Full characterization of an IncR plasmid harboring qnrSI recovered from a VIM-11-producing Pseudomonas aeruginosa

Abstract Metallo-p-lactamases (MBL) producing Pseudomonas aeruginosa isolates have been well characterized. Quinolones are commonly used in the treatment of carbapenem-resistant P. aeruginosa infections; however, data about PMQR in this species are scarce. The objective of this study was to report the simultaneous presence of qnrS and blaV-M-n in P. aeruginosa, and to characterize the qnrS-harboring plasmid. Thirty-eight carbapenem-resistant P. aeruginosa isolates were recovered from a hospital in Buenos Aires during 2012. Screening forMBL was assessed by the double disk synergy test using EDTA and carbapenem discs. Plasmid DNA extraction was performed by a method using phenol-chloroform. PCR followed by sequencing was carried out to determine each MBL and PMQR allele. PCR-BseGI-RFLP was performed to detect aac-(6')-Ib-cr. The gyrA-QRDR was sequenced in those PMQR-harboring isolates. Plasmid incompatibility groups and addiction systems were characterized by PCR. The PMQR-carrying plasmid was sequenced using Illumina technology, annotated using RAST and manually curated. Eleven/38 isolates were VIM producers (blaVIM-2 and blaVIM-11) while 1/38 harbored blaIMP-13. One isolate harbored blaVIM-11 and the PMQR qnrSI; however, both markers were located in different plasmids. The qnrSí-harboring plasmid (pP6qnrS1) was 117 945 bp in size, presented 154 CDS and corresponded to the IncR group. In addition to qnrSI, it harbored several aminoglycoside resis-tance markers. Although pP6qnrS1 was non-conjugative, it presented an oriT which made it possible for this plasmid to be transferable. This is the first report on P. aeruginosa carrying both blaVIM-11 and qnrSI, plus the first detection of an IncR plasmid in Argentina.

Resumen Las quinolonas son comúnmente utilizadas en el tratamiento de las infecciones producidas por Pseudomonas aeruginosa resistentes a carbapenems (PARC); aun así, la información sobre la resistencia a quinolonas mediada por plásmidos (PMQR) en esta especie es escasa. El objetivo de este trabajo fue reportar la presencia simultánea de los genes qnrS y blaVIM-11 en PARC y caracterizar el plásmido portador de qnrS. Durante 2012 se recuperaron 38 PARC en un hospital de Buenos Aires. El tamizaje para detectar producción de metalo-beta-lactamasas (MBL) se llevó a cabo mediante sinergia de doble disco utilizando EDTA y carbapenems. El ADN plasmídico fue extraído utilizando fenolcloroformo. Para determinar los alelos de los genes implicados en la síntesis de MBL y de PMQR, se llevó a cabo PCR-secuenciación. Para la detección de aac-(6')-Ib-cr se realizó PCR-BseGI-RFLP. En aquellos aislamientos portadores de PMQR se secuenció el gen gyrA. Los grupos de incompatibilidad y sistemas de adicción fueron caracterizados por PCR. El plásmido portador de PMQR fue secuenciado completamente y curado manualmente. De 38 aislamientos, 11 fueron productores de VIM (blaVIM-2 y blaVIM-11), mientras que uno contenía blaIMP-13. Si bien un aislamiento fue portador de blaVIM-11 y de qnrSI, dichos marcadores se encontraban en distintos plásmidos. El plásmido portador de qnrSI (pP6qnrS1) presentó un tamaño de 117.945 pby 154 secuencias codificantes (CDS); este correspondió al grupo de incompatibilidad IncR. Además de qnrSI, el plásmido portaba diversos marcadores de resistencia a aminoglucósidos. Aun cuando pP6qnrS1 no resultó conjugativo, presentó un oriT, de modo que posiblemente sea transferible. Este es el primer informe acerca de PARC portadora de blaVIM-11 y de qnrSI en simultáneo, además, es la primera descripción de un plásmido IncR en Argentina.

Complete sequencing of an IncX3 plasmid carrying blaNDM-5 allele reveals an early stage in the dissemination of the blaNDM gene.

Purpose: The aim of the present study was to perform molecular characterisation of the blaNDM plasmids and to understand the mechanism of its spread among pathogenic bacteria. Materials and Methods: Seventy-six non-repetitive carbapenem-resistant isolates which were collected during Nov 2011 to April 2013 from four hospitals in Chennai were analyzed for the presence of the blaNDM gene by PCR. Further, the genetic context of the blaNDM gene was analyzed by PCR specifi c to ISAba125 and bleMBL gene. One of the blaNDM plasmid was completely sequenced in the Illumina HiSeq platform. Results: Twenty-three isolates consisting of 8 Escherichia coli, 8 Klebsiella pneumoniae, 3 Klebsiella oxytoca, 3 Acinetobacter baumanii and 1 Pseudomonas aeruginosa were found to carry the blaNDM gene. In 18 isolates the blaNDM gene was associated with a bleMBL gene and the ISAba125 element. The complete sequencing of pNDM-MGR194 revealed an IncX3 replication type plasmid, with a length of 46,253 bp, an average GC content of 47% and 59 putative ORFs. The iteron region contained the blaNDM5 gene and the bleMBL, trpF and dsbC genes downstream and an IS5 inserted within the ISAba125 element upstream. Conclusion: This is the fi rst report where the blaNDM gene insertion in a plasmid is not accompanied by other resistance gene determinants. These observations suggest that the IncX3 plasmid pNDM-MGR194 is an early stage in the dissemination of the blaNDM.