Comparative analysis of genome sequences covering the seven cronobacter species

Abstract

Background: Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages.

Methodology/principal findings: We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes.

Conclusions/significance: Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types.

Figure 1. Phylogentic analysis of Cronobacter spp. based on whole genome sequencing.

(A) A maximum likelihood phylogram based on 138,143 SNPs identified from a concatenated alignment of 1117 core genes (879,768 nucleotides) present in single copies in all strains. The numbers on the nodes represent the bootstrap support from 1000 replicates (in percentage). (B) A majority rule consensus tree generated by summarizing individual 1117 core gene trees. The numbers on the internal nodes indicate the fraction (in percentage) of gene trees which support the partition of the taxa into the two sets. (C) A Clonal Frame (CF) analysis based on randomly selected 99 core loci. The consensus tree was obtained from 2 CF runs with 200,000 MCMC iterations. The numbers on the nodes represent the bootstrap support in percentage.

Figure 2. Maximum Likelihood tree based on the concatenated sequences (3,036 bp) of the seven MLST loci.

The tree is drawn to scale using MEGA, with 1000 bootstrap replicates.

Figure 3. Graph representing the rarefaction of the Cronobacter pan genome, generated using the 14 Cronobacter genomes included in this study.