Acinetobacter Baumannii Double Ring Of The Aminoglycoside Resistance Phenomenon

Acinetobacter baumannii is an important opportunistic pathogen responsible for severe nosocomial infections, especially in intensive-care-unit (ICU) patients. The majority of epidemic origins are resistant organism, and treatment has become difficult because many strains are resistant to a wide range of antibiotics, including broad-spectrumβ-lactams, aminoglycosides, and fluoroquinolones.Aminoglycoside antibiotics are widely used in clinical settings, especially for treatment of life-threatening infections caused by gram-negative bacteria. These agents bind to the highly conserved A-site of the 16S rRNA of the prokaryotic 30S ribosomal subunits, interfering with the protein synthesis with subsequent bacterial death. The mechanisms of resistance to aminoglycosides mainly include aminoglycoside modifying enzymes, ribosomal alterations, efflux of the agents by extrusion pump, or altered permeability leading to reduced uptake.The main aim of present study was(1) to explore the presence of resistance genes in Acinetobacter baumannii which is double-circle resistant to aminoglycoside antibiotic in K-B methods; (2) to analyze the expression levels of resistance genes mRNA among strains induced and non-induced by amikacin; and (3) to study the accuracy of the susceptibility test of Acinetobacter baumannii to aminoglycoside antibiotic detected by VITEK 2 Compact method and the reason of detection error. The integrase gene and the variable region of class integron were analyzed by PCR and RFLP. The DNA sequencing was used to identify the gene cassettes of variable integrons. Meanwhile, the four frequent types of genes coding aminoglycoside-modifying enzyme and three kinds of 16S rRNA methylase genes-armA, rmtB and rmtC were amplified by PCR. And the expressions of positive gene in drug-induced strains and non-induced strains were respectively measured by PT-PCR. The result showed that Class I integrons were found in 85.7%(48/56) double-circle resistance strains. The RFLP and the DNA sequencing indicated that the cassettes carried in variable region of integrons were aacA4-catB-aadAl, aacCl-orpX-aadAl and dfrA17-aadA5. The aminoglycoside-modifying enzyme gene-aacA4, aacCl and aacC2 were respectively found in 36,10, and 5 strains, but aphA6 had not been detected. Besides, the armA was found in all strains, but the rmtB and rmtC were not detected. And none of the 16S rRNA methylase genes was found in the susceptible and common resistant strains. The result of PT-PCR revealed that the expression level of armA gene mRNA was significantly up-regulated when the strains were induced by amikacin. The error of result tested by VITEK 2 Compact were mainly found in strains which are double-circle resistant in K-B method, and almost all the strains are identified to be susceptible.Thus, we conclude that the main reason mediating the double-circle resistance of Acinetobacter baumannii to aminoglycoside antibiotic is not the variable region of class I integron but. The methylase, which was produced by inducible expression of armA methylated 16S rRNA. Subsequently, the methylation hampered binding of amikacin to the 30S ribosomal subunits. In addition, because the turbidity of these armA positive strains was low in liquid medium, the densitometric scan of VITEK 2 Compact can't distinguish whether the strains were inhabited and erroneously identified the growth was inhabited. Therefore, the error affected the accuracy of clinical report.