Genomic Epidemiology of C2/H30Rx and C1-M27 Subclades of Escherichia coli ST131 Isolates from Clinical Blood Samples in Hungary

Abstract

Extended-spectrum β-lactamase-producing Escherichia coli ST131 has become widespread worldwide. This study aims to characterize the virulome, resistome, and population structure of E. coli ST131 isolates from clinical blood samples in Hungary. A total of 30 C2/H30Rx and 33 C1-M27 ST131 isolates were selected for Illumina MiSeq sequencing and 30 isolates for MinION sequencing, followed by hybrid de novo assembly. Five C2/H30Rx and one C1-M27 cluster were identified. C1-M27 isolates harbored the F1:A2:B20 plasmid in 93.9% of cases. Long-read sequencing revealed that bla_CTX-M-27 was on plasmids. Among the C2/H30Rx isolates, only six isolates carried the C2-associated F2:A1:B- plasmid type. Of 19 hybrid-assembled C2/H30Rx genomes, the bla_CTX-M-15 gene was located on plasmid only in one isolate, while in the other isolates, ISEcp1 or IS26-mediated chromosomal integration of bla_CTX-M-15 was detected in unique variations. In one isolate a part of F2:A1:B- plasmid integrated into the chromosome. These results suggest that CTX-M-15-producing C2/H30Rx and CTX-M-27-producing C1-M27 subclades may have emerged and spread in different ways in Hungary. While bla_CTX-M-27 was carried mainly on the C1/H30R-associated F1:A2:B20 plasmid, the IncF-like plasmids of C2/H30Rx or its composite transposons have been incorporated into the chromosome through convergent evolutionary processes.

Keywords: C1-M27; C2/H30RX; Escherichia coli; ST131; blaCTX-M-15; blaCTX-M-27; long-read sequencing; whole genome sequencing (WGS).

Figure 1

Maximum likelihood phylogeny of 63 ESBL-producing E. coli ST131 isolates and their genetic characteristic reconstructed by IQtree. Legend: Rectangles of different colors indicate clusters in the phylogenetic tree, and the star symbol indicates bootstrap values (LRT: >0.8 and UF bootstrap > 0.95). Hybrid genome assembly is indicated by a yellow background. In the table, the cells indicate the absence (grey) or presence of certain antibiotic-resistance genes (green). The symbol Δ indicates the mutation in chromosomally mediated colistin resistance, * the mutations in quinolone resistance determining region. The features show the profile of C1-M27 and C2/H30Rx isolates by health care institution, year of isolation, FAB formula, and other Inc types.

Figure 2

Linear sequence comparison of the genetic context (range 22,117 to 23,084 bp) of bla_CTX-M-15 on chromosome and plasmid. Legend: Genes are grouped and color-coded according to function. The color-coded arrows indicated genes correspond to the circles in the top right corner of the figure. Grey arrows indicate genes with no similarity. Colorful wavy links represent the sequence identity of homologous gene groups identified by Clinker. Groups are indicated as “A–I” next to the designated isolate name and genome sequence.

Figure 3

BRIG representation of eleven IncF plasmids carrying bla_CTX-M-27 in ESBL-producing Escherichia coli ST131 C1-M27 isolates. Legend: The comparisons are made in reference to pEC4. The inner rings show GC content (black) and GC skew (purple/green). The remaining rings show BLAST comparisons of eleven IncF plasmids carrying bla_CTX-M-27 of C1-M27 E. coli. The outer ring highlights the genes of pEC4, shown in different colors. The genomic features of annotated genes are indicated and color-coded in red. AMR genes are indicated in black and MGE in blue.

Figure 4

Linear sequence comparison of the genetic environment of bla_CTX-M-27. Legend: Genes are grouped and color-coded according to the function of the genes. The color-coded arrows indicated genes correspond to the circles in the top right corner of the figure. Grey arrows indicate genes with no similarity. Colorful wavy links represent the sequence identity of homologous gene groups. (A) Genetic composition of 11 plasmids containing bla_CTX-M-27. (B) Genetic environment of EC3 with Regions. Regions are indicated R on the figure. Three similar regions of resistance genes were present in the plasmid genetic context. Region I. consists of bla_CTX-M-27 and Region II. consists of tetA, aph(6)-Id, aph(3″)-Ib, sul2. Region III. consists of dfrA17, aadA5, qacEΔ1, sul1, and mph(A).