| ObjectiveTo study the homology of extensively drug-resistant Acinetobacter baumannii(XDRAB)and the ability to form biofilms,and to further understand the prevalence of Acinetobacter baumannii in the hospital;To investigate the adhesion ability,biofilm forming ability of 5 clinically available peptides antibiotics(polymyxin B,polymyxin E,vancomycin,bacitracin,teicoplanin)to XDRAB isolates,and its effect on gene expression(aba I,omp A,bfm R,bfms,csu E,pga A)of biofilm forming;To evaluate the synergisitic activities of 5 peptides antibiotics in combination with antibiotics against XDRAB in the planktonic and biofilm cells in vitro.To research the activity of 3 kinds of traditional Chinese medicine monomers(baicalin,berberine hydrochloride,quercetin dihydrate)with 4 kinds of antibacterial agents(imipenem,meropenem,polymyxin B,tigecycline)on the biofilm formation ability of XDRAB and to investigate the synergistic anti-biofilm activity of the combinations.Methods1.According to the guidelines from the Clinical and Laboratory Standards Institute(CLSI),Acinetobacter baumannii ATCC 19606,Staphylococcus aureus ATCC29213,Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 used as quality control strains in antimicrobial susceptibility testing(AST)were obtained from the American Type Culture Collection(USA)to determine the Minimum Inhibitory Concentrations(MICs)of 17 kinds of antibacterial drugs on clinically isolated Acinetobacter baumannii.2.Using multilocus sequence typing(MLST)and enterobacterial intergenic repeat consensus sequence PCR(ERIC-PCR)to type and detect the XDRAB and analyze its epidemiological characteristics.3.The growth of 5 peptides antibiotics(polymyxin B,polymyxin E,vancomycin,bacitracin,teicoplanin),3 traditional Chinese medicine monomers(baicalin,berberine hydrochloride,quercetin dihydrate),4 antibiotics(imipenem,meropenem,tigecycline,polymyxin B)against XDRAB within 24 h was determined by turbidimetric method.The concentrations that did not inhibit bacterial growth were selected as the working4.concentrations of subsequent experiments.5.The biofilm formation ability of Acinetobacter baumannii was determined by crystal violet staining.To study the effects of five peptides antibiotics on the biofilm formation ability of XDRAB at working concentrations.Using the chessboard crystal violet staining to investigate the synergistic anti-biofilm effects of traditional Chinese medicine monomers combined with antibiotics against XDRAB.6.Using colony plate counting method to study the effects of 5 peptides antibiotics on the adhesion ability of XDRAB at working concentrations.7.Real-time quantitative polymerase chain reaction(q RT-PCR)was used to study the expression of biofilm formation genes(aba I,omp A,bfm R,bfms,csu E,pga A)of 5peptides antibiotics at working concentrations of XDRAB.8.The checkerboard method,Fractional Inhibitory Concentration Index(FICI)and Fractional Biofilm Inhibitory Concentration Index(FBIC)were used to evaluate the synergistic effects of each of the 5 peptides antibiotics in combination with one of the five antibiotics in the planktonic and biofilm cells of XDRAB.Results1.The distribution of 45 clinical Acinetobacter baumannii isolates was mainly in the intensive care department(17 isolates,37.7%)and the respiratory intensive care unit(16 isolates,35.5%).Referring to the CLSI2020 standard,37 XDRAB isolates were detected,accounting for more than 80%.8 MDRAB isolates,accounting for 17.7%.Among them,The minimum inhibitory concentration(MIC)results of 45 Acinetobacter baumannii against 17 kinds of antibacterial drugs showed that the intermediate rate of levofloxacin was 8.9%,the sensitivity rate of minocycline was 8.9%,and the sensitivity rate of polymyxins was 86.7%,the intermediate rate was 13.3%,the sensitivity rate to tigecycline was 100%,and all other antibiotics were resistant.2.The 37 XDRABs all belonged to the IC2 cloning complex(CC92)of the international cloning group,among which 34 strains were ST208,followed by ST195 and ST368.ERIC-PCR typing results showed that 37 extensively drug-resistant Acinetobacter baumannii were mainly divided into two types,including type I(22strains)and type II(15 strains).3.Among the 45 clinical isolates of Acinetobacter baumannii,37 strains had biofilm-forming ability,accounting for 82.2%.Among them,the extensively drug-resistant Acinetobacter baumannii isolates were classified as strong positive(4strains),moderately positive(7 strains),weakly positive(19 strains)and negative(7strains)according to the biofilm formation ability.4.5 peptides antibiotics(polymyxin B,polymyxin E,vancomycin,bacitracin,teicoplanin)inhibited the biofilm formation of XDRAB strains(P<0.05)under0.25μg/m L,0.25μg/m L,8μg/m L,8μg/m L and 8μg/m L respectively.Among them,the inhibition rate of polymyxin E was more than 50%,and the effects could be ranked as:polymyxin E > polymyxin B > vancomycin > teicoplanin > bacitracin.5 peptides antibiotics had different degrees of inhibition on the adhesion of XDRAB strains at working concentrations(P<0.05).the effects could be ranked as: polymyxin E >teicoplanin > polymyxin B > bacitracin > vancomycin.5 peptides antibiotics had different degrees of inhibition on the expression of biofilm formation genes(aba I,omp A,bfm R,bfms,csu E,pga A)in XDRAB strains at working concentrations.Among them,vancomycin and bacitracin can significantly inhibit the expression of biofilm formation genes(P<0.01),polymyxin B and polymyxin E inhibited the biofilm formation mainly by inhibiting the expression of omp A,bfm R and bfm S(P<0.01).5.There was no significant difference in the antibacterial effects of the five peptides antibiotics against XDRAB in the planktonic and biofilm cells,only the drug resistance of the latter was further enhanced.Polymyxin B and polymyxin E combined with imipenem showed 100% synergy in the planktonic cell,and polymyxin E with rifampicin,bacitracin with imipenem or meropenem showed 100% additive effects.In the biofilm cell,100% synergistic effects were detected when polymyxin B or polymyxin E combined with azithromycin,vancomycin combined with azithromycin,rifampicin,bacitracin with azithromycin or rifampicin,teicoplanin with tigecycline or rifampicin all showed 100% additive effects.6.Baicalin,berberine hydrochloride,quercetin dehydrate,imipenem,meropenem,polymyxin B and tigecycline were at 128 μg/m L,32 μg/m L,64 μg/m L,4 μg/m L,2μg/m L,0.25 μg/m L and 0.0625 μg/m L did not inhibit the growth of bacteria within 24 hours.Using this concentration as the basis for multiple dilutions,three concentration gradients of high,medium,and low are set.Compared with the model control group,3traditional Chinese medicine monomers,4 antibacterial agents,which single use or combined use at high concentrations could significantly inhibit the biofilm formation of XDRAB(P<0.05),and the combination was better than that of single use(P<0.05),showing different degrees of synergistic anti-biofilm activities.Among them,the 9combinations of tigecycline and baicalin could significantly inhibit the biofilm formation of XDRAB.The combination of 0.015625 μg/m L tigecycline and 32 μg/m L baicalin had the smallest dose.Conclusion1.The detection rate of XDRAB in our hospital was as high as 80%,and the clinically isolated XDRAB had strong biofilm forming ability,and CC92 was the major clonal complex prevalent in nosocomial infections of XDRAB.2.5 peptides antibiotics had different degrees of inhibition on the adhesion,biofilm formation and the expression level of biofilm forming genes(aba I,omp A,bfm R,bfms,csu E,pga A)of XDRAB at working concentrations.3.5 peptides antibiotics combined with 5 other antibiotics had different degrees of synergistic or additive antibacterial effect on XDRAB in planktonic and biofilm cells,especially based on polymyxins,combined with carbapenems or azithromycin is ideal for treating both forms of XDRAB infection.4.3 traditional Chinese medicine monomers(baicalin,berberine hydrochloride,quercetin dihydrate),4 antibacterial agents(imipenem,meropenem,polymyxin B,tigecycline)had different degrees of inhibition on the biofilm formation of XDRAB when used alone or in combination,and the best combination of anti-biofilm effects was baicalin with tigecycline. |
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