Pan-genome diversification and recombination in Cronobacter sakazakii, an opportunistic pathogen in neonates, and insights to its xerotolerant lifestyle

Abstract

Background: Cronobacter sakazakii is an emerging opportunistic bacterial pathogen known to cause neonatal and pediatric infections, including meningitis, necrotizing enterocolitis, and bacteremia. Multiple disease outbreaks of C. sakazakii have been documented in the past few decades, yet little is known of its genomic diversity, adaptation, and evolution. Here, we analyzed the pan-genome characteristics and phylogenetic relationships of 237 genomes of C. sakazakii and 48 genomes of related Cronobacter species isolated from diverse sources.

Results: The C. sakazakii pan-genome contains 17,158 orthologous gene clusters, and approximately 19.5% of these constitute the core genome. Phylogenetic analyses reveal the presence of at least ten deep branching monophyletic lineages indicative of ancestral diversification. We detected enrichment of functions involved in proton transport and rotational mechanism in accessory genes exclusively found in human-derived strains. In environment-exclusive accessory genes, we detected enrichment for those involved in tryptophan biosynthesis and indole metabolism. However, we did not find significantly enriched gene functions for those genes exclusively found in food strains. The most frequently detected virulence genes are those that encode proteins associated with chemotaxis, enterobactin synthesis, ferrienterobactin transporter, type VI secretion system, galactose metabolism, and mannose metabolism. The genes fos which encodes resistance against fosfomycin, a broad-spectrum cell wall synthesis inhibitor, and mdf(A) which encodes a multidrug efflux transporter were found in nearly all genomes. We found that a total of 2991 genes in the pan-genome have had a history of recombination. Many of the most frequently recombined genes are associated with nutrient acquisition, metabolism and toxin production.

Conclusions: Overall, our results indicate that the presence of a large accessory gene pool, ability to switch between ecological niches, a diverse suite of antibiotic resistance, virulence and niche-specific genes, and frequent recombination partly explain the remarkable adaptability of C. sakazakii within and outside the human host. These findings provide critical insights that can help define the development of effective disease surveillance and control strategies for Cronobacter-related diseases.

Keywords: Accessory genome; Antibiotic resistance; Core genome; Cronobacter sakazakii; Pan-genome; Recombination.

Fig. 1

Pan-genome structure and phylogeny of C. sakazakii. a Distribution of pairwise ANI values. b The number of unique genes that are shared by any given number of genomes or unique to a single genome. Numerical values for each gene category are shown in Additional file 6: Table S3. c The size of the core genome (purple line) and pan-genome (green line) as more genomes are added. The list of core genes is listed in Additional file 7: Table S4. d The number of unique genes, i.e., genes unique to individual strains (orange line) and new genes, i.e., genes not found in the previously compared genomes (light blue line) as more genomes are added. e Gene presence-absence matrix showing the distribution of genes present in each genome. Each row corresponds to a branch on the tree. Each column represents an orthologous gene family. Dark blue blocks represent the presence of a gene, while light blue blocks represent the absence of a gene. The phylogeny reflects clustering based on presence or absence of accessory genes. The colors on the tip of each branch reflect the BAPS clustering. f Contour plots of pairwise distances between genomes in terms of their core genome divergence (measured by SNP density distance across the core genome) and the difference in their accessory genomes (measured by the Jaccard distance based on the variation in the gene content of their sequences) calculated using popPUNK [24]. g The midpoint-rooted maximum likelihood phylogenetic tree was calculated using sequence variation in the core genome alignment. Outer rings show the BAPS cluster, geographical origin, and ecological source. Scale bar represents nucleotide substitutions per site

Fig. 2

Distribution of antibiotic resistance and virulence genes in C. sakazakii and related species. Columns and gene names are colored according to related functions, except for those with distinct functions (colored in green). The midpoint-rooted maximum likelihood phylogenetic tree was calculated using sequence variation in the core genome alignment of the entire genus (n = 1942 genes). Scale bar represents nucleotide substitutions per site

Fig. 3

Recombination in C. sakazakii. (a) A phylogenetic network of the core genome generated using SplitsTree. Scale bar represents nucleotide substitutions per site. Colored dots represent BAPS clusters and are identical to those in Fig. 1a. (b) Correlation profile (circles) calculated from the core genomic alignment by mcorr. Model fit is shown as a solid line. (c) Frequency histograms showing the distributions of the three recombination parameters for all pairs of genomes. The red vertical lines indicate the means. (d) Genes that have undergone recent or ancestral recombination. Horizontal axis shows the estimated number of ancestral recombinations, and vertical axis shows the estimated number of recent recombinations. For visual clarity, names of some of the genes with known function are shown