The Correlation Between BlaOXA-23 Copy Number And Carbapenem Resistant Level In Acinetobacter Baumannii

Objective:Verify the correlation between blaOXA-23 copy number and carbapenem resistant level in Acinetobacter baumannii.Methods:Two Acinetobacter baumannii strains,XH386 and XH731,were employed in this study and PCR screening was performed to ensure the absence of other carbapenemase encoding genes.Two strains each processing single copy of blaOXA-23 which was carried by a chromosomally-located transposon Tn2009(XH386)or Tn2006(XH731).A 14-day serial passage examination was designed in this study and the Mueller-Hinton(MH)Broth supplemented 16 ?g/ml imipenem was used for culturing the two strains.Same strains were also cultured in imipenem-free broth and used as a control.The entire experiment was repeated three times,independently,as biological replicates.Antimicrobial susceptibility testing was performed using agar dilution method to determine the MIC of imipenem of each strain after serial passage.blaOXA-23 copy number and expression level were detected using qRT-PCR.The growth rate was estimated based on the OD600 curve of each strain.The complete genomes of replicate strains for both XH386 and XH731 on Day 14 were sequenced on PacBio RS II sequencer and compared with the genomes prior to imipenem treatment.Results:The initial imipenem MIC of both XH386 and XH731 were 24 ?g/ml.During the serial passage,the MIC for XH386 increased to 64-80 ?g/ml sharply immediately after imipenem treatment,but the MIC for XH731 ascended to 48-64 ?g/ml more gradually.The blaOXA-23 mRNA expression level of XH386 and XH731 were higher than respective control and reached peak level at the midanaphase of serial passage,exhibiting an unstable trajectory.The copy number of blaOXA-23 increased during the serial passage,but the number of days required for the MIC and copy number to reach their maximum were not identical,the former was prior to the later,generally.Ultimately,the copy number of blaOXA-23 in XH386 and XH731 varied between 3-4 and 1-2 respectively but remained stable at 1 in control.The growth rate of XH386 and XH731 were lower than control.The complete genomes of replicate strains for both XH386 and XH731 on Day 14 were sequenced and compared with the genomes prior to imipenem treatment and the blaOXA-23 copy number was basically consistent with that measured by qRT-PCR.The blaPXA-23-carrying transposon was the basic unit of multiplication.For XH386,the single-copy Tn2009 increased to four copies via tandem duplication.For XH731,Tn2006,which was carried by Tn6022,duplicated at a novel genomic integration site.Tn2009 and Tn2006 were flanked by two copies of ISAbal,whose orientation was same in the former but inversely orientated in the later.Conclusion:During the serial passage,MIC,blaOXA-23 copy number and expression level increased variously but synchronized with one another,other factor also contribute to increase the resistant level of carbapenem.Carbapenem resistance in CRAB is multifactorial,blaOXA-23 copy number was,but not the sole determinant.blaOXA-23 multiplication was achieved by transposons duplication but the mechanisms were different.The same orientation of ISAbal elements surrounding Tn2009 provided the homology for recombination and multiplication was achieved by non-equal homologous recombination.The ISAbal elements around Tn2006 were inversely orientated and multiplication was achieved by replicative transposition.Generally,Tn2009 seemed duplicate more efficiently than Tn2006.