title: Targeting relaxase genes for classification of the predominant plasmids in Enterobacteriaceae.
abstract: Plasmids are the main vectors of antimicrobial drug resistance and virulence genes, especially in Enterobacteriaceae. Identification and classification of plasmids is essential for analysis of their distribution. The most widely used typing method is PCR-based replicon typing (PBRT). A new classification scheme based on relaxase gene typing has been described recently. We propose a practical application of this method, with the development of a multiplex PCR set targeting relaxase genes found on plasmids most frequently encountered in Enterobacteriaceae. This method, here called "plasmid relaxase gene typing" (PRaseT), was validated with 60 transconjugants and transformants harboring various replicon types. The method was tested with 39 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1-7 replicons as well as with 17 plasmids non-typeable using PBRT; all replicons were tested in parallel with PBRT for comparison. Six multiplex PCRs and one simplex PCR, including 24 pairs of primers, recognized plasmids of groups A/C, B/O, colE, FIA, FIB, FIC, FV, FIIk, HI1, HI2, I1, K, L/M, N, P1α, Q1, U, W, X1, X2, X3 and X4. There was perfect correlation between PRaseT and PBRT results in 31/39 (79.5%) clinical isolates. Moreover, 11/17 (64.7%) plasmids non-typeable by PBRT could be typed by PRaseT. Our set of multiplex PCRs showed high sensitivity and specificity for the classification of resistance plasmids. It has proved complementary to the widely used PBRT and will improve the monitoring of plasmid distribution in every-day practice.
authors.affiliation: Université Pierre et Marie Curie-Paris 6, Faculté de Médecine, Site Saint-Antoine, Laboratoire de Bactériologie, ER8, Paris, France. Electronic address: compain.fabrice@wanadoo.fr.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Compain
authors.affiliation: Université Pierre et Marie Curie-Paris 6, Faculté de Médecine, Site Saint-Antoine, Laboratoire de Bactériologie, ER8, Paris, France. Electronic address: agatepoisson@gmail.com.
authors.forename: Agathe
authors.initials: A
authors.lastname: Poisson
authors.affiliation: Institut Pasteur, Unité des Bactéries Pathogènes Entériques, Centre National de Référence des Escherichia coli, Shigella et Salmonella, WHO Collaborating Centre for Reference and Research on Salmonella, Paris, France. Electronic address: slehello@pasteur.fr.
authors.forename: Simon
authors.initials: S
authors.lastname: Le Hello
authors.affiliation: INSERM, UMR722, Université Paris-Diderot, 75018 Paris, France. Electronic address: catherine.branger@lmr.aphp.fr.
authors.forename: Catherine
authors.initials: C
authors.lastname: Branger
authors.affiliation: Institut Pasteur, Unité des Bactéries Pathogènes Entériques, Centre National de Référence des Escherichia coli, Shigella et Salmonella, WHO Collaborating Centre for Reference and Research on Salmonella, Paris, France. Electronic address: fxweill@pasteur.fr.
authors.forename: François-Xavier
authors.initials: FX
authors.lastname: Weill
authors.affiliation: Université Pierre et Marie Curie-Paris 6, Faculté de Médecine, Site Saint-Antoine, Laboratoire de Bactériologie, ER8, Paris, France; Laboratoire de Bactériologie, Hôpital Saint-Antoine, Assistance Publique des Hôpitaux de Paris, Paris, France. Electronic address: guillaume.arlet@upmc.fr.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Arlet
authors.affiliation: Université Pierre et Marie Curie-Paris 6, Faculté de Médecine, Site Saint-Antoine, Laboratoire de Bactériologie, ER8, Paris, France; Laboratoire de Bactériologie, Hôpital Saint-Antoine, Assistance Publique des Hôpitaux de Paris, Paris, France. Electronic address: dominique.decre@upmc.fr.
authors.forename: Dominique
authors.initials: D
authors.lastname: Decré
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D004706:Endodeoxyribonucleases / Q000235:genetics*; D004755:Enterobacteriaceae / Q000235:genetics*; D060005:Genotyping Techniques; D010957:Plasmids / Q000145:classification*; D012680:Sensitivity and Specificity
chemical_list: D004706:Endodeoxyribonucleases
keywords: Classification; Multiplex PCR; Plasmid; Relaxase; Replicon typing