Mobile Insertion Cassette Elements Found in Small Non- Transmissible Plasmids in Proteeae May Explain qnrD Mobilization Thomas Guillard 1,2,3 , Antoine Grillon 3 , Christophe de Champs 1,3 , Ce ´ line Cartier 3 , Janick Madoux 3 , Be ´ atrice Berc ¸ot 2,5 , Anne-Laure Lebreil 1 , Alain Lozniewski 4 , Jacques Riahi 5 , Ve ´ ronique Vernet-Garnier 1,3 , Emmanuelle Cambau 2,5 * 1 EA 4687, UFR Me ´ decine SFR CAP-Sante ´ Universite ´ de Reims Champagne-Ardenne, Reims, France, 2 EA3964, PRES Sorbonne Paris Cite ´ Universite ´ Paris Diderot-Paris 7, Paris, France, 3 Laboratoire de Bacte ´riologie-Virologie-Hygie `ne, CHU Reims - Ho ˆ pital Robert Debre ´, Reims, France, 4 Laboratoire de Bacte ´riologie, CHU Nancy - Ho ˆ pital Central, Nancy, France, 5 Laboratoire de Bacte ´ riologie, AP-HP Groupe Hospitalier Lariboisie `re - Saint Louis, Paris, France Abstract qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36–60% different from the other Qnr (A, B, C, S and VC). Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri) the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM) in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic) element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i) the small non-transmissible qnrD- plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii) these plasmids are maintained in Proteeae being a qnrD reservoir (iii) the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv) they can recombined with larger multiresistant plasmids conjugated in Proteeae. Citation: Guillard T, Grillon A, de Champs C, Cartier C, Madoux J, et al. (2014) Mobile Insertion Cassette Elements Found in Small Non-Transmissible Plasmids in Proteeae May Explain qnrD Mobilization. PLoS ONE 9(2): e87801. doi:10.1371/journal.pone.0087801 Editor: Ulrike Gertrud Munderloh, University of Minnesota, United States of America Received August 29, 2013; Accepted December 30, 2013; Published February 4, 2014 Copyright: ß 2014 Guillard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: TG was partially funded by an annual grant from Universite ´ de Reims Champagne-Ardenne (EA 4687) and the study was found by an annual grant from Universite ´ Paris Diderot (EA 3964). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Emmanuelle.cambau@lrb.aphp.fr Introduction Quinolones inhibit replication and transcription by inhibiting the bacterial type II topoisomerases, DNA gyrase and topoisom- erase IV [1]. Quinolones attach to the DNA-topoisomerase complex, which becomes irreversible, leading to immobilization of the enzymes resulting in bacteriostasis and to the release of DNA double-strand breaks that activate the SOS system and produce the ‘‘poison’’ effect responsible for the intense bactericidal action of quinolones [2]. Fluoroquinolones (the main subgroup of quinolones) are currently among the most heavily prescribed antimicrobials in the world because of their pharmacodynamic and pharmacokinetic properties [1]. They are very potent, especially for treatment of urinary tract infections due to Enterobacteriaceae [1,2], and as a consequence of their intense use, quinolone resistance rate has increased much for the last years [1,3,4]. Quinolone resistance mechanisms are multiple such as those reducing permeability of the bacterial wall, increasing efflux, reducing target affinity, producing inactivating enzymes and target protection proteins. Clinical resistance mostly results from the combination of several mechanisms [1]. Most of these mechanisms are chromosome-mediated [1,2] but plasmid-mediated genes have been described for a decade [5]. qnr genes were the first plasmid- mediated quinolone resistance genes described in 1998 [6]. Qnr proteins are pentapeptide repeat proteins that protect DNA gyrase and topoisomerase IV from quinolone binding. Six families of plasmid-mediated qnr gene were described: qnrA, qnrB, qnrC, qnrD and qnrS [7–10]. Several alleles have been reported for each gene: 7 alleles of qnrA, 73 of qnrB, 1 of qnrC, 2 of qnrD, 9 of qnrS and 6 qnrVC. Alleles are numbered consecutively in a qnr library (http://lahey.org/qnrStudies, last update December 08, 2013). Most of qnr genes were detected in Enterobacteriaceae, where they located on large conjugative multi-resistance plasmids, such as pMG252 described in the original Klebsiella pneumoniae qnrA1- PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e87801