| ObjectiveTo investigate the drug resistance mechanism of extensively-drug-resistant Acinetobacter baumannii to carbapenems from the aspects of drug resistance phenotype,carbapenemase-related genotype,efflux pump and outer membrane protein,biofilm formation and so on.The possible drug resistance mechanism was studied to supply some theoretical basis for clinical prevention,treatment and infection control.Methods1.Retrospective investigation of drug resistance of clinical isolates of Acinetobacter baumannii;identification of strains by automatic microbial identification drug susceptibility analyzer,and detection of susceptibility to commonly used antibiotics by broth dilution method;according to CLSI 2016 standard,strains were divided into polymyxin-only or minocycline-sensitive as the main manifestations of the broad.Follow up experiments were carried out in two groups of extensively-drug-resistant Acinetobacter baumannii(XDRAb,25 strains)and common Ab(23 strain).2.Modified EDTA double disk synergy method and carbapenem deactivation method were used for detection of metal ?-lactamases and carbapenem enzyme in two groups of strains;double-disk synergistic assay and modified three-dimensional test were respectively used for detection of chromosome and plasmid-mediated AmpC enzyme.3.The efflux pump phenotypes of XDRAb and common Ab groups were detected by CCCP combined with antibiotics inhibition test,and the expression or deletion of outer membrane protein of Acinetobacter baumannii was detected by SDS-PAGE electrophoresis.4.The biolfilm formation ability of Acinetobacter baumannii was determined by crystal violet staining before and after the addition of zinc lactate and stannous fluoride,and the sensitivity of the inhibitor combined with imipenem or meropenem to Acinetobacter baumannii was detected by checkerboard dilution.5.Polymerase chain reaction(PCR)was used to detect the translocation of blaOXA-23.blaOXA-24,blaTEM,blaAmpC,blaVIM,blaNDM and efflux pump-related drug-resistant genes ade A,B,regulatory genes ade R,S,outer membrane protein-related CarO,OprD genes.The amplified products were observed and photographed by agarose gel electrophoresis.The expression of outer membrane protein gene and efflux system gene was detected by the method of real-time quantitative RT-PCR,6.Some positive products of PCR amplification were extracted and sequenced in two directions.The sequences were spliced and analyzed by homology alignment to determine whether Acinetobacter baumannii carries the genes related to drug resistance and to find out whether there are missense mutations.Results1.Extensively drug-resistant Acinetobacter baumannii accounted for 62.5%of clinical isolates,and the resistance rate of common Ab to imipenem and meropenem was 39.6%.XDRAb was mainly distributed in ICU,respiratory medicine,neurology and brain surgery.2.Among 25 strains of XDRAb,21 strains of XDRAb and 23 strains of ordinary Ab were negative for carbapenemase,21 strains of XDRAb and 23 strains of ordinary Ab were positive for metallo-beta-lactamase,and the other 23 strains of XDRAb were plasmid-mediated AmpC,while the other 23 strains of common Ab were all negative.3.Among 25 strains of XDRAb and 23 strains of ordinary Ab,16 strains and 8 strains(X2 = 4.090,P = 0.043)were positive for efflux pump phenotype by IMP combined with CCCP inhibition test,and 14 strains and 6 strains(X2 = 4.410,P = 0.036)were positive for efflux pump phenotype by MEM combined with CCCP inhibition test.4.The biofilm formation rate of XDRAb and Ab was 96.3%and 95.8%,and the positive rate of biofilm inhibition test was 77.9%and 69.6%,respectively.There was no significant difference between the two groups(P>0.05).The resistance ratio of XDRAb to carbapenems decreased significantly before and after inhibiting biofilm formation,while the resistance of common Ab to carbapenems decreased not apparently.5.No metallo-beta-lactamase-related resistance gene blaVIM,blaNDM and efflux pump-regulating gene adeR were detected in 25 strains of XDRAb and 23 strains of ordinary Ab.The detection rates of the other nine target drug-resistant genes except blaTEM were statistically significant(P<0.05).The relative expression of efflux pump gene mRNA in 11 strains of XDR-Ab and 9 strains of ordinary Ab was detected by qRT-PCR.The relative expression levels of adeA,adeB and outer membrane protein CarO mRNA in XDRAb group were significantly higher than those in normal Ab group(P<0.001).6.Sequencing results of some positive PCR products indicated that the nucleotide homology of adeA gene was 91%in strains N9 and N11,OprD gene was 95%in strains N17,and the nucleotide homology of all the other strains were 98%-100%.Conclusion1.The clinical isolates of Acinetobacter baumannii in this region mainly come from ICU,respiratory medicine and other departments.The drug resistance of Acinetobacter baumannii is serious and the isolation rate of extensively drug-resistant Acinetobacter baumannii is high.2.The main mechanism mediating carbapenems resistance in Acinetobacter baumannii was plasmid-mediated persistent high-yield of AmpC enzyme,followed by efflux pump ade ABC and outer membrane protein CarO,carbapenem hydrolase,chromosome-mediated AmpC enzyme and metallo-beta-lactamase-mediated resistance.3.BlaOXA-23,blaAmpC,adeA,adeB,carO are the main genes that mediate the carbapenem resistance of Acinetobacter baumannii,but no metallo-beta-lactamase-related resistance genes are detected.The changes of gene sequences encoding efflux pump and outer membrane proteins may be related to carbapenem resistance.4.Biofilm formation is a common biological characteristic of Acinetobacter baumannii,and the mechanism of drug resistance mediated by it has no statistical significance. |
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