Bacteriological Analytical Manual

Chapter 29: Cronobacter

Authors: Yi Chen, Nancy E. Miranda, Kun C. Liu, Jeremi S. Mullins, Keith Lampel, and 

Thomas Hammack 

Revision History: 

November 2023: 

1. Removed the use of SmartCycler for qPCR.

2. Removed the requirement to perform qPCR without IAC and to perform qPCR on two 

aliquots per sample.

3. Updated the 7500 software version. 

4. Added ROX to the qPCR assay and revised the 7500 FAST analysis settings on threshold 

and baseline. 

5. Revised the stock concentrations and volumes of each qPCR component without 

changing their final concentrations. 

6. Added extra wash buffer to the pellet to improve the quality of the resultant DNA extract.

7. Added temperature and time tolerance ranges for incubation of enrichment and agars. 

8. Added optional use of CCI agar and ESIA agar incubated at their respective temperatures 

as well as R&F agar incubated at 41.5°C. 

9. Added more figures and detailed phenotype description of the colony morphologies on 

different agars.

10. Removed the option to use RAPID ID 32E for cultural confirmation.

11. Revised the chapter with updated nomenclature of Cronobacter and non-Cronobacter

species.

12. Updated other part of the chapter to improve clarity and to provide detailed instructions 

with additional texts and figures.

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. 

March 2012: New Chapter (This chapter has replaced the method for Isolation and Enumeration 

of Enterobacter sakazakii from Dehydrated Powdered Infant Formula. 
2

Contents

Introduction..................................................................................................................................... 3

Method ............................................................................................................................................ 5

References..................................................................................................................................... 22

Appendix....................................................................................................................................... 24
3

Introduction

Cronobacter is a Gram-negative rod within the family Enterobacteriaceae (9). The organism was called 

"yellow-pigmented Enterobacter cloacae" until it was renamed Enterobacter sakazakii in 1980 (6). 

Urmenyi and Franklin reported the first two known cases of meningitis caused by E. sakazakii in 1961 

(16). Subsequently, cases of meningitis, septicemia, and necrotizing enterocolitis due to E. 

sakazakii have been reported worldwide (12). 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 (11). While a reservoir for E. sakazakii is unknown, a growing number of reports suggest 

powdered infant formulas as a vehicle for infection (17).

Between 2008 and 2014, evidence obtained using amplified fragment length polymorphism, phenotypic 

arrays, automated ribotyping, 16S rRNA gene sequencing, DNA-DNA hybridization, multilocus 

sequence typing and genome-scale phylogeny has resulted in a nomenclature change (9, 10, 14). E. 

sakazakii was reclassified into a new genus, Cronobacter, comprising seven species 

including Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter 

muytjensii, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti. 

Cronobacter dublinensis contains three subspecies (Table 1) (8, 9, 10, 14). 
4

Table 1. Biochemical tests for the identification of Cronobacter and differentiation of species and 

subspecies of the genus Cronobacter (8, 9, 10, 14). Biochemical testsa C. sakazakii C. malonaticus C. turicensis

C. universalis

C. muytjensii

C. condimenti

C. dublinensis subsp. 

dublinensis

C. dublinensis subsp. 

lactaridi

C. dublinensis subsp. 

lausannensis

Motility +b V + V + − + + +

Indole production − − − − + + + + V

Dulcitol − − + + + − − − −

Lactulose + + + + + − + + −

Malonate − + V + + + + − −

Maltitol + + + + − − + + −

Palatinose + + + V V − + + +

Putrescine + V + − + − + + V

Melezitose − − + + − − + − −

Turanose + + + − V − + V −

myo-Inositol V V + + + − + + −

cis-Aconitate + + + V + − + + +

trans-Aconitate − + − − + − + + +

4-Aminobutyrate + + + V V − + + +

1-0-Methyl a-Dglucopyranoside+ + + + − + + + +

D-Sorbitol − − − − − − − − −

D-Sucrose + + + + + + + + +

Oxidase − − − − − − − − −

Hydrolysis of 4-NP 

α-Dglucoside

+ + + + + + + + +

Lysine decarboxylase − − − − − − − − −

Ornithine 

decarboxylase

+ + + + + + + + +

Methyl Red − − − − − − − − −

Voges-Proskauer test + + + + + + + + +

D-Arabitol − − − − − − − − −

a

The biochemical tests listed in the bottom ten rows are from ISO 22964:2017 and detailed procedures 

are described in the standard. They can be used for the differentiation of Cronobacter from other genera.

b

+, > 10% Positive; V, 20-80% positive; −, < 10% positive.
5

Method

The method described here contains both a real-time PCR method (13) for rapid screening and a 

cultural method for the detection/isolation of Cronobacter spp. (3). The qPCR targets 

Cronobacter partial macromolecular synthesis operon: the ribosomal protein S21 (rpsU) gene 3' 

end and the DNA primase (dnaG) gene 5' end (13). 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 qPCR and chromogenic agars. The cultural portion of this method is 

a complete detection/isolation method, so it can be used as a standalone method if qPCR 

technology is unavailable. The qPCR portion of the method is a screening method, whose 

positive results should always be confirmed with the cultural method. The qPCR method may be 

used to confirm pure cultures as Cronobacter spp. A flowchart of the entire procedure is 

described in Figure 1. This method was validated in pre-collaborative and collaborative studies 

(1, 2).

The inclusivity of this method was determined by analyzing 52 different Cronobacter strains 

representing the seven 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 51 non-Cronobacter strains based on 

taxonomy in 2023. 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.
6

Figure 1. Flowchart of the complete procedure.
7 

A. Equipment and materials

1. Balance with capacity of 2 kg and sensitivity of 0.1 g 

2. Incubator, 36 ± 2°C 

3. Sterile Erlenmeyer flasks with polyethylene screw caps equipped with Teflon liners, 2 

L 

4. Sterile sample bags, 2 L or 4 L 

5. Micropipette and tips to dispense 1 μL, 100 μL, 150 μL, and 200 μL volumes 

6. Pipets, 1, 5, and 10 mL, graduated in 0.1 mL units 

7. Sterile inoculating loops, 3 mm loop size

8. Glass or plastic spreading rods (e.g., hockey stick) 3-4 mm diameter with 45-55 mm 

spreading area

9. Sterile utensils for sample handling (see BAM Chapter 1)

10. Centrifuge with a swinging bucket rotor, capable of 3,000 × g

11. Microcentrifuge, capable of 15,000 × g

12. Centrifuge tubes, polypropylene, 50 mL tubes with conical bottoms; 1.5 mL 

microcentrifuge tubes 

13. Vortex mixer 

14. Water bath, capable of 100°C 

15. Heating block, capable of 100°C

16. Thermometer to check the temperature of heating block/water bath.

17. Thermal cyclers: Applied BiosystemsTM 7500 Fast real-time PCR system 

(ThermoFisher Scientific, Inc., Waltham, MA), 

18. 96-well microwell plate

19. Optical adhesive covers

20. Petri dishes, plastic, sterile, 15 × 150 mm 

21. VITEK® 2 Compact (bioMerieux, Hazelwood, MO 63402) 

22. Qubit 4 Fluorometer (ThermoFisher Scientific, Waltham, MA)

23. Green Fluorescent Protein (GFP) strain, Cronobacter sakazakii G-78 

(Microbiologics, Saint Cloud, MN)

B. Media and reagents

1. Phosphate-buffered saline (BAM R59) 

2. Buffered peptone water (BPW) (BAM M192) 

3. Brilliance Enterobacter sakazakii agar (DFI formulation) (BAM M201, or Cat. No. 

CM1055, Oxoid, Lenexa, KS). After the plates have been poured and dried upside 

down in the dark at room temperature for up to 48 h, they 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 oC in the dark for up to 2 weeks. Media made from another 

manufacturer that have the same formulation can be used after verification following 

guidelines of microbiology management and QA/QC management of each laboratory. 

4. Enterobacter sakazakii chromogenic plating agar (R&F agar) (BAM M202, Cat. No. 

M-0700, R & F Laboratories, Downers Grove, IL). The plates should be poured, dried 

and stored similar to DFI agar. The final pH value of the prepared agar media should 

range between 6.8 and 7.1. The prepared plates can be stored for up to 60 days. 

5. Chromogenic Cronobacter Isolation (CCI) agar (BAM M203, or Cat. No. CM1122, 

Oxoid). The plates should be poured, dried and stored similar to DFI agar. 
8 

6. Enterobacter sakazakii Isolation Agar (ESIA) (BAM M204, or Cat. No. CM1134, 

Oxoid, Lenexa, KS). Prepare media according to the instructions on the packaging 

label. The plates should be poured, dried and stored similar to DFI agar. 

7. PrepManTM Ultra sample preparation reagent (Cat. No. 4318930, Thermo Fisher 

Scientific, Waltham, MA)

8. iQTM Supermix qPCR master mix (Cat. No. 170-8860, Bio-Rad, Hercules, CA 

94547). 2 × mix contains 100 mM KCl, 40 mM Tris-HCl, pH 8.4, 0.4 mM each 

dNTP, 50 U/mL iTaqTM DNA polymerase and 6 mM MgCl2. 

9. VITEK® 2 GN ID Card (Cat. No. 21341, bioMérieux) 

10. PlatinumTM Taq DNA Polymerase (Cat. No. 10966-018, ThermoFisher Scientific, 

Waltham, MA)

11. ROX passive reference dye (Cat. No. 12223012, ThermoFisher Scientific, Waltham, 

MA). Reagent from another manufacturer can be used after verification following 

guidelines of microbiology management and QA/QC management of each laboratory. 

12. Primers and probes (Table 2). qPCR primers are commercially synthesized with basic 

desalt purification and then reconstituted using PCR grade water to 100 μM for 

prolonged storage. They are diluted to 2.5 to 40 μM working stock concentrations. 

qPCR probes are commercially synthesized with HPLC purification and reconstituted 

to 2.5 to 5 μM in single use aliquots using 1× PCR grade low-EDTA Tris-EDTA (TE, 

10 mM Tris, 0.1 mM EDTA, pH 8.0) buffer to avoid repeated freezing/thawing. 

Primers and probes need to be stored frozen (-20 to -70°C). Discard leftover thawed 

probes and avoid repeating freeze-thawing. 

13. Internal amplification control DNA (4). Internal amplification control (IAC) DNA is 

constructed by generating a 200 bp sequence that is synthesized and inserted into a 

pZErO-2 vector or other vector and transformed into Escherichia coli pDMD801 or 

other competent strain. Partial sequence representation containing the internal control 

is shown below (GenBank accession no. FJ357008, Figure 2). The internal 

amplification control DNA (IAC in Figure 2) 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) or equivalent from the 

transformed Escherichia coli cells following manufacturer’s instructions and 

quantified by Qubit fluorometer. To avoid repeated freezing/thawing, plasmids can be 

diluted to small aliquots and single use aliquots in PCR grade 1 × low-EDTA TE 

buffer containing 10 mM Tris and 0.1 mM EDTA at pH 8.0. The IAC DNA fragment 

can also be commercially synthesized and diluted in TE buffer to a stock solution 

which will reliably provide a Ct of no less than 24 when Cronobacter DNA extracted 

from 40 mL overnight BPW cultures are present and no more than 32 with water 

negative controls. 
9

Table 2. Primers and probes for the qPCR assay.

Oligos Name Sequences (5’ to 3’)

Cronobacter

forward

CronoF GGGATATTGTCCCCTGAAACAG

Cronobacter

reverse

CronoR CGAGAATAAGCCGCGCATT

Cronobacter

probe

CronoP 6FAMAGAGTAGTAGTTGTAGAGGCCG

TGCTTCCGAAAG-TAMRA

Internal 

control forward

IACF CTAACCTTCGTGATGAGCAATCG

Internal 

control reverse

IACR GATCAGCTACGTGAGGTCCTAC

Internal 

control probe

IACP Cy5-

AGCTAGTCGATGCACTCCAGTCC

TCCT-Iowa Black RQ-Sp.

Figure 2. Illustration of the internal amplification control DNA. The 200 bp IAC is 

highlighted in grey (Complete sequence,

TACAGCACCCTAGCTTGGTAGAATCGATCAGCTACGTGAGGTCCTACGACGAT

CGCCAAGCATGCCCTAGCTAAGATGCATCGATTGCTCATCACGTACGTTAGGT

CGACTAGGAGGACTGGAGTGCATCGACTAGCTAAGATGGTTCGATTGCTCATC

ACGAAGGTTAGGTCGACTACGAACGAGTCGTACGTTAGGTT).
10

C. 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 ± 0.2 g of the powdered infant formula and add to 2 L 

sized Erlenmeyer flasks or 2 to 4 L sized sterile sample bags.

4. Add 900 mL (1:10 dilution) of sterile buffered peptone water (BPW) pre-warmed to 

36 ± 2°C and gently shake by hand until the powder is uniformly suspended. Incubate 

for 24 ± 2 h at 36 ± 2°C.

5. Thoroughly mix the enrichment mix and remove three aliquots of 40 mL each from 

the incubated sample and place them into three 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 μL of phosphate buffered saline (PBS) by 

vortexing at maximum speed for at least 20 sec. One of the aliquots will be used for 

qPCR. Two of the aliquots will be used for culture confirmation if necessary.

 Note: One aliquot can be prepared first for qPCR screening. The other two aliquots 

can be prepared and centrifugated after obtaining qPCR positive results. The 

enrichment cultures can be stored at 4°C while waiting for the qPCR result.

D. qPCR screening of Cronobacter

1. DNA extraction. Transfer 200 μL suspended cells (in PBS) to a 1.5 mL 

microcentrifuge tube and add another 600 μL of PBS to mix. Centrifuge at 3,000 × g

for 5 min. 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. Use sterile cotton tips or 

equivalent tools to remove the precipitates on the side wall of the centrifuge tube if 

necessary. Add 400 μL of PrepManTM 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 μL of the supernatant into a new tube for qPCR analysis.

2. Include a positive control (prepared by 1:10 dilution of DNA prepared from a pure 

culture of Cronobacter strain, e.g., ATCC 29544, or GFP strain, Cronobacter 

sakazakii G-78) and a no template (water) control in each qPCR run. The qPCR is 

designed to detect very low level of Cronobacter cells. If after enrichment, large 

amounts of cells are grown, the qPCR could yield high FAM signals (e.g., Ct < 12). 

One option is to dilute the DNA (1:10 or 1:100) and rerun the qPCR.
11

3. qPCR with ABI 7500 Fast Thermal Cycler (software version 2.3). Prepare qPCR 

reactions from the reaction components and final concentrations listed in Table 3. 

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 ROX as reference dye, FAM as reporter dye for 

Cronobacter probe and Cy5 as reporter dye for IAC probe, and TAMRA as 

quencher for Cronobacter probe and none as quencher for IAC probe. Assign 

appropriate sites on the cycler block.

c. In "Run Method", set reaction volume per well to 25 μL. Select the qPCR 

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. 

 Table 3. qPCR reaction components for ABI 7500 Fast with IAC.

Component Volume/reaction Final Concentration

iQ Supermix 12.5 μL 50 mM KCl, 20 mM TrisHCl, 0.2 mM each dNTP,

0.625 U iTaq DNA polymerase 

and 3 mM MgCl2

ROX 0.05 μL (25 μM solution) 50 nM

CronoF 0.25 μL (40 μM stock 

solution)

400 nM

CronoR 0.25 μL (40 μM stock 

solution)

400 nM

CronoP 1.5 μL (5 μM stock solution) 300 nM

IACF 0.25 μL (15 μM stock 

solution)

150 nM

IACR 0.25 μL (15 μM stock 

solution)

150 nM

IACP 1.5 μL (2.5 μM stock 

solution)

150 nM

IAC DNA 1 μL (3 ×103 plasmid copies 

per μL)

3000 copies

Taq 

Polymerase

0.25 μL (10 U/μL) 2.5 U

MgCl2 1.5 μL (50 mM solution) 3 mM

DNA extract 

or control

2 μL for screening, 1 μL for 

confirmation

2 μL for screening, 1 μL for 

confirmation

PCR grade 

water

Appropriate amount to reach 

25 μL

Appropriate amount to reach 25 

μL
12

4. Data analysis. Set the threshold and baseline according to Table 4. The threshold 

values are based on the plot of ΔRn vs Cycle. ROX is required for screening qPCR 

and recommended for confirmatory qPCR. Confirmatory PCR can be performed 

without ROX if it is not available; threshold of 250,000 and the baseline setting 

described in Table 4 should be used. Sample screenshots of the graph and table view 

are shown in Figure 3. If the qPCR result is positive, the sample is considered PCRpositive and proceed to the culture confirmation (Section E). If the qPCR result is 

negative, the sample is considered negative, and analysis is halted.

DNA extracts that have Ct values for FAM and demonstrate sigmoidal amplification 

curves are considered PCR-positive. The multicomponent plot in 7500 FAST software 

displays individual signals of FAM, CY5, ROX and TAMRA which can be used to 

check non-typical plots. Review of IAC reactions is not required when the FAM 

reaction is positive. If there is no Ct value in FAM for a DNA extract, or if there is Ct 

value but the curve is not the typical sigmoidal shape, the IAC for that DNA extract 

must be analyzed:

a. The DNA extract is considered negative if there is Ct value in Cy5 and the curve 

for Cy5 is sigmoidal.

b. If there is no Ct value in Cy5, then there is possible inhibitory substance in the 

sample and qPCR result is invalid. The DNA extracts need to be diluted (1/10, in 

PCR grade water) or centrifuged for further purification, and the qPCR should be 

repeated; or directly proceed with culture analysis (Section E).

 Table 4. Threshold and baseline settings for Cronobacter and IAC reactions.

qPCR targets Threshold Baseline 

FAM-Cronobacter for 

screening

0.15 Initially set to auto baseline for 

all samples. For any sample, if 

the baseline start < 3, manually 

change the baseline start to 3; 

if the baseline end < 15, 

manually change the baseline 

end to 15.

FAM-Cronobacter for 

confirmation

0.25 The same as above

Cy5-IAC for both screening 

and confirmation

0.05 3 to 20
13

(a)

(b)
14 

(c)

(d) 

Figure 3. Sample screenshots of the 7500 Fast results. (a) defining samples and targets, (b) 

assigning targets to each sample. (c) adjusting baseline of individual well. (d) amplification 

curves, thresholds and Ct values of Cronobacter and IAC.

E. Isolation of Cronobacter

For qPCR-positive samples and samples that show inhibition in the qPCR screening, 

streak a loopful of suspended cells (obtained in C8) onto the surface of two DFI (M201) 

(5) and two R&F agars (M202) (15) with sterile inoculation loops as described in Figure 

1. In addition, spread 100 μL aliquots of suspended cells evenly onto each of the two DFI 

chromogenic agars and two R&F Cronobacter chromogenic plating agars with sterile 

spreading rods. Incubate the agar plates at 36 ± 1°C for 24 ± 2 h. Observe plates for 

typical or suspect colonies of Cronobacter (Figure 4). 

Optionally, with sterile inoculation loops, streak a loopful of suspended cells onto the 

surface of one CCI agar (M203) (8) or one ESIA agar (M204) (7) and/or one R&F agar 
15

(Figure 1). Incubate the optional CCI or R&F agar plates at 41.5 ± 1°C for 24 ± 2 h. 

Incubate the optional ESIA agar plate at 44 ± 1°C for 24 ± 2 h. 

Optionally, the plates can be incubated for another 18 to 24 h to increase the size of the 

colonies and improve color development for some strains. 

If the cultures overgrow on the plates, streak a 3 mm loopful (10 μL) of lawn materials to 

at least three quadrants of a new plate for isolation of single colonies. If isolated colonies 

from streak plates are picked for further purification or direct confirmation, spread plates 

of the same aliquot may have confluent growth and may not need to be sub-cultured. 

Plates can be kept for up to 3 days at 4°C during the investigation of confluent growth.

Use UV illumination to check the GFP control strain G-78. G-78 appears pale to yellow 

green on DFI and CCI agars, light green on ESIA agar and blue-black on R&F agar.

F. Identification of Cronobacter

Presumptive Cronobacter colonies on DFI and CCI agar appear either dark green, pale 

green or yellow green. Some colonies only have a green center with a white/yellow 

border. Rare strains appear mostly yellow with very weak green center (Figure 4a, 4b, 4f 

and 4g). 

Presumptive Cronobacter colonies on R&F agar appear blue to black, or blue to grey 

with the red background. Rare strains can be light grey when grown at 36°C (Figure 4c 

and 4d). The red background can appear purplish red with different strains or under 

different light conditions. Cronobacter does not change the color of R&F agar, but the 

presence of background microflora can change the color of R&F agar from red to 

yellow, which may make Cronobacter colonies appear green to black (Figure 4e). 

Presumptive Cronobacter colonies on ESIA agar appear dark blue to green and blue to 

green. Some strains have very light green color and thus appear mostly blue, especially 

for isolated colonies (Figure 4h, 4i and 4j). 

Some strains have very small colonies on any of the agars after 24 h incubation. Pay 

attention to suspect colonies with weak green color. Typical colonies are 2-3 mm in 

diameter, but some rare strains have colonies of 1 mm in diameter. 
16

(a) Cronobacter colonies on DFI agar on a dark background. The colors can be green 

(upper left), pale green (upper right) and yellow green (bottom left). Rare strains can have 

very small colonies (bottom right).

(b) Mixed Cronobacter colonies on DFI agar on a dark background. Rare strains can appear 

yellow with very weak green center. The zoomed pictures make colonies appear larger than 

they are.
17

(c) Cronobacter colonies on R&F agar. The colors can be blue grey (upper right) and 

blue black (bottom right). Rare strains can be light grey when grown at 36°C (upper 

left) or have small colonies (bottom left).

(d) Mixed Cronobacter colonies on R&F agar.
18

(e) Cronobacter mixed with background flora on R&F agar. The background flora 

changes the background color of the agar from red to yellow in most areas, 

and Cronobacter colonies appear green to black against yellow background.

(f) Cronobacter colonies on CCI agar on a light background. The colors can be dark green 

(upper left), green (upper right), pale green (bottom left) and yellow green (bottom right).
19

(g) Mixed Cronobacter colonies on CCI agar on a dark background. Rare strains can have 

very small colonies.

(h) Cronobacter colonies on ESIA agar on a dark background. The colors can be dark blue 

green (upper), blue green with isolated colonies more blue and heavy growth areas more 

green (bottom left). Some strains have very light green color and appear mostly blue (bottom 

right).
20

(i) Same two plates in the bottom of (h) on a light background. The colonies appear 

greener than those on a dark background.

(j) Mixed Cronobacter colonies on ESIA agar on a dark background. Rare strains have light 

blue color.

Figure 4. Colors and morphologies of Cronobacter strains.

G. PCR confirmation

Prepare DNA from at least one isolated, suspect colony from each chromogenic agar 

plate if available or a TSA plate used for further purification. Plates can be kept at 4°C 

for not more than 24 h before DNA extraction. Transfer one colony to 150 μL of PCR 

grade dH2O contained in a 1.5 mL plastic centrifuge tube and heat for 5 min in a boiling 

water bath or heating block set to 100°C. Chill the tubes in ice and centrifuge at 15,000 ×

g for 2 min. Use 1 μL of this lysed material as DNA template for the real-time PCR assay 

above (Section D). If a colony is picked from a chromogenic agar plate directly for PCR 

confirmation, subculture the colony on TSA in case VITEK 2 confirmation is needed for 

that colony. 

H. Biochemical confirmation

With a sterile inoculating loop, pick at least one suspect Cronobacter colony from each 

chromogenic agar plate if available, further purify on TSA and confirm using VITEK 2 

biochemical identification system according to the manufacturer's instructions. 
21 

When qPCR is used initially to confirm suspect isolates, secondary confirmation of the 

same colony by VITEK 2 is highly recommended. When VITEK 2 is used initially to 

confirm suspect isolates and yields positive identification of the Cronobacter sakazakii

group, secondary confirmation of the same colony by qPCR is required. When VITEK 2 

is used initially to confirm suspect isolates and yields negative identification of 

Cronobacter, secondary confirmation of the same colony by qPCR is not required. 

The biochemical tests listed in Table 1 can be used for the identification of Cronobacter. 

Other commercial biochemical test kits can be used if properly validated. Franconibacter

and Siccibacter share some biochemical characteristics with Cronobacter and thus should 

be included when validating other biochemical test kits. 

 

I. Optional: enumeration of Cronobacter

Use the three-tube three-level Most Probable Number (MPN) procedure (BAM Manual,

Appendix 2; Most Probable Number Determination from Serial Dilutions: 

https://www.fda.gov/food/laboratory-methods-food/bam-appendix-2-most-probablenumber-serial-dilutions). Aseptically weigh out in triplicate, 100 g, 10 g and 1 g of the 

powdered infant formula and add to 2 L, 250 mL and 125 mL size Erlenmeyer flasks or 

similar size sample bags, 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. 

J. Subtyping of Cronobacter

Confirmed Cronobacter isolates can be further characterized by whole genome 

sequencing using standardized GenomeTrakr methods and submit data to GenomeTrakr. 
22

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. ISO/TS 22964:2006 | IDF/RM 210: 2006. Milk and milk products — Detection of 

Enterobacter sakazakii

8. ISO 22964:2017. Microbiology of the food chain — Horizontal method for the detection 

of Cronobacter spp.

9. 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.

10. Joseph, S.; Cetinkaya, E.; Drahovska, H.; Levican, A.; Figueras, M.J.; Forsythe, S.J. 

2012. Cronobacter condiment sp. Nov., isolated from spiced meat, and Cronobacter 

universalis sp. Nov., a species designation for Cronobacter sp. genomospecies 1, 

recovered from a leg infection, water and food ingredients. Int. J. Syst. Evol. 

Microbiol. 62: 1277-1283

11. Lai, K.K. 2001. Enterobacter sakazakii infections among neonates, infants, children, and 

adults. Case reports and a review of the literature. Medicine (Baltimore). 80:113-122.

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

Food Microbiol. 34:103-113.

13. Seo, K.H. and R.E. Brackett. Rapid, specific detection of Enterobacter sakazakii in infant 

formula using a real-time PCR assay. J. Food Prot. 68:59-63.

14. Stephan, R., Grim, C.J., Gopinath, G.R., Mammel, M.K., Sathyamoorthy, V., Trach, 

L.H., Chase, H.R., Fanning, S., and B.D. Tall. Re-examination of the taxonomic status of 

Enterobacter helveticus, Enterobacter pulveris and Enterobacter turicensis as members 
23

of the genus Cronobacter and their reclassification in the genera Franconibacter gen. 

nov. and Siccibacter gen. nov. as Franconibacter helveticus comb. Nov., Franconibacter

pulveris comb. Nov. and Siccibacter turicensis comb. Nov., respectively. Int. J. Syst. 

Evol. Microbiol. 64: 3402-3410

15. 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.

16. Urmenyi, A.M. and A.W. Franklin. 1961. Neonatal death from pigmented coliform 

infection. Lancet. 1:313-315.

17. World Health Organization. Enterobacter sakazakii and other microorganisms in 

powdered infant formula: meeting report. 2004.
24

Appendix

Table A. Inclusivity testing results for Cronobacter.

Original 

Lab

Original ID Organism Source Country 

Origin

DFIa R&Fb RealTime 

PCR

UCDc/UZ

Hd/NRCe

E265 C. malonaticus milk powder Malaysia Positive Positive Positive

ILSIf F6-036 C. sakazakii Environment

(Milk powder)

Malaysia Positive Positive Positive

ILSI F6-038 C. sakazakii Environment

(Milk powder)

Holland Positive Positive Positive

ILSI F6-040 C. sakazakii Environment

(Milk powder)

Holland Positive Positive Positive

UCD/UZH

/NRC

E464 C. dublinensis Environment

(Milk powder)

Zimbabwe Positive Positive Positive

ATCCg;

NCTCh

ATCC 29544;

NCTC 11467

C. sakazakii human (throat) unknown Positive Positive Positive

FDAi 607 C. sakazakii unknown unknown Positive Positive Positive

UCD/UZH

/NRC

E515 C. dublinensis water Switzerland Positive Positive Positive

UCD/UZH

/NRC

1330T C. condimenti spiced meat Slovakia Positive Positive Positive

ATCC ATCC 12868 C. sakazakii unknown unknown Positive Positive Positive

ATCC ATCC 51329 C. muytjensii unknown unknown Positive Positive Positive

HCSCj; 

FDA

SK90 C. sakazakii clinical

(children's hospital)

Canada Positive Positive Positive

UCD/UZH

/NRC

E632 C. sakazakii food USA Positive Positive Positive

HCSC HPB 2848 C. sakazakii clinical Canada Positive Positive Positive

HCSC HPB 2873 C. sakazakii clinical Canada Positive Positive Positive
25

Original 

Lab

Original ID Organism Source Country 

Origin

DFIa R&Fb RealTime 

PCR

HCSC HPB 2874 C. sakazakii clinical Canada Positive Positive Positive

UCD/UZH

/NRC

H. Muytjens

(Prague 72 26248)

C. sakazakii unknown Czech 

Republic

Positive Positive Positive

UCD/UZH

/NRC

H. Muytjens 52 C. malonaticus milk powder Australia Positive Positive Positive

UCD/UZH

/NRC

H. Muytjens 58 C. sakazakii milk powder Belgium Positive Positive Positive

UCD/UZH

/NRC

H. Muytjens 15 C. sakazakii milk powder Denmark Positive Positive Positive

UCD/UZH

/NRC

H. Muytjens 8 C. sakazakii milk powder France Positive Positive Positive

UCD/UZH

/NRC

H. Muytjens 35 C. sakazakii milk powder Russia Positive Positive Positive

UCD/UZH

/NRC

H. Muytjens 26 C. sakazakii milk powder Russia Positive Positive Positive

UCD/UZH

/NRC

H. Muytjens

(Nijmegen 15)

C. sakazakii neonate Holland Positive Positive Positive

UCD/UZH

/NRC

H. Muytjens

(Nijmegen 21)

C. sakazakii neonate Holland Positive Positive Positive

CDCk CDC 5960-70 C. dublinensis human (blood) USA Positive Positive Positive

CDC CDC 3523-75 C. malonaticus human (bone 

marrow)

USA Positive Positive Positive

NCTC NCTC 9238 C. sakazakii human

(abdominal pus)

UK Positive Positive Positive

NCTC NCTC 9529 C. universalis water UK Positive Positive Positive

ATCC ATCC BAA893 C. sakazakii unknown USA Positive Positive Positive

ATCC ATCC BAA894 C. sakazakii unknown USA Positive Positive Positive

CDC CDC 996-77 C. sakazakii human (spinal fluid) USA Positive Positive Positive
26

Original 

Lab

Original ID Organism Source Country 

Origin

DFIa R&Fb RealTime 

PCR

CDC CDC 1058-77 C. malonaticus human (breast 

abscess)

USA Positive Positive Positive

CDC CDC 407-77 C. sakazakii human (sputum) USA Positive Positive Positive

CDC CDC 3128-77 C. sakazakii human (sputum) USA Positive Positive Positive

CDC CDC 9369-75 C. sakazakii unknown USA Positive Positive Positive

UZH z3032 C. turicensis neonate (meningitis) Switzerland Positive Positive Positive

HCSC

ILSI

SK81

F6-023

C. sakazakii human Canada Positive Positive Positive

ILSI; 

RADl

F6-029 C. sakazakii neonate Holland Positive Positive Positive

ILSI 01-10-2001; F6-034 C. sakazakii clinical USA Positive Positive Positive

ILSI 8397; F6-043 C. sakazakii clinical USA Positive Positive Positive

CDC;

ILSI

CDC 289-81;

F6-049

C. malonaticus clinical USA Positive Positive Positive

CDC;

ILSI

CDC 1716-77;

F6-052

C. sakazakii human (blood) USA Positive Positive Positive

ILSI;

RAD

F6-032;

H. Muytjens 7

C. sakazakii milk powder Uruguay Positive Positive Positive

UCD CFS112 C. sakazakii milk powder Ireland Positive Positive Positive

UCD CFS349N C. sakazakii milk powder New 

Zealand

Positive Positive Positive

UCD CFS352N C. sakazakii milk powder New 

Zealand

Positive Positive Positive

UCD ES187 C. dublinensis milk powder Ireland Positive Positive Positive

CDC CDC 9363-75 C. sakazakii stool USA Positive Positive Positive

CDC CDC 4963-71 C. sakazakii stool USA Positive Positive Positive

CDC CDC 1895-73 C. malonaticus human (faeces) USA Positive Positive Positive

RFm ES626 C. sakazakii rice flour USA Positive Positive Positive
27

a Positive of DFI shows green or light green colony as Cronobacter

b Positive of R&F shows blue-green-grey-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: Nestlé 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
28

Table B. Exclusivity testing results for Cronobacter.

Original 

Lab

Strain ID Organism Source DFIa R&Fb Real-Time PCR

ATCCc 13047 Enterobacter cloacae spinal fluid Negative Negative Negative

ATCC 13048 Klebsiella aerogenes sputum Negative Negative Negative

ATCC 13182 Klebsiella oxytoca Pharyngeal 

tonsil

Negative Negative Negative

ATCC 13880 Serratia marcescens pond water Negative Negative Negative

ATCC 14485 Streptococcus thermophilus unknown No growth No growth Negative

ATCC 15469 Edwardsiella tarda faeces Negative Negative Negative

ATCC 23055 Acinetobacter calcoaceticus unknown Negative Negative Negative

ATCC 23216 Leclercia adecarboxylata drinking 

water

Negative Negative Negative

ATCC 23373 Enterobacter cloacae corn Negative Negative Negative

ATCC 25408 Citrobacter koseri throat Negative Negative Negative

ATCC 25830 Morganella morganii patient with 

summer 

diarrhea

Negative Negative Negative

ATCC 25922 Escherichia coli clinical isolate Negative Negative Negative

ATCC 27028 Citrobacter koseri blood culture Negative Negative Negative

ATCC 27982 Phytobacter ursingii IV fluid Negative Negative Negative

ATCC 49008 Pantoea agglomerans foodstuff Negative Negative Negative

ATCC 29013 Klebsiella pneumoniae blood Negative Negative Negative

ATCC 29944 Providencia rettgeri unknown Negative Negative Negative

ATCC 27853 Pseudomonas aeruginosa unknown Negative Negative Negative

ATCC 33110 Kluyvera intermedia water Positive Negative Negative

ATCC 33731 Lelliottia amnigena unknown Negative Positive Negative

ATCC 35030 Enterobacter cloacae unknown Negative Negative Negative

ATCC 13472 Bacillus cereus unknown No growth No growth Negative
29

Original 

Lab

Strain ID Organism Source DFIa R&Fb Real-Time PCR

ATCC 33105 Serratia ficaria Calimyrna fig Negative Negative Negative

ATCC 35314 Enterobacter cancerogenus Blood Negative Positive Negative

ATCC 33028 Pluralibacter gergoviae urine Negative Negative Negative

ATCC 33420 Proteus vulgaris clinical isolate Negative Negative Negative

ATCC 33650 Escherichia hermanii human toe Negative Negative Negative

ATCC 15246 Alcalgenes faecalis unknown Negative Negative Negative

ATCC 33832 Pseudescherichia vulneris unknown Negative Negative Negative

ATCC 29212 Enterococcus faecalis unknown No growth No growth Negative

ATCC 10054 Micrococcus luteus unknown No growth No growth Negative

ATCC 43864 Citrobacter freundii unknown Negative Negative Negative

ATCC 51713 Buttiauzella noakiae unknown Positive Positive Negative

ATCC 25741 Pediococus acidilactici unknown No growth No growth Negative

ATCC 49141 Enterobacter ludwigii clinical Negative Negative Negative

ATCC 8090 Citrobacter freundii unknown Negative Negative Negative

ATCC 9789 Bacillus licheniformis milk No growth No growth Negative

UZH/UCD/

NRC

E904; 05-01-120 Enterobacter hormaechei milk powder Negative Positive Negative

ATCC 35956 Enterobacter asburiae human Negative Negative Negative

ATCC 49163 Enterobacter hormaechei blood Negative Positive Negative

LMGh; 

UZH

LMG 23730 Siccibacter turicensis fruit powder Positive Negative Negative

LMG; 

UZH

LMG 23731 Siccibacter turicensis fruit powder Positive Negative Negative

LMG; 

UZH

LMG 23732 Franconibacter helveticus fruit powder Positive Positive Negative

LMG; 

UZH

LMG 23733 Franconibacter helveticus fruit powder Positive Positive Negative

LMG; 

UZH

LMG 24057 Franconibacter pulveris fruit powder Positive Positive Negative
30 

Original 

Lab

Strain ID Organism Source DFIa R&Fb Real-Time PCR

LMG; 

UZH

LMG24058 Franconibacter pulveris fruit powder Positive Negative Negative

FDAi Salmonella Cubana milk Negative Negative Negative

FDA Yp 1313 Yersinia pseudotuberculosis unknown Negative Negative Negative

FDA Ye 37 Yersinia enterocolitica unknown Negative Negative Negative

FDA 2457T Shigella flexneri clinical Negative Negative Negative

FDA Shigella sonnei clinical Negative Negative Negative

Falsepositive8 / 51 8 / 51 0 / 51

a

 Positive of DFI shows green colony as Cronobacter 

b

 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: Nestlé 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 CollectionExternal Link Disclaimer, Ghent, Belgium 

i FDA: K. Lampel, FDA-CFSAN, College Park, MD, USA