Characterization of Novel Integrons, In1085 and In1086, and the Surrounding Genes in Plasmids from Enterobacteriaceae, and the Role for attCaadA16 Structural Features during attI1 × attC Integration.

Dongguo Wang,Jiayu Chen,Zhihui Liu,Wei Hou,Jiao Feng,Dongsheng Zhou,Linjun Yang,Zhe Yin

doi:10.3389/fmicb.2017.01003

Abstract

Novel class 1 integrons In1085 and In1086, containing the class D β-lactamase -encoding gene blaOXA, were identified in clinical enterobacterial strains. In this study, we aimed to characterize the genetic contexts of In1085 and In1086, with the goal of identifying putative mechanisms of integron mobilization. Four plasmids, approximately 5.3, 5.3, 5.7, and 6.6 kb, from 71 clinical Enterobacteriaceae strains were found to contain class 1 integrons (In37, In62, In1085, and In1086, respectively). Two of these plasmids, pEco336 and pNsa292, containing In1085 and In1086, respectively, were further characterized by antibiotic susceptibility testing, conjugation experiments, PCR, sequencing, and gene mapping. The OXA-type carbapenemase activities of the parental strains were also assessed. The results revealed that the novel integrons had different genetic environments, and therefore demonstrated diverse biochemical characteristics. Using evolutionary inferences based on the recombination of gene cassettes, we also identified a role for attCaadA16 structural features during attI1 × attC insertion reactions. Our analysis showed that gene cassette insertions in the bottom strand of attCaadA16 in the correct orientation lead to the expression the encoded genes from the Pc promoter. Our study suggests that the genetic features harbored within the integrons are inserted in a discernable pattern, involving the stepwise and parallel evolution of class 1 integron variations under antibiotic selection pressures in a clinical setting.

Highlights

MATERIALS AND METHODSGene cassettes (GCs) are small, mobile elements containing one or more genes and recombination sites, and are often contained within larger genetic structures known as integrons
Integrons can be assigned into three classes (1, 2, and 3) based on their integrase gene sequences, with class 1 integrons most often associated with antimicrobial resistance in clinical bacterial isolates (Huang et al, 2015)
Integrons can evolve rapidly through the acquisition, storage, and rearrangement of genes embedded in their GCs, and, by mobilization to conjugative plasmids, they play a crucial role in increasing multidrug resistance amongst clinical bacterial strains (Escudero et al, 2016)

Summary

Introduction

MATERIALS AND METHODSGene cassettes (GCs) are small, mobile elements containing one or more genes and recombination sites, and are often contained within larger genetic structures known as integrons. The mechanisms of integration and excision of GCs are well described, with integrations known to occur at attI × attC recombination sites (MacDonald et al, 2006; Loot et al, 2012), and excisions requiring attC × attC recombination sites, which occur in single-stranded sequences and activate the folded bottom strand (bs) (Bouvier et al, 2005, 2009) Despite their mobility, GC rearrangements resulting in novel GC arrays rarely occur, it is assumed that integrases could rearrange GCs, generating integron variants, under antibiotic selective pressure (Bouvier et al, 2005, 2009; Barraud and Ploy, 2015). More than 130 different GCs (less than 98% identical) containing antibiotic resistance genes have been identified, along with many other GCs containing genes of unknown function (Partridge et al, 2009)

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