Rapid Identification of Different Escherichia coli Sequence Type 131 Clades

Abstract

Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extended-spectrum-β-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n = 12), B (n = 12), and C, including subclades C1-M27 (n = 16), C1-nM27 (n = 20), C2 (n = 17), and other C (n = 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n = 54), B (n = 23), and C (n = 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with bla_CTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.

Keywords: Escherichia coli; assay development; beta-lactamases; clonality; whole-genome sequencing.

FIG 1

Agarose gel electrophoresis of the ST131 clade PCR amplicons. Lanes: M, 100-bp DNA ladder; 1, strain SNEC15, clade A; 2, strain KFEC6, clade B; 3, strain KFEC8, subclade C1-M27; 4, strain SNEC5, subclade C1-nM27 (C1-non-M27); 5, strain ONEC14, clade C2; 6, strain KSEC7, subclade C0; 7, strain BRG210, subclade I1; 8, strain BRG28, non-ST131; 9, no-template control. All strains underwent WGS except for BRG28.

FIG 2

Yearly rates of ST131 clades and subclades in Japanese ESBL-producing E. coli. Rates of ST131 and subclades C1-M27 and C2 increased yearly (P < 0.001 each). In 2014, the rates of clades A and B and subclades C1-M27, C1-non-M27, and C2 were 3, 1, 31, 15, and 24%, respectively.

FIG 3

Recombination-free core SNP-based phylogeny of 401 ST131 genomes. This maximum-likelihood phylogenetic tree is rooted by using the clade A isolates. A total of 6,881 core SNP sites were used after excluding the 18,693 core SNP sites that were located within the 1,955,761-bp recombination region. Branches that had >90% bootstrap support from 100 replicates are highlighted in blue. The bootstrap values for the root of subclades I1 and C3 were 100 and 99%, respectively. Genomes of the WGS collection isolates are marked in red. Eleven genomes (marked in blue) were sequenced to investigate their phylogeny in addition to the 390 genomes (see Fig. S1 in the supplemental material).