Like many bacterial genera, the taxonomy of Cronobacter has evolved and expanded as more sensitive molecular- and sequence-based tools have developed. In this study, we performed two genome-scale sequence analyses to discern the taxonomic relationships of extant Cronobacter species, namely ANI and genome-scale alignment and phylogenetic reconstruction using syntenic, orthologous chromosomal sequence. The taxonomic reclassification by Iversen et al.
, which relied on both DNA studies and on results from biochemical tests, was confirmed by both analyses. We found that the ANI results from this study were more meaningful in discerning relationships between pairs of Cronobacter species that are more distantly related as compared to DNA-DNA hybridization
. This is most likely a reflection of the differences in the range of meaningful values for each analysis.
We were able to confirm the presence or absence of eight of the genetic determinants of the biochemical characteristics used previously for Cronobacter biotyping (Table
3); namely, indole (tryptophanase), dulcitol (galactitol), malonate, myo-inositol, and two genomic regions that are likely responsible for utilization of 4-aminobutyrate and production of α-methyl glucoside (Farmer biotype 15
), as well as those biotyping traits contained in the core genome of these eight strains, utilization of palatinose and putrescine (Additional file
1: Table S1). The distribution of these operons and genes were in complete agreement with the biochemical results and species description reported by Iversen et al.
. Inositol fermentation has recently been proposed as a marker of pathogenicity for Cronobacter, based on the presence of the inositol monophosphatase gene (suhB) in pathogenic strains
. In this study, we found that this gene, which is seemingly ubiquitous and highly conserved among the Enterobacteriaceae, is a component of the Cronobacter core genome (Additional file
1: Table S1). Additionally, we found that the inositol utilization operon (GR29, Table
3, Additional file
2: Table S2) was present, and functional
, in the genomes of strains isolated from the environment (water sources), Cuni NCTC 9529T, Cdubdub LMG 23823T, Cdublac LMG 23825T, and absent in the genomes of pathogenic strains, Cmal LMG 23826T and Csak BAA-894.
Using comparative genomics, we were able to define the syntenic Cronobacter core genome for the eight species genomes analyzed in this study, which is approximately 77% of the total protein coding sequences, on average, per genome. This value is considerably higher as compared to the core genome content of other genera. In fact, the core genus genome size of Cronobacter is comparable to the core genome size of certain bacterial species
[33, 34], and considerably larger than that of the related E. coli. This is a reflection of the phylogenetic closeness of this genus, as shown in the ANI results (Table
2), and indicative of a more “closed” Cronobacter genome.
The core genome size is considerably higher than that reported by Kucerova et al.
, 1,899 genes, which incorporated four of the six strains used in this study. This discrepancy is best explained by the divergent evolution of the genus to form two distinct clades (Figure
1). This divergent evolution would undoubtedly have a significant impact on the efficiency of hybridization of probes designed from the sequence of Csak BAA-894 to DNA from strains of Cdub and Cmuy, resulting in the smaller reported core genome size
. With regard to the genomic regions revealed by the comparative genomic hybridization analysis of Csak BAA-894, reported by Kucerova et al.
, we classified 12 of the 15 reported genomic regions as part of the Cronobacter mobilome. They included T6SS clusters, GR1, 2 (ESA_0292-00302), 9, and 15 (repFIB plasmid-associated T6SS); putative prophages, GR3, 4, 6, 10, 11, 12; transposons, GR7, 14; and O-antigen gene clusters, GR5 (Additional file
2: Table S2, from
The comparably large core genome size and overall high sequence identity within the genus support the hypothesis that these two clades have evolved as a result of sympatric speciation; however, the divergence of the two clades indicates that they are under different evolutionary pressures, to some degree. In addition to the divergent evolution of shared genome content, several non-core genomic regions were found to be either present (or absent) in one of the two species-complex clades. For example, the Cdub-Cmuy clade has acquired 13 genomic regions that are not present in the Csak-Cmal-Cuni-Ctur clade (Figure
An examination of the dispensable gene content of each species provides clues as to differences in environmental niches and pathogenicity, such as virulence potential. Among putative virulence-related properties, the presence and diversity of appendages within the Cronobacter genus is intriguing. Many causative agents of meningitis, such as Neisseria meningitidis, Haemophilus influenzae, and Pseudomonas aeruginosa encode for type IV pili (TFP), necessary to colonize in the face of shear forces of blood flow, associated with the capillary beds of the blood–brain barrier. The Cronobacter core genome also contains the genes that encode a TFP. Bioinformatically, the presence of a TFP, as opposed to a T2SS, was hypothesized due to the presence of the TFP-unique gene, pilT, and the genetic organization of the pilQ loci, similar to the TFP of P. aeruginosa. Several chaperone-usher fimbriae were also present in the core genome. Additionally, seven other chaperone-usher fimbriae were differentially distributed among the eight genomes analyzed in this study (Table
3), including a P pilus homologue (GR9, missing in Csak BAA-894), which is a prominent virulence factor of uropathogenic E. coli, also a causative agent of neonatal meningitis. Csak BAA-894 and Cmal LMG 23826T harbor a unique type I fimbriae (GR82), which is absent in the other genomes, and Csak BAA-894 also harbors a β class CU fimbriae (GR76), shared with Cdublac LMG 23825T, and a second, unique type I fimbriae (GR126, which corresponds to GR8 of Kucerova et al.
). Of interest is the finding that Cmuy ATCC 51329T possesses only one additional fimbriae operon, in addition to those encoded in the genus core genome. Some genomes also harbored curli biosynthesis genes, homologous to curli of E. coli and tafi fimbriae of Salmonella. Although implicated directly in cell-cell contact and biofilm formation, these organelles likely contribute to the colonization of Cronobacter, after initial cell attachment has taken place. We hypothesize that this operon was a component of the ancestral core, and has been lost in all strains of C. sakazakii and C. muytjensii (data not shown).
In addition to appendages potentially involved in adhesion, several type V(a), or autotransporter, secretion loci are present in the genomes of the Cronobacter analyzed in this study, which are annotated as hemolysin, adhesin, outer membrane autotransporter barrel, filamentous hemagglutinin, large exoproteins, etc. They are found as accessory genomic regions (GRs 4, 16, 21, 50, 101, 123), and present as single genes or pairs of genes in the core genome (Additional file
2: Table S2). Of particular interest is GR123, found exclusively in Csak BAA-894, and GR118, found in the three routinely isolated pathogenic species, Ctur, Cmal, and Csak. GR123 contains two putative invasins and an eae homologue and may constitute a pathogenicity island. This region was found to be present in the genomes of three neonatal intensive care unit (NICU) outbreak strains (including Csak BAA-894) and absent in the C. sakazakii type strain, ATCC 29544, which was isolated from a child’s throat (defined as a component of cluster 3, Additional file
1: Table S1, from
Also interesting is the presence of two genomic regions (GRs 127 and 129) involved in the utilization of sialic acid in the genome of Csak BAA-894. Sialic acid is a generic term for a family of derivatives of the nine carbon sugar acid, neuraminic acid, which are found at surface-exposed end positions of eukaryotic, primarily animal, tissues. Many pathogens have evolved to either coat their surfaces with sialic acid derivatives, in order to evade the innate immune response, or to use this biopolymer as a nutrient source
. In addition to Csak BAA-894, we found that 55 out of 57 strains of C. sakazakii are able to utilize N-acetyl-neuraminic acid, a derivative of sialic acid (by Biolog PM Microarray, data not shown). Conversely, no other Cronobacter strains were able to utilize this substrate, except four of six C. turicensis strains (data not shown).
It has been hypothesized that the environmental niche of Cronobacter is as a plant commensal
. Accordingly, we found several genomic features, both in the Cronobacter core- and pan-genome, which would be beneficial for an organism to possess in this habitat. For example, the Cronobacter core genome contains the maltose transporter operon, malGFE- malKlamBmalM, repressor, malT, and α-glucosidases that can hydrolzye maltose to two glucose molecules. Maltose is primarily restricted to plants, particularly seed tissues. An operon for the transport and hydrolysis of isomaltulose is also present in the core genome of Cronobacter, in agreement with the taxonomic description of Iversen et al.
, and previously reported by Lehner et al.
. Isomaltulose (palatinose), also used in the original Cronobacter biotyping scheme, is a disaccharide of glucose and fructose and a component of honey and sugar cane. Additionally, we found the following characteristics in the core Cronobacter core genome: utilization of arabinogalactan, a major component of plant gums; transport and utilization of xylose, a precursor to hemicellulose; galacturonate, the principal component of pectin; albicidin, a phytotoxin of Xanthomonas spp., resistance; β-carotene pigmentation, and several α and β glucosidases.
It is of interest to find that an albicidin resistance protein coding gene was found as a core genome component. Albicidin is a bacteriocin-like molecule that degrades DNA gyrase, both of bacterial and chloroplast origins
. Speculatively, Cronobacter possessing a gene promoting resistance to the action of albicidin adds further evidence for a plant-associated evolutionary history, as well as, the impartation of a competitive edge to Cronobacter survival in a mixed organism environment where competition is controlled through the action of bacteriocin expression. In addition to these conserved features, several other genomic regions and operons were found that have putative functions for plant association, or homologies to proteins from plant commensals. These include GR95/117 of Cmuy ATCC 51329 and Cuni NCTC 9529; GR70, metabolism of pyroxidine/pyroxidal (vitamin B6), of which green plants and grains and nuts contain high amounts; GR72, maltose derivative metabolism; GR73, galactose (glycoside-pentoside-hexuronide) homologue permease (possible role in cellulose degradation); GR92, mannanase; GR102, L-rhamnose ABC transporter; and GR107 of Ctur z3032.
Several inherent properties of Cronobacter have been proposed as mechanisms that aid the bacteria in survival and persistence in dried foods, such as PIF, food powders, and spices. Chief among these have been enhanced heat resistance, as compared to other enterics and contaminating microorganisms. However, most studies have reported variable results in terms of heat resistance at the strain level, and cross-tolerance to other environmental stressors, such as pH and water activity. One consistent finding is an unusually high resistance to dry stress
. Accordingly, we found several genomic determinants, which would be beneficial in a dry or low water activity environment, including cellulose biosynthesis (bcs and yhj) operons, colanic acid EPS, capsular biosynthesis operon (kps), an environmental persistence capsule (yih, GR3), and curli (GR55). Recently, it has been reported that the synergistic expression of the yih operon encoded capsule, cellulose and curli or tafi provides resistance to desiccation stress in Salmonella[40, 41]. We hypothesize that the same genetic determinants, combined with other capsular and EPS operons, likely play a similar role in the environmental persistence and desiccation resistance in Cronobacter. Although not all Cronobacter produce curli, those species possess several other fimbriae which could substitute in this adhesin and/or scaffolding role. In addition to this extracellular matrix, we also found two operons, present in all Cronobacter genomes, that encode transporters involved in osmoprotection, yeh and bet operons, with homology to plant commensals and pathogens, such as Burkholderia and Erwinia species.