Global genomic epidemiology of bla GES-5 carbapenemase-associated integrons

Abstract

Antimicrobial resistance (AMR) gene cassettes comprise an AMR gene flanked by short recombination sites (attI and attC or attC and attC). Integrons are genetic elements able to capture, excise and shuffle these cassettes, providing 'adaptation on demand', and can be found on both chromosomes and plasmids. Understanding the patterns of integron diversity may help to understand the epidemiology of AMR genes. As a case study, we examined the clinical resistance gene bla _GES-5, an integron-associated class A carbapenemase first reported in Greece in 2004 and since observed worldwide, which to our knowledge has not been the subject of a previous global analysis. Using a dataset comprising all de-duplicated NCBI contigs containing bla _GES-5 (n=104), we developed a pangenome graph-based workflow to characterize and cluster the diversity of bla _GES-5-associated integrons. We demonstrate that bla _GES-5-associated integrons on plasmids are different to those on chromosomes. Chromosomal integrons were almost all identified in Pseudomonas aeruginosa ST235, with a consistent gene cassette content and order. We observed instances where insertion sequence IS110 disrupted attC sites, which might immobilize the gene cassettes and explain the conserved integron structure despite the presence of intI1 integrase promoters, which would typically facilitate capture or excision and rearrangement. The plasmid-associated integrons were more diverse in their gene cassette content and order, which could be an indication of greater integrase activity and 'shuffling' of integrons on plasmids.

Keywords: GES; Pseudomonas aeruginosa; beta-lactamase; mobile genetic elements.

Fig. 1.. A schematic of an integron. Adapted from Gillings 2017 [75] and Ghaly 2017 [11]. Shown is an integrase in the reverse orientation with a P_c and P_int. This can lead to transcriptional interference [8]. Also shown are three gene cassettes (c₁, c₂ and c₃), flanked by the attachment sites (attI, attC₁, attC₂ and attC₃). Gene cassette c₂ has been excised above the integron. The expression of the gene cassettes generally decreases with distance from the promoter P_c.

Fig. 2.. NCBI metadata distributions for the 104 bla_GES-5-positive contigs. (a) Shows ISO region and (b) isolate genus by sampling year. Note, no contigs were sampled in 2011, so this year is omitted from the x-axis.

Fig. 3.. Exploration of flanking sequence diversity. (a) Upstream Flanker output, from 100 bp flanking sequences to 10 000 bp flanking sequences in 100 bp windows. For each length, tile colours represent clusters found multiple times, and grey values represent unique clusters. Colours do not compare across lengths, only within lengths. (b) Downstream Flanker output, as described for the upstream flanking sequences.

Fig. 4.. 10 000 bp flanking sequence analysis for bla_GES-5. (a) Clustering-ordered PanGraph blocks (pastel colours) overlaid with CDS annotations (black outlined arrows) and integron-specific annotations: intI1 in blue, intI3 in green, attI in orange and attC in red. Numbers to the left indicate duplicates, with boundaries indicating where the coloured PanGraph blocks begin/end. (b) P_int and P_c presence (black) or absence (white). (c) intI1 SNV profiles, corresponding to the blue intI1 annotations in panel (a). (d) bla_GES-5 SNV profiles. (e) Contig origin (chromosomal, plasmid or na). (f) Contig genus distribution. (g) Contig ISO region distribution. (h) Contig sampling year distribution. Smaller circles indicate one isolate, and larger circles indicate two isolates.