http://www.fda.

gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2893
78.htm
Bacteriological Analytical Manual
Chapter 29
Cronobacter
BAM Table of Contents

Authors: Yi Chen, Keith Lampel, and Thomas Hammack

Revision History:

March 2012: New Chapter (This chapter has replaced the method for Isolation and
Enumeration of Enterobacter sakazakii from Dehydrated Powdered Infant Formula
(available as archived content)).

April 2012: Sections D.1.a, D.1.b, D.2.3; Correction: The fluorescence is recorded at
the end of each annealing step, not at the end of each extension step.

Introduction
Cronobacter is a Gram-negative rod within the family Enterobacteriaceae (7). The organism
was called "yellow-pigmented Enterobacter cloacae" until it was renamed Enterobacter
sakazakii (6) in 1980. Urmenyi and Franklin reported the first two known cases of meningitis
caused by E. sakazakii in 1961 (11). Subsequently, cases of meningitis, septicemia, and
necrotizing enterocolitis due to E. sakazakii have been reported worldwide (9). Although
most documented cases involve infants, reports describe infections in adults as well. Overall,
case-fatality rates vary considerably with some as high as 80 percent (8). While a reservoir
for E. sakazakii is unknown, a growing number of reports suggest powdered infant formulas
as a vehicle for infection (12).
Recently obtained evidence using amplified fragment length polymorphism, phenotypic
arrays, automated ribotyping, 16S rRNA gene sequencing and DNA-DNA hybridization has
resulted in a nomenclature change. E. sakazakii was reclassified into a new genus,
Cronobacter, comprising five species including Cronobacter sakazakii gen. nov.,
Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii
sp. nov., and Cronobacter dublinensis sp. nov. These species have all been previously
involved in clinical cases. One additional new species was also proposed, Cronobacter
genomospecies I (Table 1) (7).
Table 1. Biochemical tests for the differentiation of species and subspecies of the genus
Cronobacter (7).

Table 1. Biochemical tests for the differentiation of species and subspecies of the genus
Cronobacter (7).
C. saka C. malon C. turic C. genomos C. muytj C. dublin C. dublin C. dublin
zakii
aticus
ensis
pecies I
ensii
ensis
ensis
ensis
subsp. subsp. subsp.
dublinen lactaridi lausanne
sis
nsis
Indole

production

Carbon source utilization:

+
+
+
+
+
+
+
V
+
+
+

+
+
V
+
V
+
+

+
V
V

Dulcitol

Lactulose +
+
Malonated
+
Maltitol +
+
Palatinose +
+
Putrescine +
V
Melezitose

Turanose +
+
myoV
V
+
+
+
Inositol
cis+
+
+
+
V
Aconitate
trans
+

+
V
Aconitate
1-0Methyl aD+
+
+
+

glucopyra
noside
4Aminobut +
+
+
V
+
yrate
+: 90% Positive; V: 20-80% positive; : 10% positive

+
+
+
+
+
+
+

+
+
+

+
V

Method
The method described here contains both a real-time PCR method for rapid screening and a
cultural method for the detection/isolation of Cronobacter spp. (3). Chromogenic agars are
used to isolate the culture for confirmation. A pre-enrichment step is used to grow the
bacteria to an amount ( 103 CFU/ mL) detectable by PCR and chromogenic agars. The
cultural portion of this method is a complete detection/isolation method, so it can be used as a
stand alone method if PCR technology is unavailable. The PCR portion of the method is a
screening method, whose positive results should always be confirmed with the cultural
method. The PCR method may be used to confirm pure cultures as Cronobacter spp. This
method was validated in pre-collaborative and collaborative studies (1,2).

The inclusivity of this method was determined by analyzing 51 different Cronobacter strains
representing the six Cronobacter species that were isolated from foods, clinical samples,
environmental surfaces, and nationally/internationally recognized culture depositories. The
origin and source of each strain are listed in the Inclusivity Table (A). Each strain was
enriched in brain heart infusion (BHI) broth and diluted in buffered peptone water (BPW) to
approximately 10 times the limit of detection. The diluted cultures were then tested according
to this method.
The exclusivity of this method was determined by testing 42 non-Cronobacter strains. The
source and origin of each strain are listed in the Exclusivity Table (B). Each strain was
enriched in BHI broth. These incubated cultures were tested according to this method.
Equipment and materials
1. Balance with capacity of 2 kg and sensitivity of 0.1 g
2. Incubator, 36 1 C
3. Sterile Erlenmeyer flasks with polyethylene screw caps equipped with teflon liners, 2
liter
4. Micropipette and tips to dispense 1 L , 100 L, 150 L, and 200 L volumes
5. Pipets, 1, 5, and 10 mL, graduated in 0.1 mL units
6. Sterile inoculating loops, 3 mm loop size
7. Glass spreading rods (e.g., hockey stick) 3-4 mm diameter with 45-55 mm spreading
area
8. Sterile utensils for sample handling (see BAM Chapter 1)
9. Centrifuge with a swinging bucket rotor, capable of 3,000 g
10. Microcentrifuge, capable of 10,000 g
11. Centrifuge tubes, polypropylene, 50 mL tubes with conical bottoms; 1.5 mL
microcentrifuge tubes
12. Vortex mixer
13. Water bath, capable of 100 C
14. Thermal cyclers: ABI Prism 7500 Fast Sequence Detection System (Life
Technologies, Inc. Carlsbad, CA 92008), SmartCycler Real-Time PCR system
(Cepheid, Sunnyvale, CA 94089)
15. Sample tubes with centrifuge adapters for SmartCycler II (minimum reaction volume
of 25 L)
16. 96-well microwell plate

17. Optical adhesive covers

18. Petri dishes, plastic, sterile, 15 150 mm
19. VITEK 2 Compact (bioMerieux, Hazelwood, MO 63402)
20. NanoDrop ND1000 (Thermal Scientific, Wilmington, DE, 19810)
Media and reagents
1. Phosphate-buffered saline (BAM R59)
2. Buffered peptone water (BPW) (BAM M192)
3. Brilliance Enterobacter sakazakii agar (DFI formulation) (Cat. No. CM1055, Oxoid,
Lenexa, KS). Prepare media according to the instructions on the packaging label.
After the plates have been poured and dried upside down at room temperature, they
can be placed in petri plate sleeves and stored upside down at 2-8 C in the dark for up
to 2 weeks.
4. Enterobacter sakazakii chromogenic plating agar (R&F agar), (Cat. No. M-0700, R &
F Laboratories, Downers Grove, IL). Prepare media according to the manufacturer's
instructions on the packaging label. After the plates have been poured, they should be
stored upside down in the dark for 48 hours at room temperature to dry the agar
surface. Then the plates can be placed in petri plate sleeves (cutting a 0.5" to 1" hole
in the sleeves to allow condensation to escape) and stored upside down at 2-8 C in
the dark for up to 45 days.
5. Oxidase test reagent (BAM R54)
6. PrepMan Ultra sample preparation reagent (Cat. No. 4318930. Life Technologies)
7. iQ Supermix PCR master mix (Cat. No. 170-8860, Bio-Rad, Hercules, CA 94547).
2mix contains 100 mM KCl, 40 mM Tris-HCl, pH8.4, 0.4 mM each dNTP, 50 U/ml
iTaq DNA polymerase and 6 mM MgCl2.
8. Rapid ID 32 E biochemical strips (bioMerieux)
9. VITEK Gram Negative Identification Card (bioMrieux)
10. Platinum Taq DNA Polymerase (Cat. No. 10966-018, Invitrogen, Carlsbad, CA
92008)
11. Primers and probes (Table 2). PCR primers are commercially synthesized with basic
desalt purification and then reconstituted using PCR grade water to 100 M for
prolonged storage. They are diluted to 40 M working stock concentrations. PCR
probes are commercially synthesized with HPLC purification and reconstituted to 2.5
M in single use aliquots using 1 PCR grade TE buffer. Primers and probes need to
be stored frozen (-20 to -70 C). Discard leftover thawed probes and avoid repeating
freeze-thawing.

Table 2. Primers and probes for the PCR assay

Oligos

Name

Sequences (5' to 3')

Cronobacter
CronoF
forward

GGGATATTGTCCCCTGAAACAG

Cronobacter
CronoR
reverse

CGAGAATAAGCCGCGCATT

Cronobacter
CronoP
probe

6FAMAGAGTAGTAGTTGTAGAGGCCGTGCTTCCGAAAGTAMRA

Internal
control
forward

InCF

CTAACCTTCGTGATGAGCAATCG

Internal
control
reverse

InCR

GATCAGCTACGTGAGGTCCTAC

Internal
InCP
control probe

Cy5-AGCTAGTCGATGCACTCCAGTCCTCCT-Iowa Black
RQ-Sp.

12. Internal Control DNA (3): Internal control (InC) DNA is constructed by generating a
198 bp sequence that is synthesized and inserted into a pZErO-2 vector and
transformed into Escherichia coli pDMD801 partial sequence representation
containing the internal control is shown below (GenBank accession no. FJ357008,
Figure 1). The internal control DNA (IAC in Figure 1) sequence is in grey, T7
promoter is represented in a box, and M13, internal control forward and reverse
primers and probe targets are represented by arrows. The plasmid is extracted by
Qiagen Plasmid Mini Kit (Cat. No. 12125, Qiagen, Valencia, CA 91355) from the
transformed Escherichia coli cells following manufacturer's instructions and
quantified by NanoDrop ND1000. The internal control DNA can also be
commercially synthesized and diluted to a stock solution which will provide a reliable
Ct of no less than 24 when Cronobacter DNA are present and no more than 34 when
Cronobacter are not present.
Figure 1. Illustration of the internal control DNA

 Preparation of infant formula samples for isolation of Cronobacter

1. Wear double gloves at all times. Change outer gloves, wipe clean the balance and
working area after processing each sample
2. Sterilize the container margins and the spoons used for sampling prior to withdrawing
the samples.
3. Aseptically weigh out 100 g of the powdered infant formula and add to 2 liter size
Erlenmeyer flasks.
4. Add 900 mL (1:10 dilution) of sterile buffered peptone water (BPW) and gently shake
by hand until the powder is uniformly suspended. Incubate for 24 2 h at 36 1 C.
5. Thoroughly mix the enrichment mix and remove four aliquots of 40 mL each from the
incubated sample and place them into four 50 mL centrifuge tubes. Centrifuge the
aliquots at 3,000 g for ten minutes in a swinging bucket centrifuge (fixed angle
centrifuges are not recommended because of problems separating the fats from the
pellet).
6. Aspirate the supernatants of each centrifuge tube.
7. Use sterile cotton swabs or equivalent tools to remove the fat precipitate on the side
wall of the centrifuge tube, if necessary.

8. Suspend the resultant pellet in 200 L of phosphate buffered saline (PBS) by

vortexing at maximum speed for at least 20 sec. Two of the aliquots will be used for
PCR. Two of the aliquots will be used for culture confirmation if necessary.
PCR screening of Cronobacter
DNA extraction. Centrifuge 200 L suspended cells (in PBS) at 3,000 g for 5 min in a 1.5
mL microcentrifuge tube. Depending on the presence and absence of bacterial cells and the
efficiency of fat removal at the previous step, there could be 4 layers after centrifugation. The
top layer is fat residues, the second layer is supernatant, the third layer is bacterial cell pellets
which are brown/yellow and the bottom pellets are milk particles. Discard the supernatants
and any fat residues of each centrifuge tube. Add 400 l of PrepMan Ultra sample
preparation reagent to each tube and vortex at maximum speed to allow complete suspension.
Heat the sample for 10 min at 100 C in a boiling water bath or heating block, then cool the
sample to room temperature for 2 min. Centrifuge the sample for 2 min at a speed of at least
15,000 g. Transfer 50 l of the supernatant into a new tube for PCR analysis. For each
DNA extract, run both PCR protocols with and without InC. If any of the PCR result is
positive, the sample is considered presumptive positive or cannot rule out (CRO) and proceed
to the culture confirmation (Section E). If both DNA extracts are negative by PCR, the
sample is considered negative and stop analysis.
Include a positive control (prepared by 1:10 dilution of a pure culture of Cronobacter strain,
i.e. E604) and a no template (water) control in each PCR run. The PCR is designed to
detect very low level of Cronobacter cells. If after enrichment, a significant large amount
cells are grown, the PCR could yield high fluorescence. One solution is to dilute the DNA
(1:10 or 1:100) and rerun the PCR.
1. Cepheid SmartCycler Thermal Cycler (software version 2.0d).
Prepare PCR reactions from the reaction components and final concentrations listed in
Table 3 and Table 4. Create a "run" on SmartCycler. Give each run a unique run name,
select dye set FCTC25, select 3-step PCR protocol as described below and assign
appropriate sites on the cycler block.
a. PCR setup without InC
PCR condition: 95 C for 3 min followed by 40 cycles of 95 C for 15 sec, 52
C for 20 sec and 72 C for 30 sec. The fluorescence is recorded at the end of
each annealing step.
1.
a. Table 3. PCR reaction components for SmartCycler without InC
Component
IQ Supermix

Volume/reaction
12.5 L

Final Concentration
50 mM KCl, 20 mM Tris-HCl, 0.2
mM each dNTP,
0.625 U iTaq DNA polymerase and 3
mM MgCl2

Component

Volume/reaction

Final Concentration

CronoF

0.5625 L (40 M stock

solution)

900 nM

CronoR

0.5625 L (40 M stock

solution)

900 nM

CronoP

2.5 L (2.5 M stock

solution)

250 nM

Taq Polymerase

0.1 L (5 U/ L)

0.5 U

DNA extract or
control

2 L

PCR grade water

Appropriate amount to reach

25 L

b. PCR setup with InC

PCR condition: 95 C for 3 min followed by 40 cycles of 95 C for 20 sec, 50
C for 60 sec and 72 C for 30 sec. The fluorescence is recorded at the end of
each annealing step.
Table 4. PCR reaction components for SmartCycler with InC
Component

Volume/reaction

Final Concentration

IQ Supermix

12.5 L

CronoF

0.5625 L (40 M stock

solution)

900 nM

CronoR

0.5625 L (40 M stock

solution)

900 nM

CronoP

2.5 L (2.5 M stock

solution)

250 nM

InCF

0.625 L (40 M stock

solution)

1000 nM

InCR

0.625 L (40 M stock

solution)

1000 nM

InCP

2.5 L (2.5 M stock

solution)

250 nM

InC DNA

50 mM KCl, 20 mM Tris-HCl, 0.2

mM each dNTP,
0.625 U iTaq DNA polymerase and 3
mM MgCl2

1 L (0.01 pg/L; equivalent 0.01 pg

to
3 103 plasmid copies per L)

Component

Volume/reaction

Final Concentration

Taq Polymerase

0.5 L (5 U/ L)

2.5 U

MgCl2

1.5 L (50 mM solution)

3 mM

DNA extract or
control

2 L

PCR grade water

Appropriate amount to reach

25 L

c. Qualitative Data Interpretation

On the SmartCycler Instrument, set the following analysis settings for FAM
and Cy5 channels. Update analysis settings if they are changed before
recording results.
1. Usage: Assay
2. Curve Analysis: Primary
3. Threshold Setting: Manual
4. Manual Threshold Fluorescence Units: FAM channel set at 20
units. Cy5 channel set at 30 units.
5. Auto Min Cycle: 5
6. Auto Max Cycle: 10
7. Valid Min Cycle: 3
8. Valid Max. Cycle: 60
9. Background subtraction: ON
10. Boxcar Avg. Cycles: 0
11. Background Min. Cycle: 5
12. Background Max. Cycle: 40
Primary fluorescence curves that cross the threshold will be recorded as
"POS" and the cycle number when it crosses the threshold will be displayed in
the "Results Table" view. Negative results are shown as "NEG". The FAM and
Cy5 channels correlate to Cronobacter and InC targets, respectively. Results
can also be viewed graphically (Figure 2).

Figure 2. Sample screenshots of the SmartCycler result (graph and table

views). The amplification curves of Cronobacter (labeled with FAM) and
internal control (labeled with Cy5) are shown in the graph. The Ct values,
positive/negative results and dye sets are shown in the result table. FCTC25
dye set contains FAM, Cy3, TxR and Cy5. Cy3 and TxR are not used in the
PCR assay.
A DNA extract is considered PCR positive if either of the PCR runs (with and
without InC) is positive.
For PCR with InC, DNA extracts that have Ct values for FAM and also
demonstrate sigmoidal amplification curves are considered CRO. If there is no
Ct value in FAM for a DNA extract, or the curve is not the typical sigmoidal
shape, the InC for that DNA extract must be analyzed:
13. The DNA extract is considered negative if there is Ct value in Cy5 and
the curve for Cy5 is sigmoidal;
14. If there is no Ct value in Cy5, then there is possible inhibitory
substance in the sample and PCR result is invalid. The DNA extracts
need to be diluted (1/10, in PCR grade water) or centrifuged for further
purification, and the PCR repeated; or directly proceed with culture
confirmation (Section E).
For PCR without InC, DNA extracts that have Ct values for FAM and also
demonstrate a sigmoidal amplification curve are considered CRO and proceed
to culture confirmation. If there are no Ct values in FAM, the DNA extract is
considered negative. If PCR without InC is negative, but the PCR with InC
shows possible presence of inhibitory substances in the sample, then further
purified DNA extracts need to be diluted and repeated with the PCR without
InC.

With both protocols, a positive template control should generate positive

signal for the PCR results to be valid. If the no template control shows
amplification, then the reaction may be contaminated and the PCR result is
invalid. Repeat PCR; or directly proceed with culture confirmation.
2. ABI 7500 Fast Thermal Cycler (software version 2.0.4)
Prepare PCR reactions from the reaction components and final concentrations listed in
Table 5 and Table 6. Create a "new experiment" on 7500 Fast. Give each experiment a
unique name. Select the following parameters:
a. In "Experimental properties", select quantitation-standard curve as the type of
experiments; Taqman reagents and standard ramp speed,
b. In "Plate Setup", select none for reference dye, TAMRA as quencher for
Cronobacter probe and none as quencher for InC probe. Assign appropriate
sites on the cycler block.
c. In "Run Method", set reaction volume per well to 25 L. Select the PCR
conditions: 95 C for 3 min followed by 40 cycles of 95 C for 15 sec, 52 C
for 40 sec and 72 C for 15 sec. The fluorescence is recorded at the end of
each annealing step. The PCR with and without InC employ the same PCR
condition.
a. PCR setup without InC
Table 5. PCR reaction components for ABI 7500 Fast without InC
Component

b.

Volume/reaction

Final Concentration

IQ Supermix

12.5 L

CronoF

0.25 L (40 M stock

solution)

400 nM

CronoR

0.25 L (40 M stock

solution)

400 nM

CronoP

3 L (2.5 M stock solution) 300 nM

Taq Polymerase

0.1 L (5 U/ L)

DNA extract or
control

2 L

PCR grade water

Appropriate amount to reach 25 L

PCR setup with InC

50 mM KCl, 20 mM Tris-HCl, 0.2 mM

each dNTP,
0.625 U iTaq DNA polymerase and 3
mM MgCl2

0.5 U

Table 6. PCR reaction components for ABI 7500 Fast with InC
Component

Volume/reaction

Final Concentration

IQ Supermix

12.5 L

50 mM KCl, 20 mM Tris-HCl, 0.2

mM each dNTP,
0.625 U iTaq DNA polymerase and 3
mM MgCl2

CronoF

0.25 L (40 M stock

solution)

400 nM

CronoR

0.25 L (40 M stock

solution)

400 nM

CronoP

3 L (2.5 M stock solution)

300 nM

InCF

0.09375 L (40 M stock

solution)

150 nM

InCR

0.09375 L (40 M stock

solution)

150 nM

InCP

1.5 L (2.5 M stock

solution)

150 nM

InC DNA

0.005 pg/L (equivalent to

1.5 103 plasmid copies per
L)

0.005 pg

Taq Polymerase

0.5 L (5 U/L)

2.5 U

MgCl2

1.5 L (50 mM solution)

3 mM

DNA extract or
control

2 L

PCR grade water

Appropriate amount to reach 25 L

To analyze the data, set auto baseline and set the threshold value to 50,000
units for both FAM and Cy5. Sample screen shots of the graph and table view
is shown in Figure 3. Follow the data interpretation criteria for the
SmartCycler to interpret the data.

Figure 3. Sample screenshots of the 7500 Fast result. The amplification curves
of Cronobacter (assigned as target 1) and InC (assigned as target 2) are shown
in the graph and the Ct values and dye sets are shown in the result table.
Isolation of Cronobacter
For CRO samples, spread 100 L aliquots of suspended cells (obtained in C8) evenly onto
each of the two DFI chromogenic agar (4) and two R&F Cronobacter chromogenic plating
agar (9) with sterile spreading rods. In addition, streak a loopful of aliquots of suspended
cells onto the surface of two DFI and two R&F agar with sterile inoculation loops. Incubate
the agar plates at 36 1 C for 18 to 24 h. Observe plates for colonial morphology typical of
Cronobacter (Figure 4). If the cultures overgrow on the plates, streak a 3 mm loopful (10 L)
of lawn materials to at least three quadrants of a new plate for isolation of single colonies.

Figure 4a. Cronobacter colonies on DFI agar.

Figure 4b. Cronobacter colonies on R&F agar. The plate on the right shows an infant
formula sample with Cronobacter and background flora. The background flora changes the
background color of the agar from red to yellow in most areas. Cronobacter colonies are
green with yellow background and black with red background.
A Identification of Cronobacter
Presumptive Cronobacter colonies on DFI agar appear either dark green, weak green,
or brownish green. Some colonies only have a green center with a white/yellow
border. Presumptive Cronobacter colonies on R&F agar appear blue to black, or blue
to grey with the red background. The red background can appear purplish red with
different strain or under different light conditions. Cronobacter does not change the
color of R&F agar, but in the presence of background microflora which change the
color of R&F agar from red to yellow, Cronobacter colonies appear blue to green
(Figure 4b).
1. Biochemical confirmation
Cultures for biochemical identification must be no older than 24 h. With a
sterile inoculating loop, pick one presumptive Cronobacter colony from each
DFI agar and R&F agar plate and confirm using Rapid ID 32 E or VITEK 2.0
biochemical identification system according to the manufacturer's instructions.
For positive identification with Rapid ID 32 E, the oxidase test must be
included.
2. PCR confirmation
Prepare DNA of presumptive positive colonies on DFI and R&F agar plates.
Add a small amount of colony material to 150 L of PCR grade dH2O
contained in a 1.5 mL plastic centrifuge tube and boil for 5 min in a boiling
water bath. Chill the tubes in ice and centrifuge at 10,000 g for 2 min. Use 1
L of this lysed material as DNA template for the real-time PCR assay with
any of the mentioned PCR protocols above.
B Optional: enumeration of Cronobacter

Use the three-tube Most Probably Number (MPN) procedure (BAM Manual,
Appendix 2; Most Probable Number Determination from Serial Dilutions; ).
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 and
proceed with sample preparation, culture isolation and identification. Calculate MPN
of Cronobacter cells/g of sample based on the number of "tubes" at each dilution in
which the presence of Cronobacter was confirmed.
C Flowchart of the complete procedure

Figure 5. Flowchart of the complete procedure

References
1.

Chen, Y., K.E. Noe, S. Thompson, C.A. Elems, E.A. Brown, K.A. Lampel, and T.S.
Hammack. 2011. Evaluation of a revised FDA method for the detection of
Cronobacter in powdered infant formula: Collaborative study. J. Food Prot. In Press.

2.

Chen, Y., T.S. Hammack, K.Y. Song, and K.A. Lampel. 2009. Evaluation of a revised
U.S. Food and Drug Administration method for the detection and isolation of

Enterobacter sakazakii in powdered infant formula: precollaborative study. J. AOAC

Int. 92:862-872.
3.

Chen, Y., K.Y. Song, E.W. Brown and K.A. Lampel. 2010. Development of an
improved protocol for the isolation and detection of Enterobacter sakazakii
(Cronobacter) from powdered infant formula. J. Food Prot. 73:1016-1022.

4.

Deer, D.M., K.A. Lampel, and N. Gonzalez-Escalona. 2010. A versatile internal

control for use as DNA in real-time PCR and as RNA in real-time reverse
transcription PCR assays. Lett. Appl. Microbiol. 50:366-372.

5.

Druggan, P. and C.Iversen. 2009. Culture media for the isolation of Cronobacter spp.
Int. J. Food Microbiol. 136:169-178.

6.

Farmer J.J., III, M.A. Asbury, F.W. Hickman. The Enterobacteriaceae Study Group
and D.J. Brenner. 1980. Enterobacter sakazakii: A new species of
"Enterobacteriaceae" isolated from clinical specimens. Intl. J. Syst. Evol. Microbiol.
30:569-584.

7.

Iversen, C., N. Mullane, B. McCardell, B.D. Tall, A. Lehner, S. Fanning, R. Stephan,

and H. Joosten. 2008. Cronobacter gen. nov., a new genus to accommodate the
biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen.
nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov.,
Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter
genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp.
dublinensis subsp. nov., Cronobacter blinensis subsp. lausannensis subsp. nov. and
Cronobacter dublinensis subsp. lactaridi subsp. nov. Int. J. Syst. Evol. Microbiol.
58:1442-1447.

8.

Lai, K.K. 2001. Enterobacter sakazakii infections among neonates, infants, children,
and adults. Case reports and a review of the literature. Medicine (Baltimore). 80:113122.

9.

Nazarowec-White, M. and J.M. Farber. 1997. Enterobacter sakazakii: a review. Int.

J. Food Microbiol. 34:103-113.

10. Restaino, L., E.W. Frampton, W.C. Lionberg, and R.J. Becker. 2006. A chromogenic
plating medium for the isolation and identification of Enterobacter sakazakii from
foods, food ingredients, and environmental sources. J. Food Prot. 69:315-322.
11. Urmenyi, A.M. and A.W. Franklin. 1961. Neonatal death from pigmented coliform
infection. Lancet. 1:313-315.
12. World Health Organization. Enterobacter sakazakii and other microorganisms in
powdered infant formula: meeting report. 2004.

Appendix
Table A. Inclusivity Testing Results for Cronobacter
Original Lab Original ID

Organism

Source Countr DFIa R&F RAPID Realb

y Origin
ID 32E Time
PCR

UCDc/UZHd/ E265
NRCe

C.
milk
malonaticus powder

ILSIf

F6-036

C. sakazakii Environ Malaysi Positi Positi Cronoba Positi

ment
a
ve
ve
cter
ve
(Milk
powder)

ILSI

F6-038

C. sakazakii Environ Holland Positi Positi Cronoba Positi

ment
ve
ve
cter
ve
(Milk
powder)

ILSI

F6-040

C. sakazakii Environ Holland Positi Positi Cronoba Positi

ment
ve
ve
cter
ve
(Milk
powder)

UCD/UZH/N E464
RC

C.
dublinensis

Environ Zimbab Positi Positi Cronoba Positi

ment
we
ve
ve
cter
ve
(Milk
powder)

ATCCg;
NCTCh

ATCC
C. sakazakii human
29544;
(throat)
NCTC 11467

FDAi

607

UCD/UZH/N E515
RC

Malaysi Positi Positi Cronoba Positi

a
ve
ve
cter
ve

unknow Positi Positi Cronoba Positi

n
ve
ve
cter
ve

C. sakazakii unknown unknow Positi Positi Cronoba Positi

n
ve
ve
cter
ve
C.
dublinensis

water

Switzerl Positi Positi Cronoba Positi

and
ve
ve
cter
ve

ATCC

ATCC 12868 C. sakazakii unknown unknow Positi Positi Cronoba Positi

n
ve
ve
cter
ve

ATCC

ATCC 51329 C.
muytjensii

unknown unknow Positi Positi Cronoba Positi

n
ve
ve
cter
ve

HCSCj; FDA SK90

C. sakazakii clinical Canada Positi Positi Cronoba Positi

(children'
ve
ve
cter
ve
s
hospital)

UCD/UZH/N E632
RC

C. sakazakii food

USA

HCSC

HPB 2848

C. sakazakii clinical

Canada Positi Positi Cronoba Positi

ve
ve
cter
ve

HCSC

HPB 2873

C. sakazakii clinical

Canada Positi Positi Cronoba Positi

Positi Positi Cronoba Positi

ve
ve
cter
ve

Original Lab Original ID

Organism

Source Countr DFIa R&F RAPID Realb

y Origin
ID 32E Time
PCR
ve

HCSC

HPB 2874

C. sakazakii clinical

ve

cter

ve

Canada Positi Positi Cronoba Positi

ve
ve
cter
ve

UCD/UZH/N H. Muytjens C. sakazakii unknown Czech Positi Positi Cronoba Positi

RC
(Prague 72 2
Republi ve
ve
cter
ve
6248)
c
UCD/UZH/N H. Muytjens C.
milk
RC
52
malonaticus powder

Australi Positi Positi Cronoba Positi

a
ve
ve
cter
ve

UCD/UZH/N H. Muytjens C. sakazakii milk

RC
58
powder

Belgium Positi Positi Cronoba Positi

ve
ve
cter
ve

UCD/UZH/N H. Muytjens C. sakazakii milk

RC
15
powder

Denmar Positi Positi Cronoba Positi

k
ve
ve
cter
ve

UCD/UZH/N H. Muytjens C. sakazakii milk

RC
8
powder

France

Positi Positi NonPositi

ve
ve
Cronoba ve
cter

UCD/UZH/N H. Muytjens C. sakazakii milk

RC
35
powder

Russia

Positi Positi Cronoba Positi

ve
ve
cter
ve

UCD/UZH/N H. Muytjens C. sakazakii milk

RC
26
powder

Russia

Positi Positi Cronoba Positi

ve
ve
cter
ve

UCD/UZH/N H. Muytjens C. sakazakii neonate

RC
(Nijmegen
15)

Holland Positi Positi Cronoba Positi

ve
ve
cter
ve

UCD/UZH/N H. Muytjens C. sakazakii neonate

RC
(Nijmegen
21)

Holland Positi Positi Cronoba Positi

ve
ve
cter
ve

CDCk

CDC 596070

C.
dublinensis

USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

CDC

CDC 352375

C.
human USA
malonaticus (bone
marrow)

Positi Positi Cronoba Positi

ve
ve
cter
ve

NCTC

NCTC 9238 C. sakazakii human UK

(abdomin
al pus)

Positi Positi Cronoba Positi

ve
ve
cter
ve

NCTC

NCTC 9529 C. genomosp water

ecies

Positi Positi Cronoba Positi

ve
ve
cter
ve

ATCC

ATCC
BAA893

C. sakazakii unknown USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

ATCC

ATCC
BAA894

C. sakazakii unknown USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

CDC

CDC 996-77 C. sakazakii human

(spinal

human
(blood)

UK

USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

Original Lab Original ID

Organism

Source Countr DFIa R&F RAPID Realb

y Origin
ID 32E Time
PCR
fluid)

CDC

CDC 105877

C.
human USA
malonaticus (breast
abscess)

Positi Positi Cronoba Positi

ve
ve
cter
ve

CDC

CDC 407-77 C. sakazakii human USA

(sputum)

Positi Positi Cronoba Positi

ve
ve
cter
ve

CDC

CDC 312877

C. sakazakii human USA

(sputum)

Positi Positi Cronoba Positi

ve
ve
cter
ve

CDC

CDC 936975

C. sakazakii unknown USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

UZH

z3032

C. turicensis neonate Switzerl Positi Positi Cronoba Positi

(meningit and
ve
ve
cter
ve
is)

HCSC
ILSI

SK81
F6-023

C. sakazakii human

Canada Positi Positi Cronoba Positi

ve
ve
cter
ve

ILSI; RADl

F6-029

C. sakazakii neonate

Holland Positi Positi Cronoba Positi

ve
ve
cter
ve

ILSI

01-10-2001; C. sakazakii clinical

F6-034

USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

ILSI

8397; F6-043 C. sakazakii clinical

USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

CDC;
ILSI

CDC 289-81; C.
clinical
F6-049
malonaticus

USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

CDC;
ILSI

CDC 171677;
F6-052

USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

ILSI;
RAD

F6-032;
C. sakazakii milk
H. Muytjens
powder
7

Uruguay Positi Positi Cronoba Positi

ve
ve
cter
ve

UCD

CFS112

C. sakazakii milk
powder

Ireland

UCD

CFS349N

C. sakazakii milk
powder

New
Positi Positi Cronoba Positi
Zealand ve
ve
cter
ve

UCD

CFS352N

C. sakazakii milk
powder

New
Positi Positi Cronoba Positi
Zealand ve
ve
cter
ve

UCD

ES187

C.
dublinensis

Ireland

Positi Positi Cronoba Positi

ve
ve
cter
ve

CDC

CDC 936375

C. sakazakii stool

USA

Positi Positi NonPositi

ve
ve
Cronoba ve
cter

C. sakazakii human
(blood)

milk
powder

Positi Positi Cronoba Positi

ve
ve
cter
ve

Original Lab Original ID

Organism

Source Countr DFIa R&F RAPID Realb

y Origin
ID 32E Time
PCR

CDC

CDC 496371

C. sakazakii stool

USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

CDC

CDC 189573

C.
human USA
malonaticus (faeces)

Positi Positi Cronoba Positi

ve
ve
cter
ve

RFm

ES626

C. sakazakii rice flour USA

Positi Positi Cronoba Positi

ve
ve
cter
ve

Positive of DFI shows green colony as Cronobacter

Positive of R&F shows blue-green-black colony as Cronobacter
c
UCD: S. Fanning, Centre for Food Safety, University College Dublin, Belfield, Dublin 4,
Ireland
d
UZH: R. Stefan, Institute for Food Safety, University of Zurich, Winterthurerstrasse 270,
CH-8057, Switzerland
e
NRC: Nestl Research Centre, Vers-Chez-les-Blanc, Lausanne, CH-1000, Switzerland
f
ILSI: R. Ivy, Food Safety Lab, Cornell University, 412 Stocking Hall, Ithaca, NY, USA
g
ATCC: American Type Culture Collection, Manassas, VA, USA
h
NCTC: National Collection of Type Cultures, London, UK
i
FDA: R. Buchanan, FDA-CFSAN, College Park, MD, USA
j
HCSC: F. Pagotto, Health Products and Food branch, Health Canada
k
CDC: Center for Disease Control, Atlanta, GA, USA
l
RAD: Department of Medical Microbiology, University of Nijmegen, Radboud, Netherlands
m
RF: L. Restaino, R&F Laboratories, Downers Grove, IL, USA
b

Table B. Exclusivity Testing Results for Cronobacter

Original Lab Strain ID

Organism

Source

DFIa

R&Fb

RAPID RealID 32E Time

PCR

ATCCc

13047

Enterobacter
cloacae

spinal
fluid

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

13048

Enterobacter
aerogenes

sputum

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

13182

Klebsiella oxytoca

Pharynge Negativ Negativ nonNegati

al tonsil e
e
Cronoba ve
cter

ATCC

13880

Serratia marcescens pond

water

ATCC

14485

Streptococcus therm unknown No gro No gro nonNegati

ophilus
wth
wth
Cronoba ve
cter

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

Original Lab Strain ID

Organism

Source

soil

DFIa

R&Fb

RAPID RealID 32E Time

PCR

ATCC

14807

Bacillus subtilis

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

15469

Edwardsiella tarda faeces

ATCC

23055

Acinetobacter calco unknown Negativ Negativ nonNegati

aceticus
e
e
Cronoba ve
cter

ATCC

23216

Leclercia
adecarboxylata

drinking Negativ Negativ nonNegati

water
e
e
Cronoba ve
cter

ATCC

25830

Morganella
morganii

patient Negativ Negativ nonNegati

with
e
e
Cronoba ve
summer
cter
diarrhea

ATCC

25922

Escherichia coli

clinical
isolate

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

27028

Citrobacter koseri

blood
culture

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

27982

Pantoea
agglomerans

IV fluid Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

29013

Klebsiella
pneumoniae

blood

ATCC

29944

Providencia rettgeri unknown Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

27853

Pseudomonas
fluorescens

unknown Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

13472

Bacillus cereus

unknown No
No
nonNegati
growth growth Cronoba ve
cter

ATCC

33105

Serratia ficaria

Calimyrn Negativ Negativ nonNegati

a fig
e
e
Cronoba ve
cter

ATCC

33420

Proteus vulgaris

clinical
isolate

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

Negativ Negativ nonNegati

e
e
Cronoba ve

Original Lab Strain ID

Organism

Source

DFIa

R&Fb

RAPID RealID 32E Time

PCR
cter

ATCC

33650

Escherichia
hermanii

human
toe

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

15246

Alcalgenes faecalis unknown Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

33832

Escherichia vulneris unknown Negativ Negativ nonNegati

e
e
Cronoba ve
cter

ATCC

29212

Enterococcus
faecalis

ATCC

10054

Micrococcus luteus unknown No

No
nonNegati
growth growth Cronoba ve
cter

ATCC

51713

Buttiauzella noakiae unknown Positive Positive nonNegati

Cronoba ve
cter

ATCC

25741

Pediococus
acidilactici

ATCC

8090

Citrobacter freundii unknown Negativ Positive nonNegati

e
Cronoba ve
cter

ATCC

9789

Bacillus
licheniformis

milk

No
No
nonNegati
growth growth Cronoba ve
cter

UZHd/UCDe/ E440
NRCf

Enterobacter
helveticus
sp. nov

milk
powder

Positive Positive nonNegati

Cronoba ve
cter

UZH/UCD/N E441
RC

Enterobacter novel
species

milk
powder

Negativ Positive nonNegati

e
Cronoba ve
cter

UZH/UCD/N E644
RC

Enterobacter
cloacae

human Negativ Negativ nonNegati

(faeces) e
e
Cronoba ve
cter

unknown No
No
nonNegati
growth growth Cronoba ve
cter

unknown No
No
nonNegati
growth growth Cronoba ve
cter

UZH/UCD/N E904; 05- Enterobacter

RC
01-120
homaechei

milk
powder

ILSIg

environm Negativ Negativ nonNegati

ent
e
e
Cronoba ve

F6-026

Enterobacter
asburiae

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

Original Lab Strain ID

Organism

Source

DFIa

R&Fb

RAPID RealID 32E Time

PCR
cter

ILSI

F6-033

Enterobacter
hormaechei

milk
powder

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

LMGh; UZH LMG

23730

Enterobacter
turicensis,
sp. nov

fruit
powder

Negativ Negativ nonPositiv

e
e
Cronoba e
cter

LMG; UZH

LMG
23732

Enterobacter
helveticus,
sp. nov

fruit
powder

Positive Negativ nonNegati

e
Cronoba ve
cter

UZH

1160/04;E Enterobacter novel

908
species

fruit
powder

Positive Negativ nonNegati

e
Cronoba ve
cter

FDAi

Salmonella Cubana milk

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

FDA

Yp 1313

Yersinia
unknown Negativ Negativ nonNegati
pseudotuberculosis
e
e
Cronoba ve
cter

FDA

Ye 37

Yersinia
enterocolitica

unknown Negativ Negativ nonNegati

e
e
Cronoba ve
cter

FDA

2457T

Shigella flexneri

clinical

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

Shigella sonnei

clinical

Negativ Negativ nonNegati

e
e
Cronoba ve
cter

FDA

False-positive
a

4 / 42

4 / 42

0 / 42

1 / 42

Positive of DFI shows green colony as Cronobacter

Positive of R&F shows blue-green-black colony as Cronobacter
c
ATCC: American Type Culture Collection, Manassas, VA, USA
d
UZH: R. Stephan, Institute for Food Safety, University of Zurich, Winterthurerstrasse 270,
CH-8057, Switzerland
e
UCD: S. Fanning, Centre for Food Safety, University College Dublin, Belfield, Dublin 4,
Ireland
f
NRC: Nestl Research Center, Vers-Chez-les-Blanc, Lausanne, CH-1000, Switzerland
g
ILSI: R. Ivy, Food Safety Lab, Cornell University, 412 Stocking Hall, Ithaca, NY, USA
h
LMG: BCCM/LMG Bacteria Collection, Gent, Belgium
i
FDA: K. Lampel, FDA-CFSAN, College Park, MD, USA
b