Genomic epidemiology and global diversity of the emerging bacterial pathogen Elizabethkingia anophelis

Abstract

Elizabethkingia anophelis is an emerging pathogen involved in human infections and outbreaks in distinct world regions. We investigated the phylogenetic relationships and pathogenesis-associated genomic features of two neonatal meningitis isolates isolated 5 years apart from one hospital in Central African Republic and compared them with Elizabethkingia from other regions and sources. Average nucleotide identity firmly confirmed that E. anophelis, E. meningoseptica and E. miricola represent demarcated genomic species. A core genome multilocus sequence typing scheme, broadly applicable to Elizabethkingia species, was developed and made publicly available (http://bigsdb.pasteur.fr/elizabethkingia). Phylogenetic analysis revealed distinct E. anophelis sublineages and demonstrated high genetic relatedness between the African isolates, compatible with persistence of the strain in the hospital environment. CRISPR spacer variation between the African isolates was mirrored by the presence of a large mobile genetic element. The pan-genome of E. anophelis comprised 6,880 gene families, underlining genomic heterogeneity of this species. African isolates carried unique resistance genes acquired by horizontal transfer. We demonstrated the presence of extensive variation of the capsular polysaccharide synthesis gene cluster in E. anophelis. Our results demonstrate the dynamic evolution of this emerging pathogen and the power of genomic approaches for Elizabethkingia identification, population biology and epidemiology.

Figure 1

Heat-map of the ANIb for each pairwise comparison (a) and intra and inter-species ANIb variability (b). Green dashed line on the top dendogram marks groups of taxa that are from the same species or have more than 95% ANIb values and therefore might be regarded as part of the same species. Black asterisks indicate strains previously described as E. meningoseptica. Black rectangles indicate the two hospital-acquired isolates from Central African Republic sequenced in this study.

Figure 2

Core- and pan-genome sizes of Elizabethkingia anophelis (a) and spectrum of frequencies for E. anophelis gene repertoires (b). The pan- and core-genomes were used to perform gene accumulation curves. These curves describe the number of new genes (pan-genome) and genes in common (core-genome) obtained by adding a new genome to a previous set. The procedure was repeated 1,000 times by randomly modifying the order of integration of genomes in the analysis. The spectrum of frequencies represents the number of genomes where the families of the pan-genome can be found, from 1 for strain-specific genes to 16 for core genes. Blue indicates accessory genes and green the genes that are highly persistent in E. anophelis. Numbers in circles above the bars indicate the number of antimicrobial resistance (AR) gene families.

Figure 3. Phylogenetic tree of the Elizabethkingia genus based on the multiple sequence alignments of the 1,546 cgMLST loci.

Black dots correspond to bootstrap supports higher than 90%.

Figure 4. Genetic organization of the putative capsular polysaccharide synthesis (cps) gene clusters in Elizabethkingia genomes.

Open reading frames (ORFs) are shown as rectangles and are drawn to scale (see scale bar). Intergenic spaces are not to scale. Three highly conserved genes, including recX, are depicted in brown. Green indicates highly conserved genes involved in capsule translocation (capD, wza, wzc). Yellow and red indicate genes wzx and wzy, respectively.

Figure 5. CRISPR-Cas type II-C organization and diversity.

(a) System organization is depicted on the top, with cas genes in brown, and the repeat-spacer array in black. Below the gene scheme, the repeat and spacer (captured MGEs) content is detailed as black diamonds and white rectangles, respectively. L corresponds to the leader sequence. Bottom line, consensus repeat sequence and degenerated repeat sequence in the 5 genomes containing such system. (b) Diversity of spacer content between the two Central African Republic hospital isolates. (c) Presence of a large MGE containing perfect matches with the two additional spacers S22 and S23.