U. S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
July 2002; Revised August 2002

Isolation and Enumeration of Enterobacter sakazakii from Dehydrated Powdered Infant Formula

Background: Enterobacter sakazakii is a gram-negative rod within the family Enterobacteriaceae, genus Enterobacter. The organism was called "yellow-pigmented Enterobacter cloacae" until 1980 when it was renamed Enterobacter sakazakii. Urmenyi and Franklin (1) reported the first two known cases of meningitis caused by E. sakazakii in 1961. Subsequently, cases of meningitis, septicemia, and necrotizing enterocolitis due to E. sakazakii have been reported worldwide. Although most documented cases involve infants, reports describe infections in adults as well (2). Overall, case-fatality rates have varied considerably with rates as high as 80 percent in some instances. While a reservoir for E. sakazakii is unknown, a growing number of reports suggest a role for powdered milk-based infant formulas as a vehicle for infection (3-5).

1. Equipment and materials

   1. Covered water bath, with circulating system to maintain temperature of 45.5 ± 0.2°C. Water level should be above the medium in immersed tubes.
   2. Immersion-type thermometer, 1-55°C, about 55 cm long, with 0.1°C subdivisions, certified by National Institute of Standards and Technology (NIST), or equivalent
   3. Incubators, 35-37°C and 24-26°C.
   4. Pipets, 1, 5, and 10 ml, graduated in 0.1 ml units
   5. Glass spreading rods (e.g., hockey stick) 3-4 mm diameter with 45-55 mm spreading area
   6. Sterile inoculating loops, 3 mm loop size
   7. Balance with capacity of 2 kg and sensitivity of 0.1 g
   8. Sterile graduated pipets, 1.0 and 10.0 ml
   9. Sterile utensils for sample handling (see BAM Chapter 1)
   10. Dilution bottles made of borosilicate glass, with stopper or polyethylene screw caps equipped with Teflon liners. 160 ml size.
   11. Sterile Erlenmeyer Flasks with polyethylene screw caps equipped with Teflon liners. 2 liter, 250 ml and 125 ml sizes. Sterile plastic ware may be substituted.
   12. Sterile screw-capped bottles with polyethylene screw caps equipped with Telflon liners. 100 ml size. Sterile plastic ware may be substituted.
   13. Quebec colony counter, or equivalent, with magnifying lens
   14. API 20E Biochemical Strips

   15. Petri dishes, plastic, sterile, 15 x 150 mm

2. Media and reagents

   1. Trypticase (Tryptic) Soy Agar (BAM M152). Prepared medium can be stored at 2-8°C for up to 4 weeks.

   2. Violet red bile glucose agar (VRBG agar): Add ingredients to 1 liter of distilled water, (add dyes as filtered 1% aqueous solutions) and heat to boiling with agitation until all ingredients are dissolved. Do not sterilize this medium further. Cool to 45°C and pour into Petri dishes, preferably of 15 cm diameter. If not used immediately, store agar in a refrigerator at 5-8°C. This medium is available commercially in dehydrated form (Oxoid, CM0485); reconstitute as directed on the container. Prepared medium can be stored at 2-8°C for up to 4 weeks.

      Yeast extract	3.0 g
      Peptone	7.0 g
      Sodium chloride	5.0 g
      Bile Salts No. 3	1.5 g
      Lactose	10.0 g
      Neutral red	0.03 g
      Crystal violet	0.002 g
      Agar	15.0 g
      Glucose	10.0g

   3. Enterobacteriaceae enrichment broth (EE broth): Only purified Ox-gall and Brilliant green should be used so as not to inhibit very low numbers of debilitated cells of the Enterobacteriaceae. Dissolve ingredients in 1 liter of distilled water, heat to boiling, and then dispense in 90 mL volumes. This medium is available commercially in dehydrated form (Oxoid CM0317); reconstitute as directed on the container. Finished media should be green in color. Prepared broth can be stored at 2-8°C for up to 4 weeks.

      Peptone	10.0 g
      Glucose	5.0 g
      Sodium monohydrogen phosphate dihydrate	8.0g
      Potassium dihydrogen phosphate	2.0 g
      Ox-gall	20.0 g
      Brilliant Green	0.015 g

   4. Oxidase test reagent (BAM R54)

3. Presumptive test for E. sakazakii bacteria: The presumptive test is based on a "three-tube" enrichment so that the low levels of the microorganism expected in this product can be detected and quantified. Presumptive testing requires a sample size of at least 333 g.

   1. Sterilize the can lid margins and the spoons used for sampling the cans prior to withdrawing the samples. Using a three-tube MPN procedure aseptically weigh out in triplicate, 100 g, 10 g and 1 g of the powdered infant formula and add to 2 liter, 250 ml and 125 ml size Erlenmeyer Flasks, respectively. Add nine parts (1:10 dilution) of sterile distilled water, prewarmed to 45°C, and gently shake by hand until the powder is uniformly suspended. See Figure 1. (Note: For ease of inoculation set up premeasured volumes of water, warm to 45°C and add directly to the flasks. 100 ml screw-cap bottles may be substituted for the 10 g and 1 g portions.). Incubate overnight at 36°C.

      

      Figure 1. Left, Unmixed; Right, Mixed. (Photograph courtesy of Sharon Edelson Mammel)

   2. Remove 10 ml from each suspension and add to 90 ml of Enterobacteriaceae enrichment broth in sterile 160 ml dilution bottles. Incubate at 36°C overnight.

   3. Gently mix each bottle and proceed with the following plating methods:

      1. Direct Spreading Method: Inoculate duplicate VRBG agar plates with each enrichment culture by spreading 0.1 ml evenly onto the surface of each plate with a sterile glass spreading rod. (Note: If the powdered infant formula is suspected to contain high numbers of E. sakazakii, the EE broth should be diluted to 10^-4 to 10^-6 with sterile EE broth before plating.)

      2. Direct Streaking Method: Inoculate duplicate VRBG agar plates with each enrichment culture by streaking a 3 mm loopful (10 µl) to at least three quadrants of the plate for isolation of single colonies. (Note: Streak plates are included as backup in case growth on the spread plates is too concentrated to contain isolated colonies).

   4. Incubate Petri dishes overnight at 36°C. Observe plates for colonial morphology typical of E. sakazakii (see below).

   5. Pick total of 5 presumptive E. sakazakii colonies from above plates and subculture each isolate by streaking onto a single Trypticase Soy Agar plate. Incubate at 25°C for 48-72 hours.

   6. Select only yellow pigmented colonies from the TSA plates and confirm using the API 20E biochemical identification system according to the manufacturer's instructions. For positive identification of E. sakazakii the oxidase test must be included. (See Table 1 below for biochemical properties of Enterobacter species).

   7. Calculate MPN (BAM Manual, Appendix 2; Most Probable Number Determination from Serial Dilutions). Calculate MPN of E. sakazakii cells/g of sample based on the number of "tubes" at each dilution in which the presence of E. sakazakii was confirmed.

4. Typical E. sakazakii colony morphology:

   1. VRBG agar: Typical colonies will appear as purple colonies surrounded by a purple halo of precipitated bile acids. See Figure 2.
      
      
      
      Figure 2. (Photograph courtesy of Sharon Edelson Mammel)
      
      
      
   2. TSA agar: Typical colonies will appear as yellow-pigmented colonies after 48-72 hr incubation at 25°C. See Figure 3.
      
      
      
      Figure 3. (Photograph courtesy of Sharon Edelson Mammel)
      
      
      

5. References:

   1. Urmenyi, A.M.C. and Franklin, A.W. 1961. Neonatal death from pigmented coliform infection. Lancet 1:313-315.

   2. Hawkins, R.E., Lissner, C.R. and Sanford, J.P. 1991. Enterobacter sakazakii bacteremia in an adult. South Med J. 84(6):793-5.

   3. Simmons, B.P., Gelfand, M.S., Haas, M., Metts, L. and Ferguson, J. 1989. Enterobacter sakazakii infections in neonates associated with intrinsic contamination of a powdered infant formula. Infect Control Hosp Epidemiol 10:398-401.

   4. van Acker, J., De Smet, F., Muyldermans, G., Bougatef, A., Naessens, A. and Lauwers, S. 2001. Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula. J Clin Microbiol 39:293-97.

   5. Biering, G., Karlsson, S., Clark, N.C., Jonsdottir, K.E., Ludvigsson, P., and Steingrimsson, O. 1989. Three cases of neonatal meningitis caused by Enterobacter sakazakii in powdered milk. J Clin Microbiol. 27(9):2054-6.

   6. Food and Agriculture Organization. 1994 Codex Alimentarius: code of hygienic practice for foods for infants and children. CAC/RCP 21-1979. Food and Agriculture Organization of the United Nations, Rome, Italy.

   7. Nazarowec-White, M. and Farber, J.M. 1999. Phenotypic and genotypic typing of food and clinical isolates of Enterobacter sakazakii. J Med Microbiol 48:559-67.

   8. Nazarowec-White, M. and Farber, J.M. 1997. Enterobacter sakazakii: a review. Int J Food Microbiol. 34:103-13.

   9. Muytjens, H.L., Roelofs-Willemse H. and Jaspar, G.H.J. 1988. Quality of powdered substitutes for breast milk with regard to members of the family Enterobacteriacae. J Clin Microbiol 26:743-746.

   10. Nazarowec-White, M., and Farber, J.M. 1997. Thermal resistance of Enterobacter sakazakii in reconstituted dried infant formula. Lett Appl Microbiol 24:9-13.

   11. Muytjens, H.L., van der Ros-van de Ripe, J. and van Druten, H.A.M. 1984. Enzymatic profiles of Enterobacter sakazakii and related species with special reference to the alpha-glucosidase reaction and reproducibility of the test system. J. Clin Microbiol. 20:684-686.

TABLE 1: TAKEN FROM REFERENCE 8

Questions and Answers on Method for E. sakazakii in Powdered Infant Formula August 2002