Formerly Enterobacter sakazakii

International Life Science Institute North America Cronobacter(Formerly Enterobacter sakazakii) Isolate Set

REID A. IVY,1 JEFFREY M. FARBER,2 FRANCO PAGOTTO,2 AND MARTIN WIEDMANN1*

1Department of Food Science, Cornell University, Ithaca, New York 14853, USA; and 2Bureau of Microbial Hazards, Sir F. G. Banting Research Centre,

Ottawa, Ontario, Canada K1A 0K9

MS 11-546: Received 13 December 2011/Accepted 21 August 2012

ABSTRACT

Foodborne pathogen isolate collections are important for the development of detection methods, for validation of intervention

strategies, and to develop an understanding of pathogenesis and virulence. We have assembled a publicly available Cronobacter(formerly Enterobacter sakazakii) isolate set that consists of (i) 25 Cronobacter sakazakii isolates, (ii) two Cronobacter malonaticusisolates, (iii) one Cronobacter muytjensii isolate, which displays some atypical phenotypic characteristics, biochemical profiles, and

colony color on selected differential media, and (iv) two nonclinical Enterobacter asburiae isolates, which show some phenotypic

characteristics similar to those of Cronobacter spp. The set consists of human (n ~ 10), food (n ~ 11), and environmental (n ~ 9)

isolates. Analysis of partial 16S rDNA sequence and seven-gene multilocus sequence typing data allowed for reliable identification

of these isolates to species and identification of 14 isolates as sequence type 4, which had previously been shown to be the most

common C. sakazakii sequence type associated with neonatal meningitis. Phenotypic characterization was carried out with API 20E

and API 32E test strips and streaking on two selective chromogenic agars; isolates were also assessed for sorbitol fermentation and

growth at 45uC. Although these strategies typically produced the same classification as sequence-based strategies, based on a panel

of four biochemical tests, one C. sakazakii isolate yielded inconclusive data and one was classified as C. malonaticus. EcoRI

automated ribotyping and pulsed-field gel electrophoresis (PFGE) with XbaI separated the set into 23 unique ribotypes and 30

unique PFGE types, respectively, indicating subtype diversity within the set. Subtype and source data for the collection are publicly

available in the PathogenTracker database (www.pathogentracker.net), which allows for continuous updating of information on the

set, including links to publications that include information on isolates from this collection.

Cronobacter spp. (formerly Enterobacter sakazakii) are

gram-negative, motile, non–spore-forming bacilli that have

been associated with disease in both neonates (6, 12, 31, 49)and adults (49). In adults, symptoms associated with

Cronobacter infection include bacteremia, wound infec-

tions, abscesses, and ulcers (32, 48). Disease due to

Cronobacter infection of neonates is rare (22); for example,

in the United States typically four to six cases in neonates

are reported per year. Symptoms that have been associated

with Cronobacter infections in neonates include meningitis,

sepsis, and necrotizing enterocolitis (31). Cronobacterinfection mortality rates in neonates can be high and have

been reported to exceed 20% (6, 22, 49, 51, 61). Powdered

infant formula (PIF) has been identified as a possible source

of Cronobacter infection in infants, and Cronobacter has

been isolated from PIF (60, 62, 67), but other sources of

infections also have been reported. Cronobacter has been

isolated from a variety of other foods and dry ingredients (4,5, 34) and from farms (59), food processing facilities (14),and other sources, including water (39).

Cronobacter spp. were formerly identified as belonging

to the species Enterobacter sakazakii, which was classified

into 16 different biochemical profile groups (biogroups) (20,39). Since the description of the genus Cronobacter (38),isolates formerly identified as E. sakazakii have been

reclassified as Cronobacter sakazakii, Cronobacter mal-onaticus, Cronobacter turicensis, Cronobacter muytjensii,Cronobacter dublinensis, and Cronobacter genomospecies

1 based on biogroup (37, 38). Recently, two new species,

Cronobacter condimenti and Cronobacter universalis(formerly C. genomospecies 1), were described (40).Studies have shown that sequence typing (e.g., 16S rDNA

sequencing and multilocus sequence typing) is more relia-

ble than biotyping for distinguishing among Cronobacterspecies (3, 58). Thus, as new species of Cronobactercontinue to be described, improved resolution of Crono-bacter taxonomy will likely require the application of

sequence-based methods.

Foodborne pathogen reference isolate collections can

serve as standardized sample sets for the development of

methods relating to detection and control and for studies on

various aspects of the biology of a given pathogen. For

example, an Escherichia coli diversity isolate collection has

been used to validate E. coli detection, subtyping, and* Author for correspondence. Tel: 607-254-2838; Fax: 607-254-4868;

E-mail: [email protected].

40

Journal of Food Protection, Vol. 76, No. 1, 2013, Pages 40–51doi:10.4315/0362-028X.JFP-11-546Copyright G, International Association for Food Protection

http://www.pathogentracker.net

characterization methods (2, 50, 56), and the SalmonellaReference Collection B has been used to study the evolution

and population genetics of Salmonella (7, 77). The

International Life Sciences Institute North America (ILSI

NA) Listeria monocytogenes Isolate Collection has been

used to study stress resistance (16), population genetics

(57), and subtyping techniques (80) for L. monocytogenes.

The objective of this study was to assemble a publicly

available collection of isolates formerly identified as E.sakazakii from various sources (i.e., clinical, food, and

environment) that laboratories and others can use for the

development of Cronobacter detection and control methods

and for the study of Cronobacter pathogenesis and biology.

MATERIALS AND METHODS

Isolates. Human (n ~ 10), food (n ~ 11), and environmental

(n ~ 9) isolates formerly identified as E. sakazakii were included

in this isolate set based on input from the ILSI NA Food

Microbiology Committee. Some of the isolates have been

described previously (28–30, 44, 46, 47, 53, 64), and others are

described for the first time here. Isolates are stored in brain heart

infusion (BHI; Difco, BD, Sparks, MD) broth with 15% glycerol at

280uC at the Food Safety Laboratory (FSL; Department of Food

Science, Cornell University, Ithaca, NY). The American Type

Culture Collection (ATCC; Manassas, VA) and other collections

also have some Cronobacter isolates available; these isolates were

not included in the ILSI NA collection because these organizations

typically do not allow redistribution of isolates.

Phenotypic characterization of isolate set. For phenotypic

characterization, isolates were plated on tryptic soy agar (TSA)

and incubated at 37 or 25uC. Colony color and morphology were

observed after incubation for 24 h at 37uC or 72 h at 25uC. Isolates

also were streaked on selective chromogenic agars, including

Oxoid chromogenic E. sakazakii agar (DFI; Thermo Fisher,

Waltham, MA) and E. sakazakii plating medium (ESPM; R&F,

Downers Grove, IL), and incubated at 37uC for 24 h. Each isolate was

tested for specific biochemical reactions as described by Iversen et al.

(38), including dulcitol and malonate utilization, indole production,

and gas production from 1-0-methyl a-D-glucopyranoside. Each isolate

also was characterized using API 20E and API 32E test strips

(bioMerieux, Hazelwood, MO) according to the manufacturer’s

instructions. API matches returned as ‘‘Enterobacter sakazakii’’ were

reported as E. sakazakii. However, phenotypic characteristics assessed

by API cannot be used to differentiate among the various Cronobacterspp. that were previously classified as E. sakazakii. To assess sorbitol

fermentation, isolates were streaked on purple agar base containing

0.5% sorbitol. Isolates also were inoculated into modified lauryl sulfate

tryptose broth (mLST) and tryptic soy broth (TSB) at a final inoculum

level of approximately 1 | 103 CFU/ml and incubated at 45uC for

24 h. Cultures were enumerated by plating appropriate dilutions on

TSA and counting colonies after 24 h of incubation at 37uC.

BAX PCR. Pure cultures were grown in mLST (Oxoid

CM0451 supplemented with 14.5 g/500 ml NaCl and 10 mg/ml

vancomycin at final concentration) for 20 to 24 h at 37uC and

diluted in BHI to yield approximately 106 CFU/ml. A 5-ml aliquot

of the diluted sample was used in the BAX System PCR assay for

E. sakazakii as per the manufacturer’s instructions.

Automated ribotyping. Ribotyping was performed using

the restriction enzyme EcoRI and the RiboPrinter Microbial

Characterization System (DuPont Qualicon, Wilmington, DE) as

previously described (9, 25). For 16 of 30 isolates, the RiboPrinter

generated DuPont identification numbers (IDs) (e.g., DUP-18775).

All DuPont IDs were confirmed by visual inspection. When the

RiboPrinter was unable to assign an existing DuPont ID (i.e., for a

new pattern with ,0.85 similarity to existing patterns in the DuPont

database), a unique type designation was assigned based on the

pattern number that had been assigned by the instrument (e.g., 235-

297-S-1) (66). Ribotype patterns also were exported from the

RiboPrinter and imported into the PathogenTracker database (www.

pathogentracker.net) to make these patterns publicly available.

PFGE. Pulsed-field gel electrophoresis (PFGE) was per-

formed using the standard Centers for Disease Control and

Prevention PulseNet protocol for Yersinia pestis (10) with slight

modifications. Isolates were grown on BHI agar plates at 37uC for

18 h. Bacterial cultures were then embedded in 1% agarose

(SeaKem Gold Agarose, Cambrex, Rockland, ME), lysed, washed,

and digested with the restriction enzyme XbaI (40 U per sample) for

at least 5 h at 37uC. Restricted agarose plugs were then placed into

1% agarose gels and electrophoresed on a CHEF Mapper XA

(BioRad Laboratories, Hercules, CA) at 6 V/cm for 20.5 h with

switch times of 1.8 to 25 s. XbaI-digested Salmonella Braenderup

(H9812) DNA was used as a reference size standard (33). Pattern

images were captured with Gel Doc and Multi Analyst software

version 1.1 (BioRad). PFGE patterns were then analyzed and

compared using the Bionumerics version 3.5 software package

(Applied Maths, Saint-Matins-Latem, Belgium). Similarity cluster-

ing analyses were performed with the Bionumerics program using

the unweighted pair group matching algorithm and the Dice

correlation coefficient with a tolerance of 1.5% (24). PFGE patterns

were exported from Bionumerics using a script developed by

Applied Maths and uploaded into the PathogenTracker database.

16S rDNA sequencing. For 16S rDNA sequencing, genomic

DNA was extracted with the Wizard genomic DNA purification kit

(Promega, Madison, WI). DNA concentration was measured with a

spectrophotometer (NanoDrop Technologies, Wilmington, DE),

and 1 mg of the template DNA was added to a 50-ml PCR mix

consisting of 41 ml of molecular biology grade water, 5 ml of 10|

PCR buffer (Invitrogen, Carlsbad, CA), 1.5 ml of 50 mM MgCl

(Invitrogen), 1 ml of 10 mM deoxynucleoside triphosphates (Roche

Diagnostics, Basel, Switzerland), 0.5 ml of Taq Polymerase

(Invitrogen), and 0.5 ml (10 mM stock solution) of both forward

(59-GGCCTAACACATGCAAGTCG-39) and reverse (59-GTA-

TTCACCGTGGCATTCTG-39) primers (Sigma-Genosys, Wood-

lands, TX). The PCR protocol consisted of 1 min of denaturation at

94uC; 45 cycles of 45 s of denaturation at 94uC, 45 s of annealing

at 55 to 62uC depending on the isolate, and 2 min of elongation at

72uC; and 10 min of elongation at 72uC. The target DNA amplicon

was approximately 1,000 bp; primers were designed by the Primer

Designer software package (Scientific and Educational Software,

Cary, NC), based on the reference strain ATCC 29544 (GenBank

accession AB004746). Sequences were submitted to the GenBank

database under accession numbers JQ936993 through JQ937022.

Partial 16S rDNA sequences were queried against type strain 16S

rDNA sequences using the ‘‘Seqmatch’’ function in the Ribosomal

Database Project (RDP) database (http://rdp.cme.msu.edu/) (13).For phylogenetic analysis, sequences were trimmed to 960

nucleotides and aligned using the DNAstar software package

(Lasergene, Madison, WI). A maximum parsimony phylogenetic

analysis with 500 bootstrap replicates was completed using PAUP*

(76). Type strain sequences of Cronobacter and closely related

genera Enterobacter and Citrobacter, which were downloaded

from RDP, also were included in the analysis. The 16S rDNA

J. Food Prot., Vol. 76, No. 1 ILSI NA CRONOBACTER COLLECTION 41

http://rdp.cme.msu.edu/

phylogenetic analysis is included in the supplementary material for

this article (Supplemental Fig. S1; all supplementary material

accessible at http://foodscience.cornell.edu/cals/foodsci/research/

labs/wiedmann/links/index.cfm).

Multilocus sequence typing. Isolates also were characterized

by sequence typing using a seven-gene multilocus sequence typing

(MLST) scheme (targeting atpD, fusA, glnS, gltB, gyrB, infB, and

pps) described previously (3). Genomic DNA was prepared using a

genomic DNA purification kit (Qiagen, Valencia, CA) followed by

PCR amplification (performed using either the PCR primers or the

sequencing primers described by Baldwin et al. (3)), purification

of PCR products, and Sanger sequencing. Consensus sequences

were aligned and trimmed using DNAstar. Sequence types were

TABLE 1. Isolates included in the ILSI NA Cronobacter strain collection

Cornell IDaHealth Canada ID,

other previous IDb SpeciescSequence

typedBiochemical panel ID (dulcitol,

malonate, indole, AMG)e PFGE typef

Cronobacter

FSL F6-023 HPB-2855, SK 81 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0037b

FSL F6-024 HPB-2871, MNW2 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0103

FSL F6-025 HPB-2878, Gd St 8 C. sakazakii 1 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0069

FSL F6-027 HPB-3198, NQ1-Environ (702) C. sakazakii 40 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0083

FSL F6-028 HPB-3231, 6 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0031

FSL F6-029 HPB-3234, 13 (Dutch 770479),

A’dam 9

C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0030a


FSL F6-034 HPB-3290, 10/1/01, Frm-TN C. sakazakii 1 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0033

FSL F6-035 HPB-3295, 2003-13-03 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0018






FSL F6-041 HPB-3414, 311 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0021a



FSL F6-044 HPB-3434, CFS-LAC C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0098

FSL F6-046 HPB-3437, 132-MBF C. sakazakii 121 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0021

FSL F6-047 HPB-3438, 111389 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0046a


FSL F6-049 HPB-3655, 289-81 C. sakazakii 13 C. malonaticus (2, z, 2, z) BOM_CSXAI.0036

FSL F6-050 HPB-3656, 322-78 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0041

FSL F6-051 HPB-3657, 1123-79 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0122

FSL F6-045i HPB-3436, CFS-SP C. sakazakii 4 Inconclusive (z, z, 2, 2) None assigned

FSL F6-030 HPB-3267, 52 C. malonaticus 62 C. malonaticus (2, z, 2, z) BOM_CSXAI.0089

FSL F6-052 HPB-3658, 1716-77 C. malonaticus 53 C. malonaticus (2, z, 2, z) BOM_CSXAI.0036a

FSL F6-031 HPB-3270, 13-PIF C. muytjensii 49j C. muytjensii (z, z, z, 2) BOM_CSXAI.0092

Enterobacter

FSL F6-026 HPB-2879, Md E. asburiae UT (2, 2, 2, z) None assigned

FSL F6-033 HPB-3287, 43 E. asburiae UT (z, z, 2, z) None assigned

a Cornell University Food Safety Laboratory isolate designation.b Health Canada IDs are listed as HPB.c Species identification was based on MLST data, except for E. asburiae for which species ID was based on 16S rDNA sequence data.d Sequence type was based on seven-gene multilocus sequence typing according to Baldwin et al. (3). UT, untypeable (for the two E.

asburiae isolates, some of the genes in the MLST scheme could not be amplified; see Table S1 in supplementary material for details).e Cronobacter species-specific biochemical assay panel described by Iversen et al. (38). For dulcitol and malonate, z indicates positive for

utilization; for indole, z indicates indole production; for AMG, z indicates acid production from 1-0-methyl a-D-glucopyranoside.f Pulsed-field gel electrophoresis was performed using the restriction endonuclease XbaI.g The first number (e.g., 306-S-3) corresponds to the ribogroup; the second number represents the DuPont (DUP) ID from the RiboPrinter

database. FSL F6-038 (3403) did not appear to be digested with EcoRI during ribotyping.h Based on sequence identity to respective type strain sequence obtained using the ‘‘seqmatch’’ function in the Ribosomal Database Project

(rdp.cme.msu.edu/).i Isolate FSL F6-045 has some atypical characteristics, including atypical biochemcial reactions, but was identified as C. sakazakii by

MLST.j Although both gltB and pps could not be amplified for FSL F6-031 in our experiments, FSL F6-031 was determined to be ST 49 based on

the Cronobacter MLST database (see isolate 530 at http://pubmlst.org/cronobacter/).

42 IVY ET AL. J. Food Prot., Vol. 76, No. 1

http://foodscience.cornell.edu/cals/foodsci/research/labs/wiedmann/links/index.cfm

http://foodscience.cornell.edu/cals/foodsci/research/labs/wiedmann/links/index.cfm

http://pubmlst.org/cronobacter/

assigned using the Cronobacter MLST database (http://pubmlst.

org/cronobacter) as described by Baldwin et al. (3). For phy-

logenetic analysis, sequences were concatenated and a phyloge-

netic tree was inferred using a maximum likelihood method

(RAxML 7.0.4 (74)) with the GTR plus CAT model of molecular

evolution and 1,000 bootstrap replicates. With the primers used,

gltB could not be amplified in two isolates (FSL F6-031 and FSL

F6-026) and pps could not be amplified in one isolate (FSL F6-

031) (see supplemental Table S1); these sequences were treated as

missing data in the phylogenetic analysis.

Internet-based access. Subtype and source data for all

isolates in the ILSI NA Cronobacter collection, including source

information, ribotype, and PFGE data, are summarized in a spread-

sheet table that can be accessed at the Cornell Web site (http://

foodscience.cornell.edu/cals/foodsci/research/labs/wiedmann/ilsi-na-

strain.cfm). The PathogenTracker database also can be searched

directly for individual isolates in the collection by use of the

appropriate FSL ID (e.g., FSL F6-023; see Table 1) to access

information.

Isolate availability. The ILSI NA Cronobacter isolate set is

maintained by the FSL. Requests for isolates or more information

should be addressed to the corresponding author of this article (M.

Wiedmann).

RESULTS AND DISCUSSION

Although reference isolate sets are available for many

foodborne pathogens, including Salmonella (7), E. coli (68),and L. monocytogenes (23), the ILSI NA Cronobacterisolate set is the first publicly accessible set of Cronobacterisolates. The set consists of various human, food, and

environmental isolates and includes a range of ribotypes and

pulsotypes. Based on a combination of MLST and 16S

rDNA sequence data and supported by phenotypic data, the

isolates in this set were classified into C. sakazakii (n ~

25), C. malonaticus (n ~ 2), and C. muytjensii (n ~ 1) and

two strains of Enterobacter asburiae, which were both

isolated from nonclinical sources. Although this isolate set

thus represents key Cronobacter species, four of the seven

known Cronobacter species (C. turicensis, C. dublinensis,C. universalis, and C. condimenti) are not currently included

in this set; representatives of these species may be added by

some users. The set described here has already been used

by multiple research groups (5, 26, 28–30, 44, 46, 47, 53,65, 79) to evaluate new detection techniques and control

methods and for the study of C. sakazakii survival or

pathogenicity. The ILSI NA Cronobacter isolate set is

Ribotypeg Source References(s) 16S rDNA sequence match (similarity)h

Cronobacter

284-S-3 (DUP-18775) Clinical 28–30, 46, 53, 64, 69, 72 C. sakazakii/C. malonaticus (99.3%)

284-S-3 (DUP-18775) Food (infant formula) 28–30, 46, 53, 64, 69, 72 C. sakazakii/C. malonaticus (99.3%)

284-S-4 (DUP-14592) Environment 79 C. sakazakii/C. malonaticus/C. dublinensis (99.4%)

304-S-4 (DUP-14594) Environment C. sakazakii/C. malonaticus/C. dublinensis (99.4%)

304-S-5 Clinical 28–30, 44, 46, 47, 53, 62 C. sakazakii/C. malonaticus (99.4%)

304-S-6 Clinical 28–30, 46, 53 C. sakazakii/C. malonaticus (99.1%)

297-S-1 (DUP-10162) Food (infant formula) 62 C. sakazakii (99.9%)

305-S-2 (DUP-14590) Clinical 26–30, 46, 53, 72 C. sakazakii/C. malonaticus (99.5%)

297-S-6 Clinical 28–30, 46, 53 C. sakazakii/C. malonaticus (99.4%)

305-S-7 (DUP-18799) Environment 28–30, 46, 53 C. sakazakii/C. malonaticus (99.4%)

305-S-8 (DUP-18787) Environment C. sakazakii/C. malonaticus (99.3%)

None (no digestion) Environment C. sakazakii/C. malonaticus/C. dublinensis (99.4%)

306-S-2 (DUP-18622) Environment C. sakazakii/C. malonaticus/C. dublinensis (99.4%)

306-S-3 Environment C. sakazakii/C. malonaticus/C. dublinensis (99.4%)

306-S-5 (DUP-18797) Environment C. sakazakii/C. malonaticus (99.8%)

306-S-6 Food (infant formula) C. sakazakii/C. malonaticus (99.4%)

305-S-7 (DUP-18799) Clinical 45 C. sakazakii/C. malonaticus/C. dublinensis (99.3%)

357-S-1 Food C. sakazakii/C. malonaticus (99.3%)

306-S-5 (DUP-18797) Food (infant formula) 27–30, 46, 53 C. sakazakii/C. malonaticus (99.7%)

309-S-8 Food (infant formula) 26, 27 C. sakazakii/C. malonaticus (100%)

357-S-5 (DUP-18620) Food (infant formula) 28–30, 44, 46, 47, 53 C. sakazakii/C. malonaticus (99.3%)

286-S-3 (DUP-18755) Clinical C. malonaticus (99.4%)

286-S-3 (DUP-18755) Clinical C. malonaticus/C. dublinensis (99.2%)

286-S-3 (DUP-18755) Clinical C. malonaticus (99.2%)

357-S-2 Food C. sakazakii/C. malonaticus/C. dublinensis (99.4%)

304-S-7 Food (infant formula) C. sakazakii/C. malonaticus (99.1%)

730-S-5 Clinical C. malonaticus (99.3%)

304-S-8 Food (infant formula) 28–30, 46, 53 C. muytjensi (99.8%)

Enterobacter

286-S-8 Environment E. asburiae (100%)

305-S-4 Food E. asburiae (99.9%)

TABLE 1. Extended


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cataloged on-line and available to qualified researchers

throughout the world. Continued use and integration of all

data available for the isolates included will facilitate further

development of a comprehensive understanding of pheno-

typic and genotypic characteristics of C. sakazakii and other

Cronobacter species.

Phenotypic description of set. The ILSI NA Crono-bacter isolate set comprises isolates formerly identified as

E. sakazakii, including clinical isolates (n ~ 10) from spinal

fluid (n ~ 4), blood (n ~ 4), brain (n ~ 1), and an

unknown human sample (n ~ 1), food isolates (n ~ 11;

including 8 isolates confirmed to be from infant formula),

and environmental isolates (n ~ 9) from processing plant

environments (Table 1). Colonies of most isolates on TSA

had typical yellow, light yellow, or beige coloration after 24

and 72 h and were moist or mucoid (Table 2). C. sakazakiiisolate FSL F6-023 produced beige colonies at 37uC and

white colonies at 25uC, whereas colonies of C. sakazakiiFSL F6-024 and FSL F6-032 were white at 37uC and yellow

at 25uC. Enterobacter spp. isolates FSL F6-026 and F6-033

were white on TSA at both temperatures (Table 2).

On DFI and ESPM, 26 of the 28 Cronobacter isolates

produced typical blue colonies (Table 2). C. malonaticusisolate FSL F6-052 produced white colonies on DFI and

blue colonies on ESPM, and C. sakazakii isolate FSL F6-

045, which also had atypical characteristics in other

phenotypic tests, produced white colonies on both media.

Enterobacter isolates FSL F6-026 and FSL F6-033 were

gray-blue on ESPM and white on DFI. All but five isolates

were negative for sorbitol fermentation; exceptions were C.sakazakii isolates FSL F6-049 and FSL F6-050, C.malonaticus isolates FSL F6-030 and FSL F6-052, and E.asburiae isolate FSL F6-026. Although other researchers

reported that some Cronobacter may not grow at 45uC (35),all isolates included here, except Enterobacter isolates FSL

F6-026 and FSL F6-033, were positive for growth at 45uCin TSB or mLST (Table 2). Future studies on larger isolate

sets that have been genotypically confirmed as Cronobacterwill be necessary to clarify the ability of different

Cronobacter species to grow at 45uC in TSB or mLST.

For 23 isolates (20 C. sakazakii, 2 C. malonaticus, and

1 C. muytjensii), API 20E indicated a $97.5% match to E.sakazakii and API 32E indicated a 99.9% match to E.sakazakii. For five C. sakazakii isolates, API 20E indicated

a 51.1% match to E. sakazakii and API 32E indicated a

99.9% match to E. sakazakii. The atypical C. sakazakiiisolate FSL F6-045 had a 97.0% match to Escherichiahermanii on API 20E and a 76.9% match to Enterobactercancerogenus on API 32E. Enterobacter isolate FSL F6-

033 had a 51.1% match to E. sakazakii with API 20E and a

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other Enterobacter isolate (FSL F6-033) had a $98.4%

match to E. sakazakii with API 20E and a 99.9% match to

E. sakazakii with API 32E. These data further indicate that

the various Cronobacter species cannot be identified using

these standard phenotypic assays.

Biochemical profiles based on four tests described by

Iversen et al. (38) correctly classified 23 of the 25 C.TA

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sakazakii isolates as C. sakazakii; these tests also classified

the two C. malonaticus isolates and the one C. muytjensiiisolate correctly (Table 1). C. sakazakii isolate FSL F6-049

was classified as C. malonaticus with these tests (Table 1).

C. sakazakii isolate FSL F6-045 produced mixed results that

were not typical of any of the described Cronobacter spp.

(Table 1); based on these and other atypical results

(Table 2), this isolate was categorized as an ‘‘atypical’’ C.sakazakii isolate. In addition to the two Enterobacterisolates included in this set that could be phenotypically

confused with Cronobacter, Escherichia vulneris and

Pantoea also have phenotypic characteristics that can cause

confusion (17) and thus may be added to this collection by

some users.

Genotypic characteristics of the isolate set. Com-

bined MLST data and 16S rDNA sequencing data allowed

us to classify the 30 isolates as C. sakazakii (n ~ 25), C.malonaticus (n ~ 2), C. muytjensii (n ~ 1), and E.asburiae (n ~ 2) (Fig. 1 and Table 1; see also Fig. S1).

With the exception of two isolates that were clearly

identified as C. sakazakii by MLST data (FSL F6-045

representing sequence type (ST) 121 and F6-049 represent-

ing ST 4, the most common C. sakazakii type in the isolate

set described here), these species assignments were

consistent with those based on the four biochemical tests

described by Iversen et al. (38). Initial characterization by

16S rDNA sequencing revealed that most Cronobacterisolates matched type strains representing more than one

Cronobacter species with the same 16S rDNA sequence

similarity score. Specifically, seven C. sakazakii isolates had

16S rDNA similarity scores (all similarity scores for these

isolates were $99.3%) identical to those of C. sakazakii, C.malonaticus, and C. dublinensis. In addition, 14 C.sakazakii isolates and one C. malonaticus isolate had 16S

rDNA scores that indicated $99.1% similarity to both C.sakazakii and C. malonaticus, and one C. sakazakii isolate

had 16S rDNA similarity scores of 99.3% for both C.malonaticus and C. dublinensis (Table 1). The remaining

five isolates had a high 16S rDNA similarity score for a

single Cronobacter species; one C. sakazakii isolate

matched C. sakazakii (99.9%), two C. sakazakii isolates

matched C. malonaticus ($99.2%), one of the two C.malonaticus isolates matched C. malonaticus (99.3%), and

the one C. muytjensii isolate matched C. muytjensii (99.8%)

(Table 1). Classification with 16S rDNA sequencing of

isolates FSL F6-026 and FSL F6-033 as E. asburiae is

consistent with the phenotypic results (i.e., white colonies

on TSA, DFI, and ESPM; Table 2). Overall, these data

clearly indicated that partial 16S rDNA sequencing does not

allow for reliable species identification in Cronobacter.

In contrast, use of a previously reported seven-gene

MLST method (3) allowed for clear classification of the 30

isolates into well-supported clades (Fig. 1; also see Table S1

for detailed MLST results). Phylogenetic analysis of the

concatenated seven-gene MLST sequences specifically

revealed one C. sakazakii clade, which is distinct from a

well-supported clade (bootstrap support [BS] ~ 99) that

includes the two C. malonaticus isolates (Fig. 1). The C.muytjensii isolate (FSL F6-031) clearly forms a distinct

clade in this tree. The 25 C. sakazakii isolates in our set

represented nine different STs; the two C. malonaticus

FIGURE 1. Maximum likelihood phylogenetic tree of concatenated atpD, fusA, glnS, gltB, gyrB, infB, and pps sequences from isolates inthe ILSI NA Cronobacter isolate set. Cornell University Food Safety Laboratory isolates are designation as FSL. For isolate FSL F6-031,data used for the tree excluded sequences for gltB and pps because these genes could not be amplified in our experiments. Data for thesetwo genes are available in the Cronobacter MLST database (http://pubmlst.org/cronobacter), in which this strain is identified as ST 49 (seeTable 1). Numerical node labels represent the percentage of bootstrap replicates (n ~ 1,000) that supported the respective node. Onlybootstrap values greater than 50 are shown. Scale represents estimated substitutions per site.


http://pubmlst.org/cronobacter

isolates represented two additional distinct STs (Fig 1; also

see Table S1). The one C. muytjensii isolate (F6-031) could

not be assigned an ST because both gltB and pps could not

be reproducibly amplified with the primers used (see Table

S1). Within the C. sakazakii group, ST 4 isolates (n ~ 14)

were grouped in a well-supported clade (BS ~ 89), as were

ST 1 isolates (n ~ 3). Identification of a large number of ST

4 isolates, which are associated with neonatal meningitis

(41, 42), is consistent with the MLST findings reported by

Baldwin et al. (3), who found that 22 of 60 C. sakazakiiisolates in their collection were ST 4. These authors also

reported that ST 8 may represent a more virulent C.sakazakii ST because seven of the eight C. sakazakii with

ST 8 were clinical isolates; the single ST 8 isolate in our set

(FSL F6-032) was a food isolate. C. sakazakii isolate FSL

F6-045, which had a number of atypical phenotypic

characteristics, was classified as ST 121, and FSL F6-049,

which was identified by biochemical profiling as C.malonaticus (Table 1), was identified as C. sakazakii ST

4. These findings are consistent with those of Baldwin et al.

(3), who also reported that phenotypic-based species

identification of Cronobacter spp. can be inconsistent with

sequence-based identification (3).Although MLST differentiated the 30 isolates in the ILSI

NA set into only 15 distinct STs, PFGE subtyping of these

isolates resulted in 30 unique pulsotypes (Fig. 2). FSL F6-

049 and FSL F6-051 differed by only two bands, as did FSL

F6-041 and FSL F6-046. However, FSL F6-041 and FSL F6-

FIGURE 2. Pulsed-field gel electrophoresis patterns for isolates in the ILSI NA Cronobacter isolate set. The dendrogram shown is basedon the XbaI restriction patterns with a similarity matrix calculated from the Dice coefficient with a tolerance of 1.5%. Both Health Canada(first four-digit identifier, e.g., 2879) and Cornell University Food Safety Laboratory (FSL) isolate designations are shown. FSL F6-026and FSL F6-033 were identified as Enterobacter asburiae, and FSL F6-045 was identified as an ‘‘atypical C. sakazakii.’’ These isolates,therefore, were not assigned a Cronobacter PFGE pattern ID.


046 had identical results in phenotypic tests (Tables 1 and 2),

whereas F6-049 and FSL F6-051 had different biochemical

results (Table 1). Ribotyping of these isolates produced 23

unique ribotypes; one isolate was not digested with EcoRI

(Fig. 3). Overall, these data indicate that the ILSI NA set

detailed here includes a diverse group of isolates.

The E. sakazakii BAX PCR was performed on each of

the isolates according to the manufacturer’s instructions. Of

the isolates confirmed to be Cronobacter by 16S rDNA

sequencing and MLST, only C. sakazakii FSL F6-045 was

BAX negative (Table 2). FSL F6-045 was also negative on

two selective media (Table 2), indicating that FSL F6-045

has some characteristics that are atypical of C. sakazakii.Enterobacter isolates FSL F6-033 and FSL F6-026 were

BAX negative as expected. Although all of the isolates in

our collection grew in mLST broth, a small percentage of

Cronobacter isolates have previously been reported to not

grow in mLST (35). These findings indicate that the use of

mLST as an enrichment medium for the Cronobacter BAX

detection method may occasionally result in a false-negative

result, and future studies on larger isolate sets that have been

genotypically confirmed as Cronobacter will be needed to

clarify the ability of various Cronobacter isolates to grow in

different enrichment media.

All isolates in the ILSI NA Cronobacter isolate set are

included in the comprehensive on-line database Pathogen-

Tracker and can be searched for by using the full isolate

designation (e.g., FSL F6-036). Information on use of any

isolate or phenotypic data for a given isolate are included

and updated continuously, including links to publications

of studies involving these isolates. This publicly accessible

on-line interface facilitates interlaboratory comparison of

results obtained with this Cronobacter isolate set.

Potential applications of the ILSI NA Cronobacterisolate set. Cronobacter spp. are emerging foodborne

pathogens that can cause a fatal disease in infants.

Therefore, control of the transmission of pathogenic

Cronobacter spp. is a priority for regulatory and public

health agencies and the PIF industry. The isolates in the

ILSI NA Cronobacter isolate set are genetically diverse.

The set comprises 23 unique ribotypes and 30 unique PFGE

types that will provide researchers with information to

develop novel Cronobacter interventions and to character-

ize various aspects of Cronobacter biology. The set also

provides considerable genetic diversity that will help

researchers evaluate new enrichment and detection tech-

niques, including DNA-based techniques. The inclusion of

two representatives of a closely related species (E. asburiae)

will allow researchers to evaluate the specificity of new

molecular detection methods. Because this isolate set

includes only three of the seven known Cronobacter spp.,

some researchers may wish to add isolates representing the

remaining four Cronobacter species.

Cronobacter spp., when detected in foods such as PIF,

are generally present at low levels (34, 61). Therefore, rapid,

sensitive, and specific techniques are needed for detection of

Cronobacter spp. in food products such as PIF. In several

recent studies, currently available enrichment or detection

media have been evaluated (18, 27, 35) or new media for

Cronobacter enrichment have been developed (1, 11).Development of new molecular detection techniques is

ongoing (21, 54, 73, 79). Diversity among Cronobacter spp.

may present a challenge for the design of new, effective

enrichment and detection methods, and an accessible set of

Cronobacter isolates could be used to validate the efficacy

of these new techniques.

The development of new, rapid, standardized, and

economical molecular subtyping methods for Cronobacterspp. will enable public health agencies to quickly detect

and identify sources of disease outbreaks (78). PFGE is

considered the ‘‘gold standard’’ of molecular subtyping and

has been used successfully to distinguish among Crono-bacter isolates (4, 58). Although our data support that fact

that PFGE allows discrimination among highly clonal

FIGURE 3. EcoRI ribotype patterns for the 30 isolates in the ILSINA Cronobacter isolate set. Ribotypes were obtained with theautomated RiboPrinter (Qualicon). Health Canada (four-digitidentifier) designations and Cornell University Food SafetyLaboratory (FSL) designations are shown for each isolate. Allribogroups carry the prefix 235. For example ‘‘306-S-5’’ standsfor ‘‘235-306-S-5.’’ Ribogroups in the DuPont database are alsoassigned a DUP designation. FSL F6-038 was not digestedwith EcoRI.


strains such as C. sakazakii ST 4, PFGE is expensive and

time-consuming and can be difficult to standardize among

laboratories. The ILSI NA Cronobacter isolate set provides

a valuable resource for assessing the discriminatory power

of new Cronobacter subtyping techniques. A new genetic

subtyping method would ideally be more economical,

reproducible, and at least as discriminatory as PFGE (i.e.,

it should group the 30 nonrelated isolates into 30 unique

subtypes) and would allow discrimination among highly

clonal Cronobacter subtypes.

The development of effective Cronobacter interven-

tions requires an in-depth understanding of the resistance of

the organism to environmental stresses. Strain-dependent

resistance to heat and desiccation (8, 19, 36, 63) and to

various antimicrobial treatments (47, 75) has been demon-

strated, and although survival under environmental stress

appears to be strain dependent (43), further studies are

needed to assess how strain diversity affects resistance to

various interventions. The ILSI NA Cronobacter isolate

set will provide the research community with a publicly

accessible set of isolates to study Cronobacter stress

resistance. Isolates in the ILSI NA Cronobacter isolate set

have recently been used to study Cronobacter attachment to

various surfaces (44, 46, 47), survival in foods (30, 45, 71),and resistance to various antimicrobials (5, 28–30).

Although recent studies have focused on Cronobactervirulence, overall the mechanisms of Cronobacter patho-

genesis are not well characterized (32). Cronobacterenterotoxin production has been reported (69, 70), and

recent studies have revealed the ability of Cronobacterisolates to attach to (55) and invade (52) mammalian cells,

including human brain cells, and to cause mortality in

neonatal mice (72). However, Cronobacter virulence

characteristics (e.g., attachment and invasion) vary by strain

(15). Validation of Cronobacter pathogenicity models will,

therefore, require the use of multiple Cronobacter strains.

The ILSI NA Cronobacter isolate set is available for use as

part of the development and validation of new models of

Cronobacter virulence, which could also facilitate the

development of novel therapeutics.

In summary, we have assembled a publicly available

isolate set of 25 C. sakazakii isolates, including 1 (i.e.,

FSL-F6-045) with a number of atypical phenotypic

characteristics, 2 C. malonaticus isolates, and 1 C.muytjensii isolate, and 2 isolates of the closely related

species E. asburiae. Isolates in this set have already been

distributed to research groups for the study of Cronobacterspp. (5, 28–30, 44, 46, 47, 53). The availability of the ILSI

NA Cronobacter isolate set will allow the continued

characterization of the organism and the development and

validation of novel detection, subtyping, and intervention

strategies for Cronobacter spp.

ACKNOWLEDGMENTS

The authors thank Pajau Vangay, Emily Wright, and Sherry Roof for

assistance with PathogenTracker, Stephen Forsythe and Esther Fortes for

help with MLST analysis, Henk C. den Bakker for assistance with

phylogenetic analyses, and Kevin Tyler for assistance with ribotyping and

PFGE. The assembly and maintenance of the ILSI NA Cronobacter isolate

set is funded by a grant from the ILSI NA to M.W. The opinions expressed

herein are those of the authors and do not necessarily represent the views of

the ILSI NA. Development of the PathogenTracker database was supported

by U.S. Department of Agriculture special research grants 2002-34459-

11758, 2003-34459-12999, and 2004-34459-14296 (to M.W.).

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