**Microbial Detection of *Enterobacter sakazakii*: Food and Clinical**

*E. sakazakii* was first associated with cases of neonatal meningitis in
1958 (11). In 1980 while proposing *E. sakazakii* as a new species of
*Enterobacteriaceae* the source of one isolate was reported to be from a
can of dried milk (4). No detail of the isolation method was described.
In 1982, during the investigation of eight neonatal meningitis cases
caused by *E. sakazakii* in the Netherlands, Muytjens, et al., 1983 (6)
recovered the organism from prepared formula but not from the formula
powder itself. In a later study, Muytjens, et al. 1988 (7) suggested
that the inability to isolate *E. sakazakii* from powdered formula may
have been due to its unequal distribution within the powdered formula or
"its presence at such a low concentration that it escaped detection by
conventional methods." Conventional methods, as described by the Food
and Agriculture Organization (FAO) can detect a lower limit of 3.0
colony forming units (CFU)/g.(5). In 1984, six strains of *E. sakazakii*
were isolated from samples of powdered formula using
deoxycholate-citrate agar incubated at 37^o^C for 48 hours; again, no
quantitative methods were used (9).

Muytjens et al., 1988 (7) described the first quantitative method for
the isolation of *E. sakazakii* from powdered infant formula. In this
method, triplicate 100, 10 and 1 gram samples of powdered formula were
mixed with 900, 90 and 9 ml, respectively, of buffered peptone water
(1:10 dilutions) at 45^o^C until completely dissolved. Following
overnight incubation at 36^o^C, 10 ml of each culture was inoculated
into 90 ml of *Enterobacteriaceae* enrichment broth (EE broth) and
incubated at 36^o^C. After overnight incubation, 1 ml from each
enrichment culture was mixed in duplicate with 20 ml of fluid violet
red-bile-glucose agar and poured into petri dishes. The agar was allowed
to solidify, then incubated overnight at 36^o^C. Suspected colonies of
*Enterobacteriaceae* were subcultured to sheep blood agar and
eosin-methylene blue agar. The API 20E system was used to identify
strains. Colonies were confirmed as *E. sakazakii* by testing for the
production of yellow colonies on nutrient agar after 48 hr at 25^o^C,
extracellular DNase production on toluidine blue agar and a positive
alpha-glucosidase reaction. The levels of *Enterobacteriaceae* present
in the formula sample were determined by the most probable number
procedure. 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 CFU/100 g. The lowest level of detection in this method was
0.36 CFU/100 g.

In 1989, Simmons et al. (10) used a similar method to isolate *E.
sakazakii* from powdered formula associated with an outbreak in a
neonatal intensive care unit located in Memphis, Tennessee. In this
study, 50, 10, and 1 gram samples of the implicated infant formula were
dissolved in 450, 90, and 9 ml of buffered peptone water, respectively.
No additional sample was available. After overnight incubation, 10 ml of
each culture was inoculated into 90 ml of EE enrichment broth, incubated
overnight, and plated onto violet red-bile-glucose agar. Suspected
colonies of E*. sakazakii* were subcultured onto MacConkey agar and
confirmed with the API-20E system. No additional biochemical tests were
described. The level of *E. sakazakii* present in the powdered formula
sample was estimated be 8 CFU/100g.

Following an outbreak of neonatal meningitis in Iceland in 1986 and
1987, *E. sakazakii* was isolated from five different lot numbers of
unopened packages of powdered infant formula; in all, five of seven
packages were positive (3). Using an amount of sterile water recommended
by the manufacturer, 500 and 1000 gram packages of powder were dissolved
and divided into 100 ml samples. Following incubation at 36^o^C for 4
hours, the samples were subcultured to blood agar and MacConkey agar
plates. No quantitative data were reported. Isolates were confirmed by
the API 20E system followed by biotyping, plasmid DNA determinations and
antibiograms.

During the investigation of a 1998 outbreak of necrotizing enterocolitis
that occurred in a neonatal intensive care unit in Brussels, Belgium
(12), van Acker et al. isolated *E. sakazakii* from liquid formula
prepared from powder (Nestle Alfare). The liquid formula was cultured by
direct plating on trypticase soy agar supplemented with 5% sheep blood,
on MacConkey agar and by culturing 1.0 ml in fastidious anaerobe broth.
This report did not indicate which of these methods produced the
positive results. The report stated that the manufacturer's quality
control data for the implicated production lot of powder indicated that
one of 5 sub-samples contained 20 coliforms per gram of powder and that
the remaining 4 sub-samples contained less than 1 coliform per gram.
These data support the hypothesis that the distribution of microbial
contaminants in a lot of powder may be heterogeneous. If the
distribution of coliforms (specifically, *E. sakazakii*) in lots of
powdered formula is typically heterogeneous, or non-random, then it may
be important to test sub-samples from each lot in order to improve the
chances of correctly determining the acceptability of the lot (5).

In the same study (12), van Acker reported that unopened cans of the
implicated Alfare powdered formula were tested for *E. sakazakii* by
inoculating 3 grams of powder directly into fastidious broth. After 48
hr incubation at 37^o^C, the enrichments were plated on tryptic soy agar
supplemented with 5% horse blood, hemin T, and NAD; on tryptic soy agar
with 5% horse blood and nalidixic acid; on MacConkey agar and on
mannitol salt agar. The organism was isolated from several unopened cans
of one of two production lots of formula in the hospital's inventory. No
attempt was made to quantify the level of *E. sakazakii* present in the
formula.

During a Canadian survey of the incidence of *E. sakazakii* in dried
infant formula (8), the method of Muytjens et al. (7) was used with only
minor modifications. The powder was suspended in sterile water and
suspect colonies from the VRBG agar plates were subcultured to TSB-YE
agar prior to confirmation using the API 20E system. No additional
biochemical tests were performed and levels of *E. sakazakii* were
determined by the most probable number technique. The sensitivity of
this method was equal to that of the original method (7). *E. sakazakii*
was isolated from 8 of 120 cans, representing 5 different manufacturers,
at a level of 0.36 CFU/100 g.

*E. sakazakii* was isolated from commercial powdered formula during the
investigation of a fatal infection in a hospitalized neonate (MMWR
Weekly, April 12, 2002/51(14):298-300). The method used by the Centers
for Disease Control and Prevention was based on the method of Muytjens,
et al. 1988 (7). One hundred grams of powdered formula was mixed with
400 ml of phosphate buffered peptone water at 45^o^C. Following
overnight incubation at 36^o^C, 1 ml of the culture was inoculated into
9 ml of brilliant green bile broth. After overnight incubation at
36^o^C, the enrichment culture was inoculated onto LesEndo agar and
incubated overnight at 36^o^C. Colonies having a green metallic sheen
were subcultured to TSA-5% sheep blood agar plates, incubated at 36^o^C
overnight then held at room temperature for 24-48 hr. Yellow pigmented
colonies were re-cultured on TSA-5% sheep blood agar to confirm yellow
pigment production. Presumptive *E. sakazakii* colonies were confirmed
with the API --20E system.

The current recommended FDA procedure was derived from the Canadian
method (8), which was based on the method of Muytjens, et al., 1988 (7).
The FDA method calls for the direct spreading or streaking of an
overnight EE broth rather than VRBG pour plates. Five presumptive
colonies are subcultured to Trypticase Soy Agar and incubated at 25^o^C
for 48-72 hours. Only yellow pigmented colonies from the TSA plates are
confirmed using the API 20E biochemical identification system. No
additional biochemical testing is recommended. The levels of *E.
sakazakii* are determined as previously described (8); the lower limit
of detection is 0.36/100g. Thus, the FDA method can detect levels of *E.
sakazakii* much lower than the recommended FAO level of 3.0 CFU/g. of
powdered infant formula (5).

*E. sakazakii* is isolated from clinical samples using standard methods
for the isolation of *Enterobacteriaceae* (1). No special media has been
developed for *E. sakazakii*, and it grows well on blood agar,
MacConkey, eosin methylene blue, deoxycholate agar and tergitol 7 agars.
Reduced plating efficiency was reported for XLD and HE agars (4). During
surveillance culturing, using conventional enteric media, in a neonatal
intensive care unit in Athens, Greece, *E. sakazakii* was isolated from
throat and rectal swabs and from tracheal aspirates (2). Isolates of *E.
sakazakii* from samples of blood and cerebrospinal fluid from cases of
meningitis have been confirmed with either the Api-20 or the Enterotube
II system (13). During an outbreak of necrotizing enterocolitis
associated with *E. sakazakii*, anal and stomach aspirates were
inoculated onto tryptic soy agar supplemented with horse blood, hemin T,
and NAD; tryptic soy agar supplemented with horse blood and nalidixic
acid; MacConkey agar and mannitol salt agar. Blood cultures were
performed using the BBL Septi-Chek system with brain heart infusion
broth bottles (12). Investigators were able to recover *E. sakazakii*
from blood, anal, and stomach sites.

In conclusion, the 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*. However, 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.*

**[References:]{.underline}**

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Tenover and R.H. Yolken (ed.), Manual of Clinical Microbiology, 7^th^
Edition, American Society for Microbiology, Washington, D.C.

2\. Arseni, A., E. Malamou-Ladas, C. Koutsia, M. Xanthou and E. Trikka.
1987. Outbreak of colonization on neonates with *Enterobacter
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3\. Biering, G., S. Karlsson, N.C. Clark, K.E. Jonsdottir, P. Ludvigsson
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5\. Food and Agriculture Organization. 1994. Codex Alimenterius code of
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6\. Muytjens, H.L., H.C. Zanen, H.J. Sonderkamp, L.A. Kollee, I.K.
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7\. Muytjens, H.L., H. Roelofs-Willemse and G.H.J. Jaspar. 1988. Quality
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family *Enterobacteriaceae*. J. Clin. Microbiol. 26: 743-746.

8\. Nazarowec-White, M. and J.M. Farber. 1997. Isolation, survival and
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9\. Postupa, R. and E. Aldova. 1984. *Enterobacter sakazakii*: A Tween
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10\. Simmons, B.P., M.S. Gelfand, M. Haas, L. Metts and J. Ferguson.
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11\. Urmenyi, A.M.C. and A.W. Franklin. 1961. Neonatal death from
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13\. Willis, J. and J.E. Robinson. 1988. *Enterobacter sakazakii*
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