pMdT1, a small ColE1-like plasmid mobilizing a new variant of the aac(6')-Ib-cr gene in Salmonella enterica serovar Typhimurium

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 similarities 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