Study Of Plasmid-mediated 16S RRNA Methylase Genes And Its Drug-resistant Transferability In Acinetobacter Baumannii Isolates From Burn Ward

Objective:The distribution of Acinetobacter baumannii(Ab) is only after the Pseudomonas aeruginosa in Gram-negative bacteria,and following the widely use of broad-spectrum antimicrobials and antibiotic selective pressure,Acinetobacter baumannii which became important pathogen of bum ward infection outbreak has increased quickly.With high efficiency,broad-spectrum,excellent pharmacokinetic properties and synergy with other antibacterial drugs,aminoglycosides have been mainly used for the threatment of infections caused by gram-negative bacteria.Both domestic and abroad research of resistance mechanism was more concentrated on the aminoglycoside-modifying enzymes.Recently,European and Japanese scholars have found a new mechanism of aminoglycosides resistance-16S rRNA methylase.These genes located on plasmids and transposons,had disseminative and diffusive characteristics.It may become a clinical problem.The nosocomial infection caused by armA,rmtB-positive Klebsiella pneumoniae and Escherichia coli was reported in Taiwan in 2005.The futher global dissemination of 16S rRNA methylase genes will be a cause of great concern in the near future.Therefore,this study was aimed to investigate the drug-resistance of Acinetobacter baumannii clinical isolated from Burn Department,to understant the incidence of 16S rRNA methylase genes which mediate high-level aminoglycoside drug-resistance and to explore its transferable mechanism,providing theoretical basis for aminoglycoside rational application and ncw drug research and development.Methods:Total of 40 Ab clinical isolates were collected from burn ward in Gansu Province People's hospital from May.2006 to Dec.2007.The sensitivity of Ab for 20 antibiotics were determinated by K-B agar diffusion.The minimal inhibitory concentrations(MIC) of amikacin,gentamycin,tobramycin,netilmicin,isepamicin and kanamycin against Ab strains were determinated by agar dilution.Five kinds of 16S rRNA methylase genes including armA,rmtA,rmtB,rmtC,rmtD were amplified by PCR,the positive PCR-products were purified and sequenced,the plasmid were extracted by alkaline lysis.The transferability of drug-resistence were determinated by conjugation and plasmid transformation tests.Results:The drug-resistence rates of 40 Ab to Imipenem,Meropenem, Cefoperazone/Sulbactam were 42.5%,45.0%,50.0%;To Cefoperazone,Aztreonam, Cefoxitin were over 90%;To six aminoglycosides antibiotics were 72.5%,72.5%, 70.0%,67.5%,70.0%,70.0%,respectively.Twenty five stains were resistant to six aminoglycosides antibiotics(62.5%),among which 10 isolates were armA-positive (40.0%);rmtA,rmtB,rmtC and rmtD-positive isolates were not found.The experiments of plasmid transformation and conjugation were successful.Ten transformants and 10 conjugons showed high-level resistance against aminoglycoside antibiotics,all of which the value of MIC＞256μg/mL carried armA gene.Conclusions:The Ab clinical isolates from burn ward in Gansu Province People's hospital have high drug-resistance.16S rRNA methylases gene exsist in Ab and locate in plasmid chromosome.They are easily disseminative,which demonstrated a very high level of resistance to various aminoglycosides.