Genotypic and phenotypic data was collected for 282 strains of Enterobacteriaceae, including 189 E. sakazakii isolates and 39 other α-glucosidase positive strains. Strains were from diverse food, clinical and environmental sources worldwide. Clinical isolates were from cases occurring over the last 25 years. At least one original strain from each of the biogroups described when the E. sakazakii species was designated were included [7].
Phenotypic data
Biochemical characteristics were derived from commercial test kits (API 20E and ID32E, bioMérieux UK Ltd.; Biolog GN2, Biolog, CA; and Microbact 24E, Oxoid UK Ltd.) and conventional manual tests as per standard protocols. Tests were performed in triplicate on separate days. Motility was determined at 37°C after 24 h and 48 h using motility medium (tryptose 10 g l-1, NaCl 5 g l-1, agar 5 g l-1, pH 7.2 ± 0.2. Acid production from carbohydrates was tested in phenol red broth base (10 g l-1 peptone, 1 g l-1 yeast extract, 5 g l-1 NaCl, 0.018 g l-1 phenol red) with addition of filter-sterilized carbohydrate solution (final concentration 0.5%). Gas production was determined by collection in Durham tubes. The methyl red test was performed at 48 h on cultures grown in MR-VP broth (VWR, 1.05712.0500). The Voges-Proskauer test was performed at 24 h by addition of 40% potassium hydroxide in water and 5% 1-naphthol in ethanol to cultures grown in MR-VP broth. Indole production was measured at 24 h by addition of Kovacs reagent (5 g p-dimethylaminobenzaldehyde, 25 ml HCl, 75 ml pentanol-1-ol) or James Reagent (70542 bioMérieux) to cultures grown in Peptone Water (CM0009 Oxoid Ltd). Nitrate reduction was measured by addition of 1% sulphanilamide in 1 M HCl and 0.02% N-1 naphthylene diamine HCl in water. Zinc dust was added to negative tubes to confirm the presence of unreduced nitrate. Constitutive metabolism of X-α-glucoside was determined by formation of blue-green colonies on media containing 5-bromo-4-chloro-3-indolyl-α, D-glucopyranoside (Chromogenic Enterobacter sakazakii medium (DFI formulation) CM1055, Oxoid Ltd.; and ESIA, AES Laboratoire, France).
Comparative 16S rDNA sequencing
This was performed by Accugenix (Newark, DE, USA) using the MicroSeq™ 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems). DNA was prepared for PCR by quick-heat lysis by removing one colony into a tube of PrepMan Ultra™ (Applied Biosystems) and placed at 99°C for 10 min. Two microlitres of genomic DNA was amplified in 50 μl of a master mixture consisting of 0.4 μM TGGAGAGTTTGATCCTGGCTCAG and TACCGCGGCTGCTGGCAC primers, 200 mM deoxynucleoside triphosphates, PCR buffer, 0.3 U of AmpliTaq DNA polymerase, and 10% glycerol. PCR conditions were 95°C for 10 min; 30 cycles each of 95°C for 30 s, 60°C for 30 s, and 72°C for 45 s; and a final step at 72°C for 10 min. Purification of the PCR product to remove excess primers and nucleotides was performed using Montage SEQ96 filter plates (Millipore). Cycle sequencing was performed with the sequencing module, and after removal of excess dyes using Montage SEQ96 filter plates (Millipore), the labelled extension products were separated on an ABI 3100 16 capillary genetic analyzer (Applied Biosystems). Partial sequencing was performed for all isolates, the length of the partial rDNA was 528 nucleotides, and in addition the full sequence for the E. sakazakii type strain (NCTC 11467) was obtained.
The data was analysed using Bionumerics (Applied Maths, Belgium) to construct Neighbour Joining trees, bootstraps were derived from 1000 replicates and the Jukes-Cantor correction applied.
The full 16S sequence was used for the representation of the secondary structure of the small subunit ribosomal RNA of E. sakazakii NCTC 11467. Nucleotide numbering follows the Reference Numbering System used for E. coli J01695 [15].
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