1. Prof. Hee Joo, Lee June, 8, 2009

2. Introduction P. aeruginosa –Nosocomial pathogen –Ability to acquire resistance to several antibiotics –Emergence of MDR P.aeruginosa Resistance to carbapenem MIC ≥16 ug/ml Amikacin MIC ≥ 32 ug/ml Fluroquinolone MIC ≥ 4 ug/ml

3. Bacterial resistance –Decreased membrane permeability –Modification of 16S RNA or ribosomal proteins –Enzymatic modification of aminoglycosides In P. aeruginosa : primarily to the aminoglycoside modifying enzyme –AAC(1), AAC(2’), AAC(3’), AAC(6’) based on the acetylation site of the aminoglycoside –AAC(6’) : most prevalent form –AAC(6’)-I : resistance to AMK but not to GM

4. AAC(6’)-Iae gene kit was developed –Two AAC(6’)-Iae negative strains were identified. –Each of these isolates contained a novel aminoglycoside aac(6’)-Iaf Report the Structure of the gene and the properties of its product

5. Materials & methods (I) Bacterial strains & plasmids –Two P. aeruginosa isolates UTI Decubitus ulcer Antimicrobial agents –AMK, IPM, arbekacin, dibekacin, aztreonam, ceftazidime, gentamicin, neomycin B & C mixture, isepamicin, netilmicin, sisomicin,KM, polymyxinB, meropenem, ofloxacin, piperacillin, piperacillin/tazobactam, tobramycin

6. Materials & methods (II) In vitro susceptibility tess : microdilution methods Serotyping : O serotype of isolates Detection of MDR P. aeruginosa using a loop- mediated isothermal amplification (LAMP) method and an agglutination tests PFGE :

7. Materials & methods (III) PCR amplification of class I integrons –PCR –Genetically mapped –Expand high fidelity PCR system was used DNA sequencing Plasmid extraction Transformation using plasmid preparation from P. aeruginosa IMCJ798 & IMCJ799

8. Materials & methods (IV) Transfer of aminoglycoside resistance Genome typing by I-Ceul digestion & southern blot hybridization Cloning of aac(6’)-1af gene Site directed mutagenesis Construction of AAC(6’)-1af-overexpressing strains Purification of recombinant AAC(6’)-1af Purification of native AAC(6’)-1af from P. aeruginosa Western blotting TLC analysis of acetylated aminoglycosides Nucleotide sequence accession number

9. Results & discussion

12. Identical

13. 552 bp (GC 34.4%)

14. Location of In123 : On a chromosome PFGE Southern hybridization 동일한 위치

15. Comparison of AAC(6’)-1af with other AAC(6’)-1 enzymes

17. Effects of aac(6’)-1af on aminoglycoside resistance

18. Acetylation activity of AAC(6’)-1af