Escherichia coli ST8196 is a novel, locally evolved, and extensively drug resistant pathogenic lineage within the ST131 clonal complex

Abstract

The H30Rx subclade of Escherichia coli ST131 is a clinically important, globally dispersed pathogenic lineage that typically displays resistance to fluoroquinolones and extended spectrum β-lactams. Isolates EC233 and EC234, variants of ST131-H30Rx with a novel sequence type (ST) 8196, isolated from unrelated patients presenting with bacteraemia at a Sydney Hospital in 2014 are characterised here. EC233 and EC234 are phylogroup B2, serotype O25:H4A, and resistant to ampicillin, amoxicillin, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, norfloxacin and gentamicin and are likely clonal. Both harbour an IncFII_2 plasmid (pSPRC_Ec234-FII) that carries most of the resistance genes on an IS26 associated translocatable unit, two small plasmids and a novel IncI1 plasmid (pSPRC_Ec234-I). SNP-based phylogenetic analysis of the core genome of representatives within the ST131 clonal complex places both isolates in a subclade with three clinical Australian ST131-H30Rx clade-C isolates. A MrBayes phylogeny analysis of EC233 and EC234 indicates ST8196 share a most recent common ancestor with ST131-H30Rx strain EC70 isolated from the same hospital in 2013. Our study identified genomic hallmarks that define the ST131-H30Rx subclade in the ST8196 isolates and highlights a need for unbiased genomic surveillance approaches to identify novel high-risk MDR E. coli pathogens that impact healthcare facilities.

Keywords: E. coli ST131 clonal complex; Escherichia coli ST8196; bla CTX-M-15; qnrB4; ST131- H30Rx.

Figure 1.

SNP phylogeny of the core genomes of sequence type representatives comprising the ST131 clonal cluster (ST131CC). Tree scale represents substitution per site. The tree also includes ST131 isolates from our extended collection [33], ST131 reference genome EC985 and representatives of the three ST131 clades A, B and C [15] highlighted in purple. The red box identifies the sub-clade with ST8196 isolates EC233 and EC234. Inset magnifies the subclade highlighted in the red box to better resolve the branching pattern and associated clade confidence scores. Clades I, II and III represent the three major clades in which the different STs within the ST131CC cluster.

Figure 2.

Pangenome analysis of isolates that clustered closely in a MrBayes analysis (Files S1 and S2). A. Magnified view of the subclade containing ST8196 isolates and other closely related ST131 genomes identified in MrBayes analysis presented in supplementary file S2. B. Genotypic feature that characterize isolates in the subclade presented in section A. Except parC which is highlighted with an asterisk (*), all genes listed in the “Resistance genes” column are known to be laterally acquired. C. BLASTn alignment (using 90% lower and 100% upper identity cutoffs) of 6 genomes against the partially completed EC234_ST8196 genome. The inner radiating bars indicate the location of genomic islands in EC234, as predicted by the SIGI-HMM module of island-viewer software. D. Graphical overview of distribution of genes that represent the core and accessory genomes of the isolates in Roary. (Core genes = genes present in 99% <= strains < = 100%), (Soft core genes = genes present in 95% <= strains < 99%, shell genes = genes present in 15% <= strains < 95%), Cloud genes (genes present in 0% <= strains < 15%). E. Genome clustering and the pangenome matrix of 8 genomes (including the outgroup, ESCFA6284_FA). Numbers at the node of the tree indicate bootstrap values.

Figure 3.

pSPRC_Ec234-FII the IncFII plasmid in EC234. A. Diagrammatic representation of gene content in the IncFII plasmid of isolate EC234 and the resistance loci in the inset above. Arrows representing gene families are colour coded. Orange arrows: genes required for plasmid transfer, light green: replication genes, dark green: class 1 integron integrase gene, red arrows: resistance genes, dark blue: insertion elements and purple: hypothetical genes. B. Mapping of raw illumina reads from ST131 in our collection (upper panel) and EC233_ST8196 and other ST131 isolates identified to be closely related to EC234 from Mr Bayes phylogeny analysis (bottom panel).

Figure 4.

Features of IncI1 plasmid pSPRC_Ec234-I in strain EC234. A. Phylogeny of complete IncI1 plasmids available in the plasmid database. Orange arrows indicate genes required for plasmid transfer and conjugation, green indicates replication genes, red arrows indicate bacteriocin resistance genes and purple arrows indicate hypothetical genes. B. EasyFig alignment of the three most closely related plasmids identified in isolate EC234.