Microbial Detection of Enterobacter sakazakii: Food and Clinical Donald H. Burr CFSAN/FDA

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.

Initial Isolation Reports of E. sakazakii 1980 - Farmer et. al. 1983 - Muytjens et. al. 1984 – Postupa and Aldova *All non-quantitative

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.

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.

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.

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.

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.

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.

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.

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.

Representative API Strip for E. sakazakii

Representative API Strip Results for E. sakazakii

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

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.

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.

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.

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

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.

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

VRBG agar: Typical colonies will appear as purple colonies surrounded by a purple halo of precipitated bile acids.

TSA agar: Typical colonies will appear as yellow-pigmented colonies after 48-72 hr incubation at 25°C

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.

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.

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.