Genome sequence of Cronobacter sakazakii BAA-894 and comparative genomic hybridization analysis with other Cronobacter species

Abstract

Background: The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified.

Methodology/principal findings: We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10-17% absence of genes.

Conclusions/significance: CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.

Figure 1. Whole genome clustering analysis showing the relationship between 10 Cronobacter isolates.

Genomic regions GR1–GR15 are marked. Clustering analysis was performed using Gene Cluster (EisenSoftware). Hierarchical clustering was performed using the average linkage method on the trinary matrix based on the CGH analysis (1 for presence, 0 for uncertain and −1 for absence/divergence of a gene). For description of strains refer to Table 1.

Figure 2. Distribution of the 15 variable regions across the C. sakazakii BAA-894 genome.

Each column represents a gene classified as absent by CGH analysis in at least one strain. The height of the columns indicate the number of strains (out of 10) in which the gene was found to be absent. The major variable regions (blue) and prophages (red) are indicated in order of their appearance in the genome of C. sakazakii BAA-894.

Figure 3. Gene maps of putative prophages.

A. Gene map of putative prophage 1. B. Gene map of putative prophage 2. C. Gene map of putative prophage 3. Annotation of the putative prophage genes is available in Table S2.