title: Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa.
abstract: BACKGROUND
  Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance.
  
  
  METHODS
  We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997.
  
  
  RESULTS
  An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses.
  
  
  CONCLUSIONS
  Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.
authors.affiliation: Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Yohei
authors.initials: Y
authors.lastname: Doi
authors.forename: Kunikazu
authors.initials: K
authors.lastname: Yamane
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kurokawa
authors.forename: Naohiro
authors.initials: N
authors.lastname: Shibata
authors.forename: Keigo
authors.initials: K
authors.lastname: Shibayama
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yagi
authors.forename: Haru
authors.initials: H
authors.lastname: Kato
authors.forename: Yoshichika
authors.initials: Y
authors.lastname: Arakawa
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics*; D020459:Genes, rRNA / Q000235:genetics*; D008780:Methyltransferases / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase