| BackgroundThe infection of multidrug-resistant Acinetobacter baumannii(MDRAB)has become a difficult problem in clinical anti-infection treatment.Although antimicrobial agents combination such as carbapenems(CBPs)and tigecycline(TGC)are the most frequently used and effective treatment options,increasingly reports of carbapenem-resistant A.baumannii(CRAB)clinical isolates all over the world,especially carbapenem and tigecycline-resistant A.baumannii(CTRAB)isolates,which is worth the medical attention.The detection rate of CTRAB clinical strains is about 5%abroad.According to a report by CHINET,the resistance rate of MDRAB to TGC has stabilized at around 8%in the past three years.Therefore,understanding the ability of Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry(MALDI-TOF MS)to rapidly identify A.baumannii complex,studying the prevalence and drug resistance mechanisms of CTRAB,it is hoped that it will help delay the useful life of alternative antimicrobial agents.Current studies have shown that the mechanism of resistance to CBPs among A.baumannii isolates is mainly to produce Carbapenem-Hydrolyzing Class D P-Lactamases(CHDLs),including OXA-23,OXA-51,OXA-24,OXA-58,OXA-143 and metallo-β-lactamases like New Delhi metallo-β-lactamase 1(NDM-1).Bacteria can acquire multidrug resistance through horizontal gene transfer,which poses a huge challenge for clinicians.The resistance mechanism of tigecycline among A.baumannii isolates was mainly active efflux pump system.The synergistic effect of carbapenemases and efflux pumps may lead to high level of resistance to antibiotics in A.baumannii.Therefore,revealing the role of carbapenemase and efflux pumps in CTRAB clinical isolates and it is useful for clinical anti-infection treatment and new drug development.Objective(1)To understand the ability of MALDI-TOF MS to rapidly identify A.baumannii complex.(2)To investigate the prevalence of CRAB and CTRAB clinical strains in our hospital.(3)To investigate the prevalence of CHDLs and MBLs genes in MDRAB clinical strains.(4)To study the role of two-component regulatory elements and RND efflux pumps family in CTRAB clinical strains.(5)To study inhibitory effect of efflux pump inhibitor Carbonyl Cyanide 3-Chlorophenylhydrazone(CCCP)for CTRAB isolates.Methods(1)A total of 140 non-repetitive A.baumannii isolates identified by the BD Phoenix-100 automatic microbiology system from Renmin Hospital of Wuhan University in 2017 were collected.MALDI-TOF MS were used to quickly identify and 16S rDNA sequencing for validation.To screen antimicrobial agents susceptibility using K-B method and BMD method was used to confirm MICs.The results were determined by referring to the standards of the CLSI and FDA of the United States in 2016,and finally using GraphPad Prism 7 software statistical analysis.(2)CHDLs genes were amplified by multiplex PCR,the MBLs and efflux pump genes were amplified by conventional PCR to investigate the local distribution of drug resistance gene.The conjugation assay was used to understand the potential for horizontal transfer of the locally endemic carbapenemase gene,and CCCP phenotype test to understand the role of reversal of efflux pump inhibitor.16S rRNA as an internal reference gene and ATCC 19606 as a reference strain,the relative quantitative 2-δδCt method was used to calculate the relative expression level of AdeB,AdeG,AdeJ genes.In addition to AdeRS sequencing analysis was used to determine the role between RND-type efflux pump and CTRAB isolates.Finally using SPSS 22 and GraphPad Prism 7 software for statistical analysis.Results(1)A total of 140 A.baumannii complex strains were collected.134 A.baumannii strains and 6 AP strains were identified by MALDI-TOF MS.However,the result of 16S rDNA sequencing showed 124 A.baumannii strains,10 AN strains and 6 AP strains.Among the 124 A.baumannii isolates,intensive care unit and respiratory specimens were the most common department and specimen type respectively.Resistance rates were for minocycline and tigecycline,at 21%and 3.2%,respectively.Others were up to 80%.(2)After the first part of the experiment,64 MDRAB strains were selected,including 60 CRAB strains and 4 CTRAB strains.Both blaOXA-23 and blaOXA-23 carbapenemase genes were amplified in both groups.However,other carbapenemase genes such as blaOXA-24,blaoxA-58,blaOXA-143 and MBLs failed to detect.The conjugation assays failed and the gene may be based on vertical transmission.In addition,the detection rates of AdeB,AdeJ and AdeG were 91.4%,97.1%and 100%respectively in the CRAB group.The positive rates of AdeSR two-component regulatory system were 91.4%and 85.7%.There is no significant difference between CTRAB group and CRAB group in detection rate of efflux pump.The mean 士 SD of AdeB,AdeJ and AdeG in CRAB group were 10.73±1.435 6.59±0.84 and 16.72 ± 2.10,respectively.The mean±SD of AdeB,AdeJ and AdeG in CTRAB group were 6.30±0.22 and 5.47±0.38,9.13 ± 0.33.Compared with CRAB group,the relative expression level of AdeB in CTRAB group increased by 29 times.There are two amino acid substitutions of AdeS by NCBI alignment,including K125N and V279A.At the CCCP working concentration of 5 μg/ml,the TGC MIC of two CTRAB decreased by 4-fold and the TGC MIC of one decreased by 2-fold.Conclusion(1)MALDI-TOF MS can quickly identify the A.baumannii complex strians.(2)Most of the A.baumannii clinical isolates isolated from our hospital are MDRAB strains and the detection rate were 3.2%and 79%for CTRAB and CRAB,respectively.Tigecycline still shows broad-spectrum activity against MDRAB strains.(3)OXA-23 carbapenemase is the main reason for the prevalence of CRAB in our hospital.(4)K125N and V279A substitution in AdeS may lead to RND-type efflux pump AdeABC overexpression in CTRAB strains.(5)CCCP partially reverses the efflux pump of CTRAB strains. |
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