Characterization of IncI1 sequence type 71 epidemic plasmid lineage responsible for the recent dissemination of CTX-M-65 extended-spectrum β-lactamase in the Bolivian Chaco region

Abstract

During the last decade, a significant diffusion of CTX-M-type extended-spectrum β-lactamases (ESBLs) was observed in commensal Escherichia coli from healthy children in the Bolivian Chaco region, with initial dissemination of CTX-M-2, which was then replaced by CTX-M-15 and CTX-M-65. In this work, we demonstrate that the widespread dissemination of CTX-M-65 observed in this context was related to the polyclonal spreading of an IncI1 sequence type 71 (ST71) epidemic plasmid lineage. The structure of the epidemic plasmid population was characterized by complete sequencing of four representatives and PCR mapping of the remainder (n = 16). Sequence analysis showed identical plasmid backbones (similar to that of the reference IncI1 plasmid, R64) and a multiresistance region (MRR), which underwent local microevolution. The MRR harbored genes responsible for resistance to β-lactams, aminoglycosides, florfenicol, and fosfomycin (with microevolution mainly consisting of deletion events of resistance modules). The bla CTX-M-65 module harbored by the IncI1 ST71 epidemic plasmid was apparently derived from IncN-type plasmids, likely via IS26-mediated mobilization. The plasmid could be transferred by conjugation to several different enterobacterial species (Escherichia coli, Cronobacter sakazakii, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, and Salmonella enterica) and was stably maintained without selective pressure in these species, with the exception of K. oxytoca and S. enterica. Fitness assays performed in E. coli recipients demonstrated that the presence of the epidemic plasmid was apparently not associated with a significant biological cost.

FIG 1

(a) Sequence comparison of plasmids pC271 and R64. Homologous segments generated by a BLASTn comparison (≥99% identity) are shown as gray blocks. Genes are represented by bars and in pC271 are classified by function into different groups, as detailed below the diagram. The location of oriT is represented by a black dot. The MRRs of pC271 and of R64 are indicated by dotted lines. (b) Structure of the MRR of pC271 (27,587 bp). Open reading frames (ORFs) are represented by thick arrows, with arrowheads showing the direction of transcription. Genes encoding resistance to antibiotics and metals are represented in red and yellow, respectively; nonfunctional ORFs (deleted or disrupted) and mobile elements are represented by pale-blue thin arrows; ORFs with known and unknown functions are drawn in green and white, respectively; insertion sequences and transposases are hatched, with the exception of IS26, shown in solid black. Triangular and square flags show inverted repeats (IRs) of Tn2 and Tn5393, respectively; the semicircular flag shows a partial IR of a Tn1721-like transposon; ISEc57 direct repeats (CACA) are indicated by diamond flags. A more detailed description of the MRR of pC271 can be found in Fig. S1 in the supplemental material. (c) Comparison of the MRRs of the four sequenced plasmids. Homologous regions (≥99% identity) are shown as gray blocks connected across lanes. The other features are as in panel b.

FIG 2

Growth kinetics of E. coli recipients harboring pC193 (IncI1 CTX-M-65-encoding plasmid), pV38-8 (IncA/C CTX-M-2-encoding plasmid), or both plasmids. (a) E. coli MG1655. (b) E. coli ER-B1 (a wild-type isolate of phylogroup B1). The data are representative of three independent experiments. The error bars represent standard deviations.

FIG 3

Growth competition between E. coli MG1655(pC193) and E. coli MG1655(pV38-8) in the absence of antibiotic selective pressure (a), in the presence of 0.5 μg/ml cefotaxime (CTX) (b), and in the presence of 8 μg/ml cefotaxime (c). The data are representative of three independent experiments. The error bars represent standard deviations.