Evidence of structural rearrangements in ESBL-positive pESI(like) megaplasmids of S.Infantis

Abstract

The increasing prevalence of pESI(like)-positive, multidrug-resistant (MDR) S. Infantis in Europe is a cause of major concern. As previously demonstrated, the pESI(like) megaplasmid is not only a carrier of antimicrobial resistant (AMR) genes (at least tet, dfr, and sul genes), but also harbours several virulence and fitness genes, and toxin/antitoxin systems that enhance its persistence in the S. Infantis host. In this study, five prototype pESI(like) plasmids, of either CTX-M-1 or CTX-M-65 ESBL-producing strains, were long-read sequenced using Oxford Nanopore Technology (ONT), and their complete sequences were resolved. Comparison of the structure and gene content of the five sequenced plasmids, and further comparison with previously published pESI(like) sequences, indicated that although the sequence of such pESI(like) 'mosaic' plasmids remains almost identical, their structures appear different and composed of regions inserted or transposed after different events. The results obtained in this study are essential to better understand the plasticity and the evolution of the pESI(like) megaplasmid, and therefore to better address risk management options and policy decisions to fight against AMR and MDR in Salmonella and other food-borne pathogens. Graphical representation of the pESI-like plasmid complete sequence (ID 12037823/11). Block colours indicate the function of the genes: red: repB gene; pink: class I integrons (IntI); yellow; mobile elements; blue: resistance genes; green: toxin/anti-toxin systems; grey: mer operon; light green: genes involve in conjugation.

Keywords: Salmonella Infantis; ESBL-producing Salmonella; antibiotic resistance; long read sequencing; megaplasmid; rearrangements.

Figure 1.

Graphical representation of the pESI-like plasmid complete sequence (ID 12037823/11). Block colours indicate the function of the genes: red: repB gene; pink: class I integrons (IntI); yellow; mobile elements; blue: resistance genes; green: toxin/anti-toxin systems; grey: mer operon; light green: genes involve in conjugation.

Figure 2.

Comparison of five complete sequences of pESI-like plasmids harbouring bla_CTX-M-1 or bla_CTX-M-65 from the following Italian isolates IDs   11024547-4,   12037823/11,   13065790-183,   14026835, and   16092401-41, using Geneious, and aligned with Mauve. Coloured squares identify similar regions based on %ID. In the figure, some key features/gene are indicated with an approximate position. From left to right: in the red and violet regions, is represented the replication region (289902..1) including the IncFII replicon; in the green region are contained the markers of pESI-like such as k88 or fim; in the pink region are included the genes of the yersiniabactin Operon, as irp1 and irp2 (99383..128414), in the orange region is located IntI1 (165385..166398) together with bla_CTX-M-1; in the dark violet region are located resistance genes as sul, tetA, qac, dfr1, or the mer Operon; in the light green region are located the IncI conjugation genes; in the yellow region is included the impABC Operon (253221..255221); in the blue region is located IntI2 (268153..269130) together with the dfr14 gene. White rectangles, for each sequence, represents the plasmid annotation .

Figure 3.

Comparison of pESI-like from ID 12037823 (CTX-M-1-positive, Italy) and pESI from CP047882 (CTX-M-1-negative, Israel), using Geneious and aligned with Mauve. Coloured squares identify similar regions based on %ID. In the figure, some resistance genes are indicated with an approximate position. White rectangles, for each sequence, represents the plasmid annotation.

Figure 4.

Comparison of pESI-like from NZ_CP016409 (CTX-M-65-positive, USA) and pESI-like from isolate ID 14026835 (CTX-M-65-positive, Italy, travel history to America), using Geneious and aligned with Mauve. Coloured squares identify similar regions based on %ID. In the figure, some resistance genes are indicated with an approximate position. White rectangles, for each sequences, represents the plasmid annotation.