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

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

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 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/1/40/1685371/0362-028x_jfp-11-546.pdf by guest on 23 September 2021

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 malonaticus isolates, (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 Cronobacter spp. were formerly identified as belonging gram-negative, motile, non–spore-forming bacilli that have to the species Enterobacter sakazakii, which was classified been associated with disease in both neonates (6, 12, 31, 49) into 16 different biochemical profile groups (biogroups) (20, and adults (49). In adults, symptoms associated with 39). Since the description of the genus Cronobacter (38), Cronobacter infection include bacteremia, wound infec- isolates formerly identified as E. sakazakii have been tions, abscesses, and ulcers (32, 48). Disease due to reclassified as Cronobacter sakazakii, Cronobacter mal- Cronobacter infection of neonates is rare (22); for example, onaticus, Cronobacter turicensis, Cronobacter muytjensii, in the United States typically four to six cases in neonates Cronobacter dublinensis, and Cronobacter genomospecies are reported per year. Symptoms that have been associated 1 based on biogroup (37, 38). Recently, two new species, with Cronobacter infections in neonates include meningitis, Cronobacter condimenti and Cronobacter universalis sepsis, and necrotizing enterocolitis (31). Cronobacter (formerly C. genomospecies 1), were described (40). infection mortality rates in neonates can be high and have Studies have shown that sequence typing (e.g., 16S rDNA been reported to exceed 20% (6, 22, 49, 51, 61). Powdered sequencing and multilocus sequence typing) is more relia- infant formula (PIF) has been identified as a possible source ble than biotyping for distinguishing among Cronobacter of Cronobacter infection in infants, and Cronobacter has species (3, 58). Thus, as new species of Cronobacter been isolated from PIF (60, 62, 67), but other sources of continue to be described, improved resolution of Crono- infections also have been reported. Cronobacter has been bacter taxonomy will likely require the application of isolated from a variety of other foods and dry ingredients (4, sequence-based methods. 5, 34) and from farms (59), food processing facilities (14), Foodborne pathogen reference isolate collections can and other sources, including water (39). 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 * Author for correspondence. Tel: 607-254-2838; Fax: 607-254-4868; example, an Escherichia coli diversity isolate collection has E-mail: [email protected]. been used to validate E. coli detection, subtyping, and J. Food Prot., Vol. 76, No. 1 ILSI NA CRONOBACTER COLLECTION 41 characterization methods (2, 50, 56), and the Salmonella previously described (9, 25). For 16 of 30 isolates, the RiboPrinter Reference Collection B has been used to study the evolution generated DuPont identification numbers (IDs) (e.g., DUP-18775). and population genetics of Salmonella (7, 77). The All DuPont IDs were confirmed by visual inspection. When the International Life Sciences Institute North America (ILSI RiboPrinter was unable to assign an existing DuPont ID (i.e., for a NA) Listeria monocytogenes Isolate Collection has been new pattern with ,0.85 similarity to existing patterns in the DuPont database), a unique type designation was assigned based on the used to study stress resistance (16), population genetics pattern number that had been assigned by the instrument (e.g., 235- (57), and subtyping techniques (80) for L. monocytogenes. 297-S-1) (66). Ribotype patterns also were exported from the The objective of this study was to assemble a publicly RiboPrinter and imported into the PathogenTracker database (www. available collection of isolates formerly identified as E. pathogentracker.net) to make these patterns publicly available. sakazakii from various sources (i.e., clinical, food, and environment) that laboratories and others can use for the PFGE. Pulsed-field gel electrophoresis (PFGE) was per- development of Cronobacter detection and control methods formed using the standard Centers for Disease Control and and for the study of Cronobacter pathogenesis and biology. Prevention PulseNet protocol for Yersinia pestis (10) with slight modifications. Isolates were grown on BHI agar plates at 37uC for Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/1/40/1685371/0362-028x_jfp-11-546.pdf by guest on 23 September 2021 MATERIALS AND METHODS 18 h. Bacterial cultures were then embedded in 1% agarose (SeaKem Gold Agarose, Cambrex, Rockland, ME), lysed, washed, ~ ~ Isolates. Human (n 10), food (n 11), and environmental and digested with the restriction enzyme XbaI (40 U per sample) for (n ~ 9) isolates formerly identified as E. sakazakii were included at least 5 h at 37uC. Restricted agarose plugs were then placed into in this isolate set based on input from the ILSI NA Food 1% agarose gels and electrophoresed on a CHEF Mapper XA Microbiology Committee. Some of the isolates have been (BioRad Laboratories, Hercules, CA) at 6 V/cm for 20.5 h with described previously (28–30, 44, 46, 47, 53, 64), and others are switch times of 1.8 to 25 s. XbaI-digested Salmonella Braenderup described for the first time here. Isolates are stored in brain heart (H9812) DNA was used as a reference size standard (33). Pattern infusion (BHI; Difco, BD, Sparks, MD) broth with 15 glycerol at % images were captured with Gel Doc and Multi Analyst software 280 C at the Food Safety Laboratory (FSL; Department of Food u version 1.1 (BioRad). PFGE patterns were then analyzed and Science, Cornell University, Ithaca, NY). The American Type compared using the Bionumerics version 3.5 software package Culture Collection (ATCC; Manassas, VA) and other collections (Applied Maths, Saint-Matins-Latem, Belgium). Similarity cluster- also have some Cronobacter isolates available; these isolates were ing analyses were performed with the Bionumerics program using not included in the ILSI NA collection because these organizations the unweighted pair group matching algorithm and the Dice typically do not allow redistribution of isolates. correlation coefficient with a tolerance of 1.5% (24). PFGE patterns were exported from Bionumerics using a script developed by Phenotypic characterization of isolate set. For phenotypic Applied Maths and uploaded into the PathogenTracker database. characterization, isolates were plated on tryptic soy agar (TSA) and incubated at 37 or 25uC. Colony color and morphology were 16S rDNA sequencing. For 16S rDNA sequencing, genomic observed after incubation for 24 h at 37uC or 72 h at 25uC. Isolates DNA was extracted with the Wizard genomic DNA purification kit also were streaked on selective chromogenic agars, including (Promega, Madison, WI). DNA concentration was measured with a Oxoid chromogenic E. sakazakii agar (DFI; Thermo Fisher, spectrophotometer (NanoDrop Technologies, Wilmington, DE), Waltham, MA) and E. sakazakii plating medium (ESPM; R&F, and 1 mg of the template DNA was added to a 50-ml PCR mix Downers Grove, IL), and incubated at 37uC for 24 h. Each isolate was consisting of 41 ml of molecular biology grade water, 5 mlof10| tested for specific biochemical reactions as described by Iversen et al. PCR buffer (Invitrogen, Carlsbad, CA), 1.5 ml of 50 mM MgCl (38), including dulcitol and malonate utilization, indole production, (Invitrogen), 1 ml of 10 mM deoxynucleoside triphosphates (Roche and gas production from 1-0-methyl a-D-glucopyranoside. Each isolate Diagnostics, Basel, Switzerland), 0.5 mlofTaq Polymerase also was characterized using API 20E and API 32E test strips (Invitrogen), and 0.5 ml (10 mM stock solution) of both forward (bioMe´rieux, Hazelwood, MO) according to the manufacturer’s 9 9 9 instructions. API matches returned as ‘‘Enterobacter sakazakii’’ were (5 -GGCCTAACACATGCAAGTCG-3 ) and reverse (5 -GTA- reported as E. sakazakii. However, phenotypic characteristics assessed TTCACCGTGGCATTCTG-39) primers (Sigma-Genosys, Wood- by API cannot be used to differentiate among the various Cronobacter lands, TX). The PCR protocol consisted of 1 min of denaturation at spp. that were previously classified as E. sakazakii. To assess sorbitol 94uC; 45 cycles of 45 s of denaturation at 94uC, 45 s of annealing fermentation, isolates were streaked on purple agar base containing at 55 to 62uC depending on the isolate, and 2 min of elongation at 0.5% sorbitol. Isolates also were inoculated into modified lauryl sulfate 72uC; and 10 min of elongation at 72uC. The target DNA amplicon tryptose broth (mLST) and tryptic soy broth (TSB) at a final inoculum was approximately 1,000 bp; primers were designed by the Primer level of approximately 1 | 103 CFU/mlandincubatedat45uCfor Designer software package (Scientific and Educational Software, 24 h. Cultures were enumerated by plating appropriate dilutions on Cary, NC), based on the reference strain ATCC 29544 (GenBank TSA and counting colonies after 24 h of incubation at 37uC. accession AB004746). Sequences were submitted to the GenBank database under accession numbers JQ936993 through JQ937022. BAX PCR. Pure cultures were grown in mLST (Oxoid Partial 16S rDNA sequences were queried against type strain 16S CM0451 supplemented with 14.5 g/500 ml NaCl and 10 mg/ml rDNA sequences using the ‘‘Seqmatch’’ function in the Ribosomal vancomycin at final concentration) for 20 to 24 h at 37uC and Database Project (RDP) database (http://rdp.cme.msu.edu/) (13). diluted in BHI to yield approximately 106 CFU/ml. A 5-ml aliquot For phylogenetic analysis, sequences were trimmed to 960 of the diluted sample was used in the BAX System PCR assay for nucleotides and aligned using the DNAstar software package E. sakazakii as per the manufacturer’s instructions. (Lasergene, Madison, WI). A maximum parsimony phylogenetic analysis with 500 bootstrap replicates was completed using PAUP* Automated ribotyping. Ribotyping was performed using (76). Type strain sequences of Cronobacter and closely related the restriction enzyme EcoRI and the RiboPrinter Microbial genera Enterobacter and Citrobacter, which were downloaded Characterization System (DuPont Qualicon, Wilmington, DE) as from RDP, also were included in the analysis. The 16S rDNA 42 IVY ET AL. J. Food Prot., Vol. 76, No. 1

TABLE 1. Isolates included in the ILSI NA Cronobacter strain collection Health Canada ID, Sequence Biochemical panel ID (dulcitol, Cornell IDa other previous IDb Speciesc typed 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), C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0030a A’dam 9 FSL F6-032 HPB-3284, 7 C. sakazakii 8 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0111 FSL F6-034 HPB-3290, 10/1/01, Frm-TN C. sakazakii 1 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0033 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/1/40/1685371/0362-028x_jfp-11-546.pdf by guest on 23 September 2021 FSL F6-035 HPB-3295, 2003-13-03 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0018 FSL F6-036 HPB-3396, 272 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0008 FSL F6-037 HPB-3402, 286 C. sakazakii 73 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0115 FSL F6-038 HPB-3403, 288 C. sakazakii 40 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0080 FSL F6-039 HPB-3404, 290 C. sakazakii 3 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0042 FSL F6-040 HPB-3410, 305 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0020 FSL F6-041 HPB-3414, 311 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0021a FSL F6-042 HPB-3420, 323 C. sakazakii 1 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0067 FSL F6-043 HPB-3428, 8397 C. sakazakii 4 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0024 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 F6-048 HPB-3439, 111392 C. sakazakii 42 C. sakazakii (2, 2, 2, z) BOM_CSXAI.0053 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/).

phylogenetic analysis is included in the supplementary material for (MLST) scheme (targeting atpD, fusA, glnS, gltB, gyrB, infB, and this article (Supplemental Fig. S1; all supplementary material pps) described previously (3). Genomic DNA was prepared using a accessible at http://foodscience.cornell.edu/cals/foodsci/research/ genomic DNA purification kit (Qiagen, Valencia, CA) followed by labs/wiedmann/links/index.cfm). PCR amplification (performed using either the PCR primers or the sequencing primers described by Baldwin et al. (3)), purification Multilocus sequence typing. Isolates also were characterized of PCR products, and Sanger sequencing. Consensus sequences by sequence typing using a seven-gene multilocus sequence typing were aligned and trimmed using DNAstar. Sequence types were J. Food Prot., Vol. 76, No. 1 ILSI NA CRONOBACTER COLLECTION 43

TABLE 1. Extended

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%) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/1/40/1685371/0362-028x_jfp-11-546.pdf by guest on 23 September 2021 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%) assigned using the Cronobacter MLST database (http://pubmlst. RESULTS AND DISCUSSION org/cronobacter) as described by Baldwin et al. (3). For phylogenetic analysis, sequences were concatenated and a phyloge- Although reference isolate sets are available for many netic tree was inferred using a maximum likelihood method foodborne pathogens, including Salmonella (7), E. coli (68), (RAxML 7.0.4 (74)) with the GTR plus CAT model of molecular and L. monocytogenes (23), the ILSI NA Cronobacter evolution and 1,000 bootstrap replicates. With the primers used, isolate set is the first publicly accessible set of Cronobacter gltB could not be amplified in two isolates (FSL F6-031 and FSL isolates. The set consists of various human, food, and F6-026) and pps could not be amplified in one isolate (FSL F6- environmental isolates and includes a range of ribotypes and 031) (see supplemental Table S1); these sequences were treated as pulsotypes. Based on a combination of MLST and 16S missing data in the phylogenetic analysis. rDNA sequence data and supported by phenotypic data, the ~ Internet-based access. Subtype and source data for all isolates in this set were classified into C. sakazakii (n ~ ~ isolates in the ILSI NA Cronobacter collection, including source 25), C. malonaticus (n 2), and C. muytjensii (n 1) and information, ribotype, and PFGE data, are summarized in a spread- two strains of Enterobacter asburiae, which were both sheet table that can be accessed at the Cornell Web site (http:// isolated from nonclinical sources. Although this isolate set foodscience.cornell.edu/cals/foodsci/research/labs/wiedmann/ilsi-na- thus represents key Cronobacter species, four of the seven strain.cfm). The PathogenTracker database also can be searched known Cronobacter species (C. turicensis, C. dublinensis, directly for individual isolates in the collection by use of the C. universalis, and C. condimenti) are not currently included appropriate FSL ID (e.g., FSL F6-023; see Table 1) to access in this set; representatives of these species may be added by information. some users. The set described here has already been used Isolate availability. The ILSI NA Cronobacter isolate set is by multiple research groups (5, 26, 28–30, 44, 46, 47, 53, maintained by the FSL. Requests for isolates or more information 65, 79) to evaluate new detection techniques and control should be addressed to the corresponding author of this article (M. methods and for the study of C. sakazakii survival or Wiedmann). pathogenicity. The ILSI NA Cronobacter isolate set is 44

TABLE 2. Phenotypic characteristics of isolates included in the Cronobacter ILSI NA strain collection V TAL. ET IVY Colony color on: Colony Colony color on TSA Growth at 45uC Previous morphology DFI (37uC, ESPM (37uC, Sorbitol BAX- FSL IDa HPB IDb designation API 20E ID% API 32E ID% 37uC, 24 h 25uC, 72 h on TSA 24 h)c 24 h)d utilization PCR TSB mLST Typical Cronobacter F6-023 2855 SK 81 98.40 99.90 Beige White Moist Blue Dark blue 2 zzz F6-024 2871 MNW2 99.90 99.90 White Light yellow Mucoid Blue Dark blue 2 zzz F6-025 2878 Gd St 8 51.10 99.90 Light yellow Light yellow Moist Blue Dark blue 2 zzz F6-027 3198 NQ1-Environ 98.40 99.90 Light yellow Yellow Moist Blue Blue 2 zzz (702) F6-028 3231 6 98.40 99.90 Light yellow Light yellow Mucoid Blue Dark blue 2 zzz F6-029 3234 13 (Dutch 98.40 99.90 Yellow Yellow Moist Blue Blue 2 zzz 770479) F6-032 3284 7 98.40 99.90 White Light yellow Mucoid Blue Dark blue 2 zzz F6-034 3290 37165 51.10 99.90 Dark media, Beige Moist Blue Dark blue 2 zzz colony F6-035 3295 2003-13-03 98.40 99.90 Dark media, Yellow Mucoid Blue Dark blue 2 zzz colony F6-036 3396 272 97.00 99.90 Light yellow Two colors Moist Blue Dark blue 2 zzz F6-037 3402 286 51.10 99.90 Light yellow Yellow Mucoid Blue Dark blue 2 zzz F6-038 3403 288 99.90 99.90 Yellow Yellow Moist Blue Dark blue 2 zzz F6-039 3404 290 51.10 99.90 Light yellow Yellow Moist Blue Dark blue 2 zzz F6-040 3410 305 98.40 99.90 Yellow Yellow Moist Blue Dark blue 2 zzz F6-041 3414 311 98.40 99.90 Yellow Yellow Moist Blue Dark blue 2 zzz F6-042 3420 323 51.10 99.90 Yellow Yellow Moist Blue Dark blue 2 zzz F6-043 3428 8397 98.40 99.90 Yellow Yellow Mucoid Blue Dark blue 2 zzz F6-044 3434 CFS-LAC 98.40 99.90 Yellow Yellow Moist Blue Blue 2 zzz F6-046 3437 132-MBF 97.50 99.90 Beige Beige Moist Blue Blue 2 zzz F6-047 3438 111389 99.90 99.90 Beigee Beige Moist Blue Blue 2 zzz F6-048 3439 111392 98.40 99.90 Yellow Yellow Moist Blue Blue 2 zzz F6-049 3655 289-81 98.40 99.90 Light yellow Light yellow Moist Dark blue Blue zzzz F6-050 3656 322-78 99.90 99.90 Light yellow Light yellow Moist Blue Blue zzzz .Fo rt,Vl 6 o 1 No. 76, Vol. Prot., Food J. F6-051 3657 1123-79 98.40 99.90 Light yellow Light yellow Moist Blue Blue 2 zzz F6-030 3267 52 98.40 99.90 Beige Light yellow Mucoid Blue Dark blue zzzz F6-052 3658 1716-77 98.40 99.90 Light yellow Light yellow Moist White Blue zzzz F6-031 3270 13 99.90 99.90 Light yellow Beige Moist, Blue Dark blue 2 zzz

mucoid Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/1/40/1685371/0362-028x_jfp-11-546.pdf by guest on 23 September 2021 September 23 on guest by http://meridian.allenpress.com/jfp/article-pdf/76/1/40/1685371/0362-028x_jfp-11-546.pdf from Downloaded J. Food Prot., Vol. 76, No. 1 ILSI NA CRONOBACTER COLLECTION 45 C

u 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

zz development of a comprehensive understanding of pheno- TSB mLST Growth at 45 typic and genotypic characteristics of C. sakazakii and other Cronobacter species. 222 PCR BAX- Phenotypic description of set. The ILSI NA Crono- bacter isolate set comprises isolates formerly identified as 22 2222 ~ z E. sakazakii, including clinical isolates (n 10) from spinal Sorbitol

utilization fluid (n ~ 4), blood (n ~ 4), brain (n ~ 1), and an unknown human sample (n ~ 1), food isolates (n ~ 11; C, u

d including 8 isolates confirmed to be from infant formula), and environmental isolates (n ~ 9) from processing plant Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/1/40/1685371/0362-028x_jfp-11-546.pdf by guest on 23 September 2021 24 h) blue blue environments (Table 1). Colonies of most isolates on TSA ESPM (37 had typical yellow, light yellow, or beige coloration after 24 C, u c and 72 h and were moist or mucoid (Table 2). C. sakazakii Colony color on:

24 h) isolate FSL F6-023 produced beige colonies at 37uC and

DFI (37 white colonies at 25uC, whereas colonies of C. sakazakii FSL 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). Colony on TSA

morphology On DFI and ESPM, 26 of the 28 Cronobacter isolates produced typical blue colonies (Table 2). C. malonaticus isolate FSL F6-052 produced white colonies on DFI and

C, 72 h blue colonies on ESPM, and C. sakazakii isolate FSL F6- u 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. Colony color on TSA sakazakii isolates FSL F6-049 and FSL F6-050, C. C, 24 h 25 u

37 malonaticus isolates FSL F6-030 and FSL F6-052, and E. Beige Beige Moist White White White White Moistasburiae White Light, gray- 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 45uC in TSB or mLST (Table 2). Future studies on larger isolate (76.9 (53.0 API 32E ID sets that have been genotypically confirmed as Cronobacter E. cancerogenus E. cancerogenus will 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.

hermannii (97.0 sakazakii and API 32E indicated a 99.9% match to E. Escherichia

as producing colonies of a ‘‘matte palesakazakii yellow’’ color on tryptic soy agar; here we report the color as beige. . For five C. sakazakii isolates, API 20E indicated a 51.1% match to E. sakazakii and API 32E indicated a (26) 99.9% match to E. sakazakii. The atypical C. sakazakii isolate FSL F6-045 had a 97.0% match to Escherichia Previous designation API 20E ID hermanii on API 20E and a 76.9% match to Enterobacter cancerogenus on API 32E. Enterobacter isolate FSL F6- 033 had a 51.1% match to E. sakazakii with API 20E and a plating medium agar. b 53.0% match to E. cancerogenus with API 32E, whereas the other Enterobacter isolate (FSL F6-033) had a $98.4%

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

Continued E. sakazakii with API 32E. These data further indicate that the various Cronobacter species cannot be identified using Cronobacter a these standard phenotypic assays. Biochemical profiles based on four tests described by F6-045 3436 CFS-SP F6-026F6-033 2879 3287 Md 43 98.40 51.10 99.90 White White Moist White Light, gray- FSL ID Cornell University Food SafetyHealth Laboratory Canada isolate isolate designation. designation. Enterobacter sakazakii DFI, Druggan-Forsythe-Iversen agar. Isolate F6-047 was reported by Gurtler TABLE 2. Atypical a b c d e Enterobacter Iversen et al. (38) correctly classified 23 of the 25 C. 46 IVY ET AL. J. Food Prot., Vol. 76, No. 1 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/1/40/1685371/0362-028x_jfp-11-546.pdf by guest on 23 September 2021

FIGURE 1. Maximum likelihood phylogenetic tree of concatenated atpD, fusA, glnS, gltB, gyrB, infB, and pps sequences from isolates in the 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 these two genes are available in the Cronobacter MLST database (http://pubmlst.org/cronobacter), in which this strain is identified as ST 49 (see Table 1). Numerical node labels represent the percentage of bootstrap replicates (n ~ 1,000) that supported the respective node. Only bootstrap values greater than 50 are shown. Scale represents estimated substitutions per site. sakazakii isolates as C. sakazakii; these tests also classified 16S rDNA similarity scores (all similarity scores for these the two C. malonaticus isolates and the one C. muytjensii isolates were $99.3%) identical to those of C. sakazakii, C. isolate correctly (Table 1). C. sakazakii isolate FSL F6-049 malonaticus, and C. dublinensis. In addition, 14 C. was classified as C. malonaticus with these tests (Table 1). sakazakii isolates and one C. malonaticus isolate had 16S C. sakazakii isolate FSL F6-045 produced mixed results that rDNA scores that indicated $99.1% similarity to both C. were not typical of any of the described Cronobacter spp. sakazakii and C. malonaticus, and one C. sakazakii isolate (Table 1); based on these and other atypical results had 16S rDNA similarity scores of 99.3% for both C. (Table 2), this isolate was categorized as an ‘‘atypical’’ C. malonaticus and C. dublinensis (Table 1). The remaining sakazakii isolate. In addition to the two Enterobacter five isolates had a high 16S rDNA similarity score for a isolates included in this set that could be phenotypically single Cronobacter species; one C. sakazakii isolate confused with Cronobacter, Escherichia vulneris and matched C. sakazakii (99.9%), two C. sakazakii isolates Pantoea also have phenotypic characteristics that can cause matched C. malonaticus ($99.2%), one of the two C. confusion (17) and thus may be added to this collection by malonaticus isolates matched C. malonaticus (99.3%), and some users. the one C. muytjensii isolate matched C. muytjensii (99.8%) (Table 1). Classification with 16S rDNA sequencing of Genotypic characteristics of the isolate set. Com- isolates FSL F6-026 and FSL F6-033 as E. asburiae is bined MLST data and 16S rDNA sequencing data allowed consistent with the phenotypic results (i.e., white colonies us to classify the 30 isolates as C. sakazakii (n ~ 25), C. on TSA, DFI, and ESPM; Table 2). Overall, these data malonaticus (n ~ 2), C. muytjensii (n ~ 1), and E. clearly indicated that partial 16S rDNA sequencing does not asburiae (n ~ 2) (Fig. 1 and Table 1; see also Fig. S1). allow for reliable species identification in Cronobacter. With the exception of two isolates that were clearly In contrast, use of a previously reported seven-gene identified as C. sakazakii by MLST data (FSL F6-045 MLST method (3) allowed for clear classification of the 30 representing sequence type (ST) 121 and F6-049 represent- isolates into well-supported clades (Fig. 1; also see Table S1 ing ST 4, the most common C. sakazakii type in the isolate for detailed MLST results). Phylogenetic analysis of the set described here), these species assignments were concatenated seven-gene MLST sequences specifically consistent with those based on the four biochemical tests revealed one C. sakazakii clade, which is distinct from a described by Iversen et al. (38). Initial characterization by well-supported clade (bootstrap support [BS] ~ 99) that 16S rDNA sequencing revealed that most Cronobacter includes the two C. malonaticus isolates (Fig. 1). The C. isolates matched type strains representing more than one muytjensii isolate (FSL F6-031) clearly forms a distinct Cronobacter species with the same 16S rDNA sequence clade in this tree. The 25 C. sakazakii isolates in our set similarity score. Specifically, seven C. sakazakii isolates had represented nine different STs; the two C. malonaticus J. Food Prot., Vol. 76, No. 1 ILSI NA CRONOBACTER COLLECTION 47 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/1/40/1685371/0362-028x_jfp-11-546.pdf by guest on 23 September 2021

FIGURE 2. Pulsed-field gel electrophoresis patterns for isolates in the ILSI NA Cronobacter isolate set. The dendrogram shown is based on 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 F6-026 and 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. isolates represented two additional distinct STs (Fig 1; also (FSL F6-032) was a food isolate. C. sakazakii isolate FSL see Table S1). The one C. muytjensii isolate (F6-031) could F6-045, which had a number of atypical phenotypic not be assigned an ST because both gltB and pps could not characteristics, was classified as ST 121, and FSL F6-049, be reproducibly amplified with the primers used (see Table which was identified by biochemical profiling as C. S1). Within the C. sakazakii group, ST 4 isolates (n ~ 14) malonaticus (Table 1), was identified as C. sakazakii ST were grouped in a well-supported clade (BS ~ 89), as were 4. These findings are consistent with those of Baldwin et al. ST 1 isolates (n ~ 3). Identification of a large number of ST (3), who also reported that phenotypic-based species 4 isolates, which are associated with neonatal meningitis identification of Cronobacter spp. can be inconsistent with (41, 42), is consistent with the MLST findings reported by sequence-based identification (3). Baldwin et al. (3), who found that 22 of 60 C. sakazakii Although MLST differentiated the 30 isolates in the ILSI isolates in their collection were ST 4. These authors also NA set into only 15 distinct STs, PFGE subtyping of these reported that ST 8 may represent a more virulent C. isolates resulted in 30 unique pulsotypes (Fig. 2). FSL F6- sakazakii ST because seven of the eight C. sakazakii with 049 and FSL F6-051 differed by only two bands, as did FSL ST 8 were clinical isolates; the single ST 8 isolate in our set F6-041 and FSL F6-046. However, FSL F6-041 and FSL F6- 48 IVY ET AL. J. Food Prot., Vol. 76, No. 1

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 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/1/40/1685371/0362-028x_jfp-11-546.pdf by guest on 23 September 2021 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 Cronobacter isolate 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 techniques, 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 FIGURE 3. EcoRI ribotype patterns for the 30 isolates in the ILSI molecular detection methods. Because this isolate set NA Cronobacter isolate set. Ribotypes were obtained with the includes only three of the seven known Cronobacter spp., automated RiboPrinter (Qualicon). Health Canada (four-digit some researchers may wish to add isolates representing the identifier) designations and Cornell University Food Safety remaining four Cronobacter species. Laboratory (FSL) designations are shown for each isolate. All ribogroups carry the prefix 235. For example ‘‘306-S-5’’ stands Cronobacter spp., when detected in foods such as PIF, for ‘‘235-306-S-5.’’ Ribogroups in the DuPont database are also are generally present at low levels (34, 61). Therefore, rapid, assigned a DUP designation. FSL F6-038 was not digested sensitive, and specific techniques are needed for detection of with EcoRI. Cronobacter spp. in food products such as PIF. In several recent studies, currently available enrichment or detection 046 had identical results in phenotypic tests (Tables 1 and 2), media have been evaluated (18, 27, 35) or new media for whereas F6-049 and FSL F6-051 had different biochemical Cronobacter enrichment have been developed (1, 11). results (Table 1). Ribotyping of these isolates produced 23 Development of new molecular detection techniques is unique ribotypes; one isolate was not digested with EcoRI ongoing (21, 54, 73, 79). Diversity among Cronobacter spp. (Fig. 3). Overall, these data indicate that the ILSI NA set may present a challenge for the design of new, effective detailed here includes a diverse group of isolates. enrichment and detection methods, and an accessible set of The E. sakazakii BAX PCR was performed on each of Cronobacter isolates could be used to validate the efficacy the isolates according to the manufacturer’s instructions. Of of these new techniques. the isolates confirmed to be Cronobacter by 16S rDNA The development of new, rapid, standardized, and sequencing and MLST, only C. sakazakii FSL F6-045 was economical molecular subtyping methods for Cronobacter BAX negative (Table 2). FSL F6-045 was also negative on spp. will enable public health agencies to quickly detect two selective media (Table 2), indicating that FSL F6-045 and identify sources of disease outbreaks (78). PFGE is has some characteristics that are atypical of C. sakazakii. considered the ‘‘gold standard’’ of molecular subtyping and Enterobacter isolates FSL F6-033 and FSL F6-026 were has been used successfully to distinguish among Crono- BAX negative as expected. Although all of the isolates in bacter isolates (4, 58). Although our data support that fact our collection grew in mLST broth, a small percentage of that PFGE allows discrimination among highly clonal J. Food Prot., Vol. 76, No. 1 ILSI NA CRONOBACTER COLLECTION 49 strains such as C. sakazakii ST 4, PFGE is expensive and set is funded by a grant from the ILSI NA to M.W. The opinions expressed time-consuming and can be difficult to standardize among herein are those of the authors and do not necessarily represent the views of the ILSI NA. Development of the PathogenTracker database was supported laboratories. The ILSI NA Cronobacter isolate set provides by U.S. Department of Agriculture special research grants 2002-34459- a valuable resource for assessing the discriminatory power 11758, 2003-34459-12999, and 2004-34459-14296 (to M.W.). of new Cronobacter subtyping techniques. A new genetic subtyping method would ideally be more economical, REFERENCES reproducible, and at least as discriminatory as PFGE (i.e., 1. Al-Holy, M. A., J. H. Shin, T. M. Osaili, and B. A. 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