title: New plasmid-mediated aminoglycoside 6'-N-acetyltransferase, AAC(6')-Ian, and ESBL, TLA-3, from a Serratia marcescens clinical isolate.
abstract: OBJECTIVE
  Enterobacteriaceae clinical isolates showing amikacin resistance (MIC 64 to >256 mg/L) in the absence of 16S rRNA methyltransferase (MTase) genes were found. The aim of this study was to clarify the molecular mechanisms underlying amikacin resistance in Enterobacteriaceae clinical isolates that do not produce 16S rRNA MTases.
  
  
  METHODS
  PCR was performed to detect already-known amikacin resistance determinants. Cloning experiments and sequence analyses were performed to characterize unknown amikacin resistance determinants. Transfer of amikacin resistance determinants was performed by conjugation and transformation. The complete nucleotide sequence of the plasmids was determined by next-generation sequencing technology. Amikacin resistance enzymes were purified with a column chromatography system. The enzymatic function of the purified protein was investigated by thin-layer chromatography (TLC) and HPLC.
  
  
  RESULTS
  Among the 14 isolates, 9 were found to carry already-known amikacin resistance determinants such as aac(6')-Ia and aac(6')-Ib. Genetic analyses revealed the presence of a new amikacin acetyltransferase gene, named aac(6')-Ian, located on a 169 829 bp transferable plasmid (p11663) of the Serratia marcescens strain NUBL-11663, one of the five strains negative for known aac(6') genes by PCR. Plasmid p11663 also carried a novel ESBL gene, named blaTLA-3. HPLC and TLC analyses demonstrated that AAC(6')-Ian catalysed the transfer of an acetyl group from acetyl coenzyme A onto an amine at the 6'-position of various aminoglycosides.
  
  
  CONCLUSIONS
  We identified aac(6')-Ian as a novel amikacin resistance determinant together with a new ESBL gene, blaTLA-3, on a transferable plasmid of a S. marcescens clinical isolate.
authors.affiliation: Department of Bacteriology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.
authors.forename: Wanchun
authors.initials: W
authors.lastname: Jin
authors.affiliation: Department of Bacteriology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan wachino@med.nagoya-u.ac.jp.
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Wachino
authors.affiliation: Department of Bacteriology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.
authors.forename: Kouji
authors.initials: K
authors.lastname: Kimura
authors.affiliation: Department of Bacteriology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Yamada
authors.affiliation: Department of Bacteriology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.
authors.forename: Yoshichika
authors.initials: Y
authors.lastname: Arakawa
mesh_terms: D000123:Acetyltransferases / Q000235:genetics* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D002855:Chromatography, Thin Layer; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D022761:Gene Transfer, Horizontal; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D010957:Plasmids / Q000032:analysis*; D017422:Sequence Analysis, DNA; D016868:Serratia Infections / Q000382:microbiology*; D012706:Serratia marcescens / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification; D001618:beta-Lactamases / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial; D000123:Acetyltransferases; C056532:aminoglycoside N(6')-acetyltransferase; D001618:beta-Lactamases
keywords: S. marcescens; amikacin resistance; antibiotic resistance genes