The biochemical profiles obtained for each strain were compared to the biogroups originally described by Farmer et al 5. Clinical strains were distributed among fourteen of the biogroups (Table 1). The defining tests were motility, Voges-Proskauer, methyl red, indole, ornithine decarboxylase, reduction of nitrate to nitrite, production of gas from D-glucose, malonate utilization and production of acid from myo-inositol and dulcitol. Where strains could not be assigned to an original biogroup, a new biogroup or subgroup was designated (Table 2). The majority of isolates (60/189) were in Biogroup 1 with the E. sakazakii ATCC 29544^T type strain. These strains were motile, produced gas from glucose, produced acid from inositol, reduced nitrate and were positive for Voges-Proskauer and ornithine decarboxylase, but negative for methyl red, indole, malonate utilization and acid production from dulcitol. Biogroup 2 (n = 42) contained isolates negative for acid production from inositol; four of these were also non-motile. Biogroup 3 (non-motile) contained six strains and Biogroup 4 (ornithine negative) contained nine strains three of which were non-motile. Biogroup 5 (n = 16) was positive for malonate utilization and six of these were non-motile. Biogroup 6 (n = 2) was positive for indole and Biogroup 7 (n = 4) was negative for gas production from glucose. Biogroup 8 (n = 7) was defined by the inability to reduce nitrate. Two of these 7 strains were positive in the malonate test, three were negative for the inositol test and two were inositol negative but malonate positive. Biogroup 9 contained 13 strains that were inositol negative and malonate positive. Biogroup 10 contained one strain that was inositol negative and indole positive, while Biogroup 11 contained one strain that was inositol negative and dulcitol positive. Biogroup 12 was also represented by only one strain, which was indole and malonate positive. The seven isolates in Biogroup 13 were negative for the Voges-Proskauer reaction, three were non-motile, one was negative for methyl red and one was negative for ornithine. Biogroup 14 (n = 5) was negative for ornithine decarboxylase and inositol, with four of these strains being positive for malonate. Biogroup 15 was positive for all the tests performed except methyl red. A new group (Biogroup 16) had to be defined to accommodate 9 strains which were inositol and dulcitol positive, but indole negative. They were malonate positive, with the exception of one strain. Two strains were non-motile and one of these was also ornithine decarboxylase negative. Acid production from α-methyl-D-glucoside was included in the original study 1 and all biogroups were reported positive for this trait with the exception of Biogroup 15. As Biogroup 15 could be distinguished from the other biogroups without the α-methyl-D-glucoside test, this was not repeated for all strains in this study.

Partial 16S rDNA sequences (528 bp) containing less than 1% undetermined positions were obtained for all strains in this study. The sequences were analysed using the maximum parsimony method, which is an evolutionary model that searches for the simplest tree that can be constructed using the fewest inferred changes between characters. The topology was optimised using simulated annealing, a heuristic that occasionally accepts a worse tree during the course of the search allowing it to escape local optima. This method is more economical than the more usual heuristic searches (stepwise addition and hill-climbing), which can require many randon re-starts, especially with large data matrices 12. In agreement with previous reports 10, the isolates used in this study were divided into four genomic groups by 16S rDNA sequence analysis (Fig 1). The majority (170/189) of the presumptive E. sakazakii isolates in this study clustered with the type strain (Fig. 1E. sakazakii cluster 1). Cluster 1 was composed of the majority of biogroups, Biogroups 1–5, 7–9, 11, 13 and 14. The 9 isolates forming the new proposed Biogroup 16 corresponded with E. sakazakii cluster 2 (Fig. 1 cluster 2), with between 1.23–1.89% 16S rDNA sequence difference from the E. sakazakii type strain. No strains representative of this biochemical profile were included in the original study by Farmer et al. 5.

The six isolates in Biogroup 15 corresponded to E. sakazakii cluster 3 (Fig. 1 cluster 3). The four strains described as E. sakazakii cluster 4 (Fig. 1 cluster 4) represent Biogroups 6, 10 and 12. For this dataset, the four partial 16S rDNA cluster groups can be distinguished biochemically using the indole, dulcitol, and inositol tests (Table 4). Cluster 1 strains are variable for inositol, negative for indole, and dulcitol; with the exception of Biogroup 11, which is dulcitol positive and inositol negative. Cluster 2 – Biogroup 16 strains are positive for inositol and dulcitol but negative for indole. Cluster 3 – Biogroup 15 is positive for inositol, dulcitol and indole. Cluster 4 strains were also positive for indole but can be distinguished from Cluster 3 as they are negative for dulcitol. One of the strains in Cluster 4 was inositol negative (Biogroup 10) and one was malonate positive (Biogroup 12). There were insufficient isolates in Cluster 4 biogroups to determine whether these biogroups could be further genomically divided.

A total of 189 E. sakazakii strains were analyzed in this study. Strains were from diverse food, clinical and environmental sources worldwide. The origin distribution of the isolates is given in Table 1. At least one strain from each of the biogroups described when the E. sakazakii species was designated were included and representative strains for each biogroup are given in Table 3. The type strain (NCTC 11467^T) was purchased from the NCTC, London, UK, as were strains NCTC 9844, NCTC 9238 and NCTC 9846. Strain NCIMB 8272 was purchased from the NCIMB, Aberdeen, Scotland. Eleven strains, CDC 5960-70, 1895-73, 0743-75, 3523-75, 9363-75, 9369-75, 0407-77, 0996-77, 1058-77, 1716-77, and 3128-77 were donated by the CDC, Atlanta, GA, USA. Strains were also kindly donated by the following: – Nestlé Research Center, Lausanne, Switzerland; Health Products and Food branch, Health Canada; Children's Hospital Los Angeles CA, USA; Northern Foods, UK; Oxoid Ltd., Basingstoke, UK; Hospital Cèské Budéjovice, Czech Republic; Institut fûr Tierärztliche Nahrungsmittelkunde Milchwissenschaften, Justus-Liebig-Universität Gießen, Germany; Nottingham City Hospital Trust, Nottingham, UK and the Department of Medical Microbiology, Radboud, Nijmegen, Netherlands. All other strains were food and environmental isolates from the culture collection at Nottingham Trent University, Nottingham, UK.

In addition to phenotypes derived from commercial biochemical test kits (Microbact GN2, Oxoid; API 20E and ID32E, BioMérieux) according to manufacturers' recommendations, the following tests were performed using conventional manual methods. Motility was determined at 37°C after 24 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 the addition of filter-sterilized carbohydrate solution (final concentration 0.5%). Gas production from D-glucose was determined by collection in Durham tubes. Malonate utilization was determined using sodium malonate broth (sodium malonate 3 g l^-1, ammonium sulphate 2 g l^-1, bromothymol blue 0.025 g l^-1, NaCl 2 g l^-1, yeast extract 1 g l^-1, dipotassium hydrogen phosphate 0.6 g l^-1, potassium dihydrogen phosphate 0.4 g l^-1). The methyl red test was performed by addition of indicator (0.1 g methyl red per 300 ml 95% ethanol) to cultures grown for 48 h in 4 ml of MR-VP broth (VWR, 1.05712.0500). The Voges-Proskauer test was performed by the addition of 40% potassium hydroxide in water and 5% 1-naphthol in (95% ethanol) to cultures grown for 24 hours in MR-VP broth. Indole production was measured by the addition of Kovacs reagent (5 g p-dimethylaminobenzaldehyde, 25 ml HCl, 75 ml pentanol-1-ol) or James Reagent (70542 BioMérieux) to cultures grown for 24 h in peptone water (CM0009 Oxoid). Nitrate reduction to nitrite was determined by the addition of 1% sulphanilamide in 1 M HCl and 0.02% N-1 naphthylene diamine HCl in water to nitrate test broths. Zinc dust was added to negative tubes of nitrate-negative strains to confirm the presence of unreduced nitrate.

Comparative 16S rDNA sequencing (528 bp) was performed as previously described 11 by Accugenix (Newark, DE, USA) using the MicroSeq™ 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems).

A maximum parsimony tree (Fig. 1) of 16S rDNA sequences was generated using Bionumerics (Applied Maths, Belgium). Gaps were not considered an extra state and the topology was optimized using simulated annealing.