powdered formula analyzed. However,
• On 3/4/03, Dr. Muytjens informed FDA that the
  quantity analyzed was 10 g.

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1984 Postupa and Aldova

• Described 4 strains of E. sakazakii from powdered
  milk and 2 strains from powdered milk infant
  formula.
• Isolated on deoxycholate-citrate agar incubated
  at 37oC for 48 hrs.
• No details on the quantity analyzed.

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Quantitative Methods Development

• 1988 - Muytjens and co-workers European
  Method
• 1997 Nazarowec-White and Farber Canadian
  Method
• 2002 - FDA Method
• Minor modifications only in the latter two
  methods Sample size and sensitivity remains the
  same.

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European Method

• First described by Muytjens et. al. in 1988.
• In referring back to their 1983 paper they
  commented that although it was not cultured from
  the formula powder itself, this might have been
  due to an unequal distribution in the powder or
  its presence at such a low concentration that it
  escaped detection by conventional methods.
  (i.e., a 10 g sample).
• Therefore, they decided to culture large
  quantities of powdered substitutes for breast
  milk for the presence of all Enterobacteriaceae
  including E. sakazakii.

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European Method

• In triplicate mix 100, 10 and 1 gram samples with
  900, 90 and 9 ml, respectively, of buffered
  peptone water at 45oC until completely dissolved.
  Incubate overnight at 36oC.
• Inoculate 10 ml from each flask into 90 ml of
  Enterobacteriaceae enrichment (EE) broth.
  Incubate overnight at 36oC.
• In duplicate, inoculate 1 ml from each enrichment
  broth into 20 ml of fluid Violet-Red-Bile Glucose
  (VRBG) agar. Incubate overnight at 36oC.

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European Method

• Suspect colonies subcultured to sheep blood and
  eosin-methylene blue agars.
• Identify strains with API-20E system.
• Additional testing for E. sakazakii included
  production of yellow colonies on nutrient agar
  after 48 hr at 25oC, production of extracellular
  DNase and a positive alpha-glucosidase reaction.

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API 20E-System                                 
                                                  
                                                 
The API 20E system has become popular for rapid
identification of members of the
Enterobacteriaceae and other Gram-negative
bacteria. The plastic strips consist of 20 small
wells containing dehydrated media components (top
row). The bacterium to be tested is suspended in
sterile saline and added to each well, then the
strip is incubated for 16-24 hours and the colour
reactions are noted as either positive or
negative. The test results can be entered into a
computer programme to identify the bacterium.
Four strips inoculated with four different
bacteria are shown in the Figure. In each case
the spectrum of results was different.
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Representative API Strip for E. sakazakii
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Representative API Strip Results for E. sakazakii
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European Method

• Levels of E. sakazakii in the sample determined
  by the most probable number (MPN) procedure.

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MPN Procedure

• Statistical method assuming that the bacteria
  are separate and the conditions of incubation
  such that every inoculum that contains even one
  viable organism will produce detectable growth.
• Based on the number of positive samples from each
  of the series of triplicate cultures of the three
  inoculation levels (100g,10g,1g).
• MPN Table provides MPN and 95 confidence
  interval.

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European Survey Results

• From 35 countries, 141 powdered formula samples
  were analyzed.
• E. sakazakii was isolated from 20 samples
  collected from 13 of the countries.
• The levels of E. sakazakii recovered ranged from
  0.36 to 66 Colony Forming Units (CFU)/100 g.
• The lowest level of detection reported in this
  method was 0.36 CFU/100 g.

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Canadian Method 1997 Nazarowec-White and Farber

• Minor modification of Dr. Muytjens method
• Dried infant formula suspended in sterile water.
• Suspect colonies from VRBG plates subcultured to
  TSB-YE agar.
• API-20E confirmation. No additional biochemicals.
• Levels determined by MPN.
• 0.36/100g sensitivity.

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Canadian Survey Results

• E. sakazakii was isolated from 8 of 120 cans,
  representing 5 manufacturers, at a level of 0.36
  CFU/100 g.

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FDA Method - 2002

• Minor modifications of the Canadian method
• Direct spreading or streaking of the overnight EE
  broth rather than VRBG pour plates.
• Five presumptive colonies subcultured to
  Trypticase Soy Agar and incubated at 25oC for
  48-72 hours.
• Only yellow pigmented colonies from the TSA
  plates are confirmed using the API 20E system.
• No additional biochemical testing is recommended.
  
• Level of detection by MPN is 0.36/100g.
• Can detect levels of E. sakazakii much lower than
  the recommended Food and Agriculture Organization
  (FAO) level of 3.0 CFU/g of powdered infant
  formula.

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Left, Unmixed Right, Mixed. (Photograph
courtesy of Sharon Edelson Mammel)

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VRBG agar Typical colonies will appear as
purple colonies surrounded by a purple halo of
precipitated bile acids.
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TSA agar Typical colonies will appear as
yellow-pigmented colonies after 48-72 hr
incubation at 25C

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Clinical Isolation

• E. sakazakii is isolated from clinical samples
  using standard methods for the isolation of
  Enterobacteriaceae.
• No special media has been developed for E.
  sakazakii.
• Grows well on blood agar, MacConkey, eosin
  methylene blue, deoxycholate agar and tergitol 7
  agars.
• Has been confirmed with either the API-20 or the
  Enterotube II system.

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Conclusions

• Procedures for isolating E. sakazakii from
  powdered formula and clinical samples follow the
  standard microbiological methods for the
  isolation of other members of the family
  Enterobacteriaceae.
• Normally, sterile clinical samples pose no major
  problems for isolating E. sakazakii.

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Conclusions - continued

• Because of the very low levels of E. sakazakii in
  powdered formula samples and its non-random
  distribution in the powder, larger quantities and
  sub-samples should be cultured for isolation.
• In both clinical and food microbiology
  laboratories, appropriate incubation times and
  temperatures should be applied in diagnostic
  tests for precise identification of E. sakazakii.