Enterobacter Sakazakii)
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International Journal of Food Microbiology 136 (2009) 159–164 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro Microarray-based comparative genomic indexing of the Cronobacter genus (Enterobacter sakazakii) B. Healy a, S. Huynh b, N. Mullane a, S. O'Brien a, C. Iversen a, A. Lehner c, R. Stephan c, C.T. Parker b,⁎, S. Fanning a,⁎ a Centres for Food Safety and Food-borne Zoonomics, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland b Produce Safety and Microbiology Research Unit, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA c Institute for Food Hygiene and Safety, VetSuisse Faculty, University of Zurich, Zurich CH-, Switzerland article info abstract Keywords: Cronobacter (Enterobacter sakazakii) is a recently defined genus consisting of 6 species. To extend our Microarray understanding of the genetic relationship between Cronobacter sakazakii BAA-894 and the other species of Comparative this genus, microarray-based comparative genomic indexing (CGI) was undertaken to determine the Genome presence/absence of genes identified in the former sequenced genome and to compare 276 selected open Cronobacter reading frames within the different Cronobacter strains. Seventy-eight Cronobacter strains (60 C. sakazakii, Enterobacter sakazakii 8 C. malonaticus,5C. dublinensis,2C. muytjensii,1C. turicensis,1C. genomospecies 1, and 1 Cronobacter sp.) representing clinical and environmental isolates from various geographical locations were investigated. Hierarchical clustering of the CGI data showed that the species grouped as clusters. The 5 C. dublinensis and 2 C. muytjensii strains examined formed distinct species clusters. Moreover, all of the C. sakazakii and 3 of 8 C. malonaticus strains formed a large cluster. The remaining C. malonaticus strains formed a sub-group within a larger cluster that also contained C. turicensis, C. genomospecies 1, and an unknown Cronobacter sp. Cronobacter sakazakii and 3 of 8 C. malonaticus strains could be distinguished from the others within the collection by the presence of 10 fimbrial related genes. Similarly, capsule and/or lipopolysaccharide (LPS) related glycosyltransferases differentiated several of the C. sakazakii strains from each other. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Microarray technology facilitates the rapid analysis of an orga- nism's genome. Array-based comparative genomic indexing (CGI) has Cronobacter, a recently described genus currently consists of 6 previously been employed to examine the genetic content of a different species; C. sakazakii, C. malonaticus, C. dublinensis, collection of clinical Campylobacter jejuni strains and to assist in the C. muytjensii, C. turicensis and C. genomospecies 1(Iversen et al., identification of distinguishing features within their genomes linked 2008). This recently defined nomenclature is the result of a polyphasic to disease outcomes (Parker et al., 2007; Quinones et al., 2008). In the taxonomic investigation aimed at re-defining this group of organisms present study, microarray-based genome indexing with an array (Iversen et al., 2007, 2008). Cronobacter spp. are described as consisting of 276 ORFs was applied to 78 well-characterized Crono- opportunistic pathogens, causing bacteremia, necrotizing enterocoli- bacter isolates cultured from food, environmental and clinical sources. tis (NEC) and meningitis in immunocompromised neonates (Mullane Bacterial gene targets included most of the functional categories within et al., 2007). More recently, it has also emerged that Cronobacter spp. the genome C. sakazakii strain BAA-894 (http://genome.wustl.edu/ may cause infections among immunocompromised adults, in parti- genome.cgi?GENOME=Enterobacter%20sakazakii&SECTION=assem- cular the elderly (Gosney et al., 2006; See et al., 2007). Due to their blies). These data extend the early characterization of these opportu- ubiquitous nature, Cronobacter spp. have been isolated from a wide nistic pathogens. variety of foods (Baumgartner et al., 2009-this issue; Friedemann, 2007; Gurtler et al., 2005). While the primary reservoir of Cronobacter has yet to be defined, plant material is believed to be the likely source 2. Materials and methods (Lehner, 2009). 2.1. Bacterial strains and growth ⁎ Corresponding authors. Fanning is to be contacted at Tel.: +353 7166096; fax: All Cronobacter strains (Table 1) were cultured on Colombia blood +353 7166091. Parker, Tel.: +1 510 559 6187; fax: +1 510 559 6162. − E-mail addresses: [email protected] (C.T. Parker), [email protected] agar at 37 °C. Bacterial strains were frozen at 80 °C for long time (S. Fanning). storage. 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.07.008 160 B. Healy et al. / International Journal of Food Microbiology 136 (2009) 159–164 − Table 1 ethidium bromide (10 mg ml 1). Two-hundred and seventy-six PCR Cronobacter strains used in this study. products ranging from 200 to 1000 bp were amplified from Clinical Environmental Food Unknown Water C. sakazakii strain BAA-894. All amplicons were purified on a Qiagen C. sakazakii 8000 robot using a Qiaquick 96-well Biorobot kit (Qiagen, Valencia, Canada E604, E654, CA), dried, and resuspended to an average concentration of 0.1–0.2 μg E655a, E655b, µl− 1 in 20 μl 50% dimethyl sulfoxide (DMSO) containing 0.3× saline- E656, E657, E880 sodium citrate (SSC, 1× SSC; 150 mM NaCl, 15 mM sodium citrate). All Czech E755, E756 Republic of the PCR amplicons were subsequently spotted in triplicate on Denmark E770 UltraGAPS slides (Corning, Lowell, MA) with an OmniGrid Accent France E834, E835, E836, E292, E772, (GeneMachines, Ann Arbor, MI) producing a final array that contained E837, E838, E839, E857, E859 a total of 276 features. The DNA was UV cross-linked to the microarray E840, E841, E842, slides with a Stratalinker at 300 mJ (Stratagene, La Jolla, CA). E843, E844, E845, E848, E849, E851, E852, E853 2.3. Preparation and fluorescent labeling of genomic DNA Germany E540 Holland E784, E787, E886 E305 Genomic DNA from 78 well-characterized Cronobacter strains was Ireland CFS101, CFS155, CFS109 ™ CFS174, CFS1001 isolated by using the Qiagen Dneasy kit (Qiagen, Valencia, CA) Malaysia E272, E274 according to the manufacturer's specifications. For each microarray New Zealand 254N hybridization reaction, genomic DNAs from the reference strain Russia E775 (C. sakazakii strain BAA-894) and a test strain were fluorescently UK E736 labeled with indodicarbocyanine (Cy5)-dUTP and indocarbocyanine Unknown E602 μ μ Uruguay E903 (Cy3)-dUTP, respectively. An aliquot (2 g) of DNA was mixed with 5 l USA ATCC29544, E823, E632, E821, E830 10× NEBlot labeling buffer containing random sequence octamer E824, E827, E828, E934, E935 oligonucleotides (NEB, Beverly, MA) and water to a final volume of E829, E893, E901 41 μl. This mixture was heated to 95 °C for 5 min and then cooled for C. malonaticus 5 min on at 4 °C. After cooling, the remainder of the labeling reaction Australia E766 components were added: 5 μl of 10× dNTP labeling mix (1.2 mM each of Czech E608 dATP, dGTP,dCTP; 0.5 mM dTTP in 10 mM Tris [pH 8.0]; 1 mM EDTA), 3 μl Republin Cy3 dUTP (25 nmol) or Cy5 dUTP (25 nmol) (GE Biosciences, Piscataway, Germany E532 NJ) and 1 μl Klenow fragment of DNA polymerase I (5000 U/ml). The Ireland CFS28 CFS10 338 Unknown E685 labeling reactions were incubated overnight at 37 °C. Labeled DNA was USA E825 purified from unincorporated label using Qiaquick PCR Cleanup kits (Qiagen, Valencia, CA) and dried by vacuum. C. dublinensis Ireland E930 UK E798 2.4. Microarray hybridization USA E791 Switzerland E515 Labeled reference and test DNAs were combined in 45 μl Pronto Zimbabwe E464 cDNA hybridization solution (Corning, Corning, NY) and heated to 95 °C for 5 min. Fifteen microliters of the hybridization mixture was pipetted C. muytjensii France E603 directly onto a microarray slide and sealed with a coverslip. The USA E793 microarray slide was then placed in a hybridization chamber (Corning) and incubated at 42 °C for 18 h. Following the hybridization step, slides C. genomospecies 1 were washed twice in 2× SSC, 0.1% [w/v] sodium dodecyl sulfate at 42 °C UK E797 for 10 min, followed by two washings in 1× SSC at room temperature for C. turicensis 10 min, and finally twice in 0.2× SSC at room temperature for 5 min. The Switzerland E866 microarray slides were dried by centrifugation at 300 ×g for 10 min before scanning. At least two hybridization reactions were performed for each test strain to ensure reproducibility. 2.2. Construction of the Cronobacter DNA microarray 2.5. Microarray data analysis DNA fragments of individual ORFs identified based on the genome Microarrays were scanned and analyzed as previously described sequence of C. sakazakii BAA-894 were amplified by using primers (Parker et al., 2006, 2007). DNA microarrays were scanned using an designed with ArrayDesigner 3.0 (Premier Biosoft, Palo Alto, CA) and Axon GenePix 4000B microarray laser scanner (Axon Instruments, Inc. purchased from MWG Biotech Inc. (High Point, NC). Each PCR reaction Union City, CA). Features and the local background intensities were (total reaction volume, 100 μl) consisted of 1× MasterAmp Taq PCR detected and quantified with GenePix 4.0 software (Axon Instruments, buffer, 1× MasterAmp Taq Enhancer, 2.5 mM MgCl2,200μM each Inc.). Poor features were excluded from further analysis if they contained dNTP, forward and reverse primers at 0.2 μM each, 0.5 U of MasterAmp abnormalities or were within regions of high fluorescent background. Taq DNA polymerase (Epicentre, Madison, WI), and approximately These data were filtered so that spots with a reference signal lower than 50 ng of genomic DNA from C. sakazakii strain BAA-894. Thermal the background plus 2 standard deviations of the background were cycling was performed with a Tetrad thermal cycler (Bio-Rad, discarded.