| Objective:To investigate the clinical isolates of drug resistance of Acinetobacter baumannii and analyze the homology and molecular epidemiology of drug-resistant A.baumannii.To explore the relationship between carbapenemase gene,insertion sequence,efflux pump gene,biofilm-related gene,and integron and drug resistance in A.baumannii.The expression differences of extensively drug-resistant Acinetobacter baumannii(XDRAB)biofilm genes were compared before and after the biofilm formation.The roles of XDRAB biofilm-related genes in both biofilm and drug resistance were analyzed.The treatment plan for XDRAB infection was screened by the combination of antibiotics and drug sensitivity tests.Methods:1.The minimum inhibitory concentration(MIC)of 17 kinds of antibacterial drugs against 59 strains of A.baumannii was determined by the microbroth method.2.Multilocus sequence typing(MLST)was used to analyze the homology of 59 strains of A.baumannii.3.The carbapenemase genes of 59 strains of A.baumannii were amplified by polymerase chain reaction(PCR),including sim,vim,imp,oxa-23,oxa-24,oxa-51,oxa-58,and the inserted sequence genes were ISAba1,ISAba1-oxa-23,ISABa1-oxa-51.Efflux pump genes: ade R,ade S,ade B,ade J,ade G;Biofilm-related genes: bfm R,bfm S,csu A/B,csu A,csu B,csu C,csu D,csu E,aba I,eps A,pga A,pga B,pga C,pga D,pgl C,bap,omp A,and Class I,II and III integrase genes: int I-1,int I-2 and int I-3,and amplified and sequenced the variable region of Class I synthase-positive strain and ISABa1-oxa-51 positive strain.4.The biofilm formation of 40 XDRAB strains was detected by crystal violet staining.The microbroth method was used to determine the MIC of 17 antibacterial drugs on 28 XDRAB strains after biofilm formation.The expression levels of the biofilm-related genes bfm R,bfm S,aba I,csu E,bap,and omp A of XDRAB in the floating state and biofilm state were detected respectively by quantitative real time PCR(q RTPCR)5.The MIC of minocycline combined with carbapenem antibiotics(imipenem and meropenem),special grade antibiotics(polymyxins and tigecycline),biofilm inhibitors(azithromycin,rifampicin,and esacridine lactate)on 12 strains of XDRAB was evaluated by microcheckerboard dilution method and fractional inhibitory concentration index(FICI).The time bactericidal curves of minocycline alone and combined with imipenem,meropenem,polymyxin,tigecycline,azithromycin,and esacridine lactate against XDRAB were plotted.Result:1.Except for polymyxin B and tigecycline,the resistance rates of strains of A.baumannii were 67.80% and 71.19% to imipenem and meropenem were 67.80% and71.19%59.32%-84.75% to other antibacterial agents,but only 1.69% to minocycline.Among the 59 strains,40(67.80%)were XDRAB,5(8.47%)were multidrug-resistant A.baumannii(MDRAB),and 14(23.73%)were sensitive.2.A total of 12 ST types were detected in 59 strains of A.baumannii,and ST208 accounted for 64.4%(38/59),belonging to the international clone complex(CC)92.Six new sequence types,ST2474,ST2484,ST2485,ST2486,ST2488,and ST2489,have been found in sensitive A.baumannii and have been uploaded to the MLST database(http: //pubmlst.org/).3.The results of drug resistance gene amplification in 59 A.baumannii strains were as follows:(1)The detection rates of carbapenemase gene: oxa-23 and oxa-51 were 71.19% and 100%,respectively.sim,vim,imp,oxa-24,oxa-58 are not detected.(2)the detection rate of the Insertion sequence gene: ISAba1 was 76.27%.The detection rate of ISAba1-oxa-51 was 5.08%,and ISAba1-oxa-23 was not detected.(3)Efflux pump genes: The detection rates of ade J and ade G were 100%,and the detection rates of ade R,ade S,and ade B were 79.66%,81.36%,and 83.05%,respectively.(4)Biofilmrelated genes: The detection rates of bfm R,bfm S,csu C,csu D,csu E,pga D,and gac S were all 100%,and the detection rates of the other 12 biofilm-related genes ranged from74.58 to 98.31%.(5)Class I,II,and III integrase genes: The detection rate of int I-1 was66.10%.int I-2,int I-3 is not detected.(6)Class I integron resistance gene boxes: aac A4,cat B8,and aad A1.(7)oxa-23,ade R,ade S,ade B,ISAba1,aba I,eps A,pgl C,omp A and int I-1 for XDRAB The detection rates of 10 drug-resistant genes were 100%,92.50%,100%,100%,100%,100%,95.00%,87.50%,100%,90.00%,respectively.The detection rates of sensitive A baumannii were 0,42.86%,50.00%,57.14%,7.14%,64.29%,7.14%,42.86%,50.00%,and 0,respectively,and the detection rates of XDRAB were higher than those of sensitive A.baumannii(all P < 0.05).4.70.00%XDRAB(28/40)could form different degrees of biofilm,including 4strains of strongly positive biofilm bacteria,3 strains of moderately positive biofilm bacteria,and 21 strains of weakly positive biofilm bacteria.The MIC of the XDRAB biofilm state was further increased than that in floating state.The expressions of biofilm-related genes bfm S,aba I and bap were significantly increased(all P < 0.05).5.The results of synergistic antibacterial experiments showed that when minocycline was combined with imipenem and meropenem,33.33% and 41.67%showed synergistic effect and 66.67% and 58.33% showed additive effect.Combined with polymyxin B and tigecycline,50.00%,and 58.33% showed synergistic effect,and50.00% and 41.67% showed additive effect;Combined with azithromycin,rifampicin,and esacridine lactate,66.67%,8.33% and 50.00% showed synergistic effect,33.33%,91.67% and 50.00% showed additive effect.The time-bactericidal experiment suggested that minocycline combined with imipenem,meropenem,polymyxin B,tigecycline and eshamaline lactate had synergistic effect,and the combination of minocycline combined with polymyxin had the best synergistic effect.Conclusion:1.The clinical isolates of A.baumannii were seriously resistant to common antibiotics,with XDRAB as high as 67.80%,but sensitive to tigecycline and polymyxin,and resistant to minocycline only 1.69%.The main prevalent ST type was ST208,and CC92 was still the main clonal complex of nosocomial infection.2.The mechanism of XDRAB resistance is complex and influenced by multiple mechanisms and genes.The carbapenemase gene oxa-23,efflux pump genes ade R,ade S,ade B,insertion sequence ISAba1,biofilm-related genes aba I,eps A,pgl C,omp A and Class I integrons were closely associated with XDRAB resistance.3.XDRAB biofilm formation rate as high as70.00%,and the bfm S,aba I,bap,and omp A genes were involved in biofilm formation.In the biofilm state,XDRAB further enhanced its resistance to imipenem,meropenem,polymyxin,tigecycline,and other antimicrobial agents by regulating the expression of two-component regulatory system bfm S,quorum-sensing system aba I,biofilm-associated protein bap,and outer membrane protein omp A.4.The combination of minocycline and 7 antibacterial agents had synergistic or additive antibacterial effects on XDRAB to varying degrees.Combined with the experimental results of the two combined regiments of microcheckerboard dilution method and time bactericidal curve,the combination regimen of minocycline with polymyxin B and meropenem was recommended,which had the best efficacy. |
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