pMdT1, a small ColE1-like plasmid mobilizing a new variant of the aac(6 ′ )-Ib-cr gene in Salmonella enterica serovar Typhimurium Marı ´a de Toro 1,2 , Irene Rodrı ´guez 3 †, Beatriz Rojo-Bezares 1 , Reiner Helmuth 3 , Carmen Torres 1,2 , Beatriz Guerra 3 ‡ and Yolanda Sa ´ enz 1 *‡ 1 A ´ rea de Microbiologı ´a Molecular, Centro de Investigacio ´n Biome ´dica de La Rioja (CIBIR), Logron ˜ o, Spain; 2 A ´ rea de Bioquı ´mica y Biologı ´a Molecular, Universidad de La Rioja, Logron ˜ o, Spain; 3 Department for Biological Safety, Federal Institute for Risk Assessment (BfR), Berlin, Germany *Corresponding author. Tel: +34-941278868; Fax: +34-941278887; E-mail: ysaenz@riojasalud.es †Present address: Servicio de Microbiologı ´a, Hospital Universitario Ramo ´n y Cajal, Madrid, Spain. ‡These authors contributed equally to this work. Received 9 October 2012; returned 30 November 2012; revised 17 December 2012; accepted 27 December 2012 Objectives: To characterize a 5.9 kb aac(6 ′ )-Ib-cr-harbouring plasmid that was detected in a clinical Salmonella Typhimurium DT104B strain. Methods: Extraction and purification of plasmid DNA and electrotransformation assays were carried out in order to obtain kanamycin-resistant transformants. MICs of several fluoroquinolones and aminoglycosides were determined. DNA sequencing was performed by primer walking on purified plasmid preparations. The new plasmid nucleotide sequence was analysed and compared with available sequences using bioinformatic tools. Results: pMdT1 is a 5.9 kb mobilizable ColE1-like plasmid that harbours aac(6 ′ )-Ib-cr4, a gene encoding a new variant of the AAC(6 ′ )-Ib-cr protein (225 amino acids). This active protein conferred resistance to tobramycin and kanamycin, and also decreased susceptibility to ciprofloxacin and norfloxacin in the transformant strain, as MICs demonstrated. The mobilization region, necessary for horizontal transfer and composed of the mobA, mobB, mobC and mobD genes, displayed a high degree of identity with those from representative ColE1-like plasmids. The basis of mobility (bom), oriT and origin of replication regions were also detected. Apart from the acetylase-encoding gene, three other open reading frames (ORFs) were determined. No similar- ities were found when the ORF1 sequence was compared with the sequences included in GenBank. The deduced ORF2 protein predicted a CopG-like structure characteristic of transcriptional regulators, and the deduced ORF3 protein was identical to macrophage stimulating factors. Conclusions: The pMdT1 is the smallest mobilizable ColE1-like plasmid containing an aac(6 ′ )-Ib-cr gene that has been described so far. Keywords: plasmid-mediated quinolone resistance, PMQR, Salmonella Typhimurium, mob genes Introduction The aac(6 ′ )-Ib gene (also named aacA4), coding for an acetyl- transferase, confers resistance to kanamycin, amikacin and tobramycin, and can be mobilized and spread by plasmids. 1,2 The aac(6 ′ )-Ib genes have mainly been found as gene cassettes within class 1 integrons, although fused genes and different var- iants, such as the aac(6 ′ )-Ib-cr gene, have also been described. 1 The AAC(6 ′ )-Ib-cr variant displays Trp102Arg and Asp179Tyr changes with respect to the reference AAC(6 ′ )-Ib (GenBank AAD22142.2), and leads to additional low-level resistance to the fluoroquinolones ciprofloxacin and norfloxacin. 2,3 Different genetic environments facilitating the dissemination of aac(6 ′ )-Ib-cr have been described. 1,4 In this sense, the first in vivo selection of the aac(6 ′ )-Ib-cr gene and the GyrA change (Ser83Tyr) in the qnrS1-positive Salmonella Typhimurium DT104B strain Se20 after ciprofloxacin treatment was described in previous work by our group. 5 This strain was resistant to nali- dixic acid, ciprofloxacin, levofloxacin, norfloxacin, ofloxacin, kanamycin, tobramycin, streptomycin and trimethoprim, and harboured five plasmids (sizes ranged from 6 to 194 kb). The aim of the present work was to characterize the ≏6 kb plasmid (designated pMdT1) in which the aac(6 ′ )-Ib-cr gene was located. # The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com J Antimicrob Chemother doi:10.1093/jac/dkt001 1 of 4 Journal of Antimicrobial Chemotherapy Advance Access published January 29, 2013 by guest on January 30, 2013 http://jac.oxfordjournals.org/ Downloaded from