Pan-genome analysis of the emerging foodborne pathogen Cronobacter spp. suggests a species-level bidirectional divergence driven by niche adaptation

Abstract

Background: Members of the genus Cronobacter are causes of rare but severe illness in neonates and preterm infants following the ingestion of contaminated infant formula. Seven species have been described and two of the species genomes were subsequently published. In this study, we performed comparative genomics on eight strains of Cronobacter, including six that we sequenced (representing six of the seven species) and two previously published, closed genomes.

Results: We identified and characterized the features associated with the core and pan genome of the genus Cronobacter in an attempt to understand the evolution of these bacteria and the genetic content of each species. We identified 84 genomic regions that are present in two or more Cronobacter genomes, along with 45 unique genomic regions. Many potentially horizontally transferred genes, such as lysogenic prophages, were also identified. Most notable among these were several type six secretion system gene clusters, transposons that carried tellurium, copper and/or silver resistance genes, and a novel integrative conjugative element.

Conclusions: Cronobacter have diverged into two clusters, one consisting of C. dublinensis and C. muytjensii (Cdub-Cmuy) and the other comprised of C. sakazakii, C. malonaticus, C. universalis, and C. turicensis, (Csak-Cmal-Cuni-Ctur) from the most recent common ancestral species. While several genetic determinants for plant-association and human virulence could be found in the core genome of Cronobacter, the four Cdub-Cmuy clade genomes contained several accessory genomic regions important for survival in a plant-associated environmental niche, while the Csak-Cmal-Cuni-Ctur clade genomes harbored numerous virulence-related genetic traits.

Figure 1

Evolutionary relationships of eight Cronobacter genomes. Neighbor-Joining phylogenetic tree based on 574,352 bp alignment of homologous sequences was computed using the Maximum Composite Likelihood method for nucleotide substitution. The bootstrap supports, as percentage, are shown next to the branches. The scale bar represents 0.01 base substitutions per site.

Figure 2

Gain and loss of putative genomic regions among eight Cronobacter genomes. Genomic regions (see Table 3 and Additional file 4: Table S2 for more details) are shown as present (+, shaded boxes) or absent (−, dashed borders), representing most likely point of gain or loss for each genome or group of genomes. The ancestral core comprises the current eight Cronobacter core genome (Additional file 3: Table S1) and GIs 1–34, 55–57, 67, 83. Underlined genomic regions are those whose current species distribution occurred through both insertions and deletions in the evolutionary history of Cronobacter.

Figure 3

Mobilome of Cronobacter spp. Putative mobilome elements from eight Cronobacter genomes were mapped on C. sakazakii ATCC BAA-894 chromosome, using the Microbial Genome Viewer [31]. Symbols on outside ring represent insertion sites of putative mobile genetic elements (Table 4, with mobile elements plotted clock-wise, from the top) – prophages, transposons and insertion sequences, and integrative-conjugative element-like elements (green circles), variable type six secretion system clusters (orange circles), genomic regions at insertion sites (red circles), and LPS (O-antigen and core OS) regions (yellow circles). Blue ring represents genes on the + strand and purple ring represents genes on the – strand. Middle rings represent COGs on + (outer) and – (inner) strands (COG functional category legend provided in lower right of figure). Innermost circle represent % G + C.