Site-Specific Recombination at XerC/D Sites Mediates the Formation and Resolution of Plasmid Co-integrates Carrying a blaOXA-58- and TnaphA6-Resistance Module in Acinetobacter baumannii.

María M Cameranesi,Adriana S Limansky,Guillermo D Repizo,Alejandro M Viale,Jorgelina Morán-Barrio

doi:10.3389/fmicb.2018.00066

Abstract

Members of the genus Acinetobacter possess distinct plasmid types which provide effective platforms for the acquisition, evolution, and dissemination of antimicrobial resistance structures. Many plasmid-borne resistance structures are bordered by short DNA sequences providing potential recognition sites for the host XerC and XerD site-specific tyrosine recombinases (XerC/D-like sites). However, whether these sites are active in recombination and how they assist the mobilization of associated resistance structures is still poorly understood. Here we characterized the plasmids carried by Acinetobacter baumannii Ab242, a multidrug-resistant clinical strain belonging to the ST104 (Oxford scheme) which produces an OXA-58 carbapenem-hydrolyzing class-D β-lactamase (CHDL). Plasmid sequencing and characterization of replication, stability, and adaptive modules revealed the presence in Ab242 of three novel plasmids lacking self-transferability functions which were designated pAb242_9, pAb242_12, and pAb242_25, respectively. Among them, only pAb242_25 was found to carry an adaptive module encompassing an ISAba825-blaOXA-58 arrangement accompanied by a TnaphA6 transposon, the whole structure conferring simultaneous resistance to carbapenems and aminoglycosides. Ab242 plasmids harbor several XerC/D-like sites, with most sites found in pAb242_25 located in the vicinity or within the adaptive module described above. Electrotransformation of susceptible A. nosocomialis cells with Ab242 plasmids followed by imipenem selection indicated that the transforming plasmid form was a co-integrate resulting from the fusion of pAb242_25 and pAb242_12. Further characterization by cloning and sequencing studies indicated that a XerC/D site in pAb242_25 and another in pAb242_12 provided the active sister pair for the inter-molecular site-specific recombination reaction mediating the fusion of these two plasmids. Moreover, the resulting co-integrate was found also to undergo intra-molecular resolution at the new pair of XerC/D sites generated during fusion thus regenerating the original pAb242_25 and pAb242_12 plasmids. These observations provide the first evidence indicating that XerC/D-like sites in A. baumannii plasmids can provide active pairs for site-specific recombination mediating inter-molecular fusions and intra-molecular resolutions. The overall results shed light on the evolutionary dynamics of A. baumannii plasmids and the underlying mechanisms of dissemination of genetic structures responsible for carbapenem and other antibiotics resistance among the Acinetobacter clinical population.

Highlights

IntroductionAcinetobacter baumannii represents nowadays a significant cause of healthcare-associated infections generally affecting immunocompromised and severely-injured patients with the global spread of a number of epidemic clonal complexes (CC) displaying multidrug-resistance (MDR) phenotypes (Peleg et al, 2008; Roca et al, 2012; Antunes et al, 2014; Wong et al, 2017)
In this work we characterized in detail the plasmids present in A. baumannii Ab242, a MDR clinical strain of the CC104 isolated in Argentina, to obtain clues into different aspects related to plasmid diversity, evolutionary dynamics, and underlying mechanisms of dissemination among the Acinetobacter population of genetic structures conferring blaOXA-58-mediated carbapenem resistance
A bi-replicon plasmid of around 25 kbp in size designated pAb242_25, was found to harbor an adaptive module carrying a blaOXA-58 gene with an upstream ISAba825 insertion which we previously reported to promote the over-expression of the CHDL gene (Ravasi et al, 2011) accompanied by a TnaphA6 transposon inserted in a lysE gene located downstream of the CHDL gene (Figure 2)

Summary

Introduction

Acinetobacter baumannii represents nowadays a significant cause of healthcare-associated infections generally affecting immunocompromised and severely-injured patients with the global spread of a number of epidemic clonal complexes (CC) displaying multidrug-resistance (MDR) phenotypes (Peleg et al, 2008; Roca et al, 2012; Antunes et al, 2014; Wong et al, 2017). MDR strains contain an arsenal of antimicrobial resistance determinants some located in chromosomal resistance islands and others in plasmids, and have shown an outstanding ability to rapidly acquire additional resistance when confronted to new antimicrobials (Peleg et al, 2008; Bertini et al, 2010; Roca et al, 2012; Ramírez et al, 2013; Antunes et al, 2014; Touchon et al, 2014; Nigro et al, 2015; Blackwell and Hall, 2017; Wong et al, 2017) It is in this context that the increasing resistance among MDR A. baumannii strains to last-resort therapeutic options such as the carbapenem β-lactams represents a most worrisome problem (Peleg et al, 2008; Roca et al, 2012; Antunes et al, 2014; Wong et al, 2017). A detailed characterization of the plasmids carried by carbapenem-resistant A. baumannii strains may help our understanding of the mechanisms of dissemination of these resistance structures, and contribute to the adoption of measures that limit the spread of antimicrobial resistance at both local and global scales

Methods

Results

Conclusion