blaOXA-48-like genome architecture among carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Netherlands

Abstract

Carbapenem-hydrolysing enzymes belonging to the OXA-48-like group are encoded by bla_OXA-48-like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective here was to investigate the characteristics, gene content and diversity of the bla_OXA-48-like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014 to 2019 in comparison with genome sequences from 29 countries. A combination of short-read genome sequencing with long-read sequencing enabled the reconstruction of 47 and 132 complete bla_OXA-48-like plasmids for E. coli and K. pneumoniae, respectively. Seven distinct plasmid groups designated as pOXA-48-1 to pOXA-48-5, pOXA-181 and pOXA-232 were identified in the Netherlands which were similar to internationally reported plasmids obtained from countries from North and South America, Europe, Asia and Oceania. The seven plasmid groups varied in size, G+C content, presence of antibiotic resistance genes, replicon family and gene content. The pOXA-48-1 to pOXA-48-5 plasmids were variable, and the pOXA-181 and pOXA-232 plasmids were conserved. The pOXA-48-1, pOXA-48-2, pOXA-48-3 and pOXA-48-5 groups contained a putative conjugation system, but this was absent in the pOXA-48-4, pOXA-181 and pOXA-232 plasmid groups. pOXA-48 plasmids contained the PemI antitoxin, while the pOXA-181 and pOXA-232 plasmids did not. Furthermore, the pOXA-181 plasmids carried a virB2-virB3-virB9-virB10-virB11 type IV secretion system, while the pOXA-48 plasmids and pOXA-232 lacked this system. A group of non-related pOXA-48 plasmids from the Netherlands contained different resistance genes, non-IncL-type replicons or no replicons. Whole genome multilocus sequence typing revealed that the bla_OXA-48-like plasmids were found in a wide variety of genetic backgrounds in contrast to chromosomally encoded bla_OXA-48-like alleles. Chromosomally localized bla_OXA-48 and bla_OXA-244 alleles were located on genetic elements of variable sizes and comprised regions of pOXA-48 plasmids. The bla_OXA-48-like genetic element was flanked by a direct repeat upstream of IS1R, and was found at multiple locations in the chromosomes of E. coli. Lastly, K. pneumoniae isolates carrying bla_OXA-48 or bla_OXA-232 were mostly resistant for meropenem, whereas E. coli bla_OXA-48, bla_OXA-181 and chromosomal bla_OXA-48 or bla_OXA-244 isolates were mostly sensitive. In conclusion, the overall bla_OXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, confirming global dissemination of bla_OXA-48-like plasmids. Variations in size, presence of antibiotic resistance genes and gene content impacted pOXA-48, pOXA-181 and pOXA-232 plasmid architecture.

Keywords: blaOXA-181; blaOXA-232; blaOXA-244; blaOXA-48; genomes.

Fig. 1.

Genetic clustering of bla _OXA-48-like plasmids in seven groups. (a) UPGMA clustering based on plasmid DNA sequences revealed seven distinct groups of bla _OXA-48-like plasmids. These groups were designated as pOXA-xxx (e.g. pOXA-48-1). Plasmids retrieved from the Netherlands are indicated in orange and international plasmids in blue. Group numbers are indicated. A heatmap shows the percentage of sequence identity, where red is 100 % identical and white 0 % identical. (b) The presence of AMR genes and replicons among the plasmids is indicated with black squares. Plasmids are depicted in rows and the AMR genes and replicons in columns. (c) As in (a) and (b) for the non-cluster plasmids.

Fig. 2.

bla _OXA-48-like plasmids have distinct molecular characteristics. (a) The number of AMR genes among the pOXA-48-like plasmid groups, (b) the G+C content (%) of the distinct pOXA-48-like plasmid groups and (c) the size (kb) of the pOXA-48-like plasmid groups. Bars, the variation per group.

Fig. 3.

Differences in bla _OXA-48-like plasmid architecture. (a) Diversity in pOXA-48-1 to pOXA-48-5 plasmid gene content. Complete plasmids were visualized in a linear way with the bla _OXA-48-like allele at starting position 1. The presence and absence of genes is indicated among representative plasmids from the plasmid groups. Colours indicate different groups of genes corresponding to different regions in the plasmid, or the variable region. Plasmid regions are labelled above the plasmid sequence. (b) Similar to (a) but displaying diversity in pOXA-181 and pOXA-232 plasmid gene content.

Fig. 4.

Distribution of chromosome- or plasmid-localized bla _OXA-48 or bla _OXA-244. (a) MST of E. coli in which chromosome- or plasmid-localized bla _OXA-48 or bla _OXA-244 alleles are indicated by different colours. (b) Similar to (a) but for K. pneumoniae . (c) The presence of AMR genes among the chromosomes analysed in this study is indicated with black squares. Chromosomes are depicted in rows and the AMR genes in columns. Antibiotic classes are indicated above the AMR genes in different colours.

Fig. 5.

Distinct integration sites of variable bla _OXA-48 and bla _OXA-244 elements in the chromosome. (a) Artificial chromosome in which the different bla _OXA-48-like insertion positions are indicated by triangles. (b) Comparison of plasmid region 1 with chromosomal insertion sites of bla _OXA-48-like. Arrows indicate ORFs of which bla _OXA-48 is depicted in red and bla _OXA-244 in light red. DR indicates the direct repeat sequence ggtaatgactccaac located upstream of IS1R. Sequence types are depicted by ST and sizes of the different insertion sequences are indicated in kilobases (kb). (c) Frequency of the DR sequence in bla _OXA-48-like plasmids and chromosomes of E. coli and K. pneumoniae .