Genomic Research On Mechanism Of Resistance And Dissemination In Pandrug-Rresistant Clinical Isolate Of Acinetobacter Baumannii W61

Acinetobacter baumannii has emerged as an important pathogen causing increasing health problems in the nosocomial setting, particularly intensive care units. an increased prevalence of drug-resistant A. baumannii isolates has been recently observed in hospitals worldwide. According to CHINET surveillance, the isolating rate of pan-drug resistant A. baumannii increased from17.0%in2009to21.4%in2010, higher than Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter spp. Multidrug and pandrug-resistant A. baumannii poses treatment challenge. Recent years, high-throughput sequencing technologies have developed rapidly, genome and comparative genomics provide insights into latest research platform in bacteriology. This study focuses on the mechanisms of multi-drug resistance and dissemination of A. baumannii clinical islate W61using microbiology, molecular biology and bioinformatics method, provide experimentation basis to use antibiotics reasonablely, avoid or reduce drug resistance and dissemination, develop new antibiotics and find new target site for antibiotics.Objective:1. To investigate the characteristic of the genome of a pan-drug resistant A. baumannii clinical isolate W61, analyze the phenotype and genotype of drug resistance in strain W61.2. To investigate the distribution of efflux systems in genome of pan-drug resistant A. baumannii clinical isolate W61, analyze the expression of13efflux pumps and2membrane proteins, explore the contribution of the efflux pumps and membrane proteins in multi-drug resistance of strain W61.3. To investigate the distribution of mobile genetic elements:integron, transposon, insertion sequences and plasmid, explore the mechanism of resistance dissemination.Methods:1. Strains, Susceptibility testing:A. baumannii W61was isolated from a hospital in Tianjin in2009, identified by routine tests, biochemistric tests and API20NE. Antibiotic susceptibility of16antibiotic agents （piperacillin/Tazobactam, cefotaxime, ceftazidime, ceftriaxone, cefoperazone, cefepime, cefoperazone/sulbactam, amikacin, gentamicin, levofloxacin, ciprofloxacin, ofloxacin, moxifloxacin, imipenem, meropenem, polymyxin） were tested by micro-dilution broth method and disk diffusion on Mueller-Hinton agar.2. High-density pyrosequencing and genome annotation:The genomic DNA of A. baumannii W61was extracted, fragmented and subjected to the complete sequencing work flow of the454genome sequencer FLX system （Roche）. Sequence assembly was performed by the454life sciences software program. The gaps were closed by sequencing PCR products. The genome sequence of W61was deposited in the GenBank data library （GenBank, Los Alamos, N.M.）. The chromosome and plasmid of A. baumannii W61were annotated by Bacterial Annotation System （BASys）, and analyzed the phenotype and genotype of drug resistance.3. Interference testing of CCCP and expression of efflux pumps and membrane proteins:The MICs of8antibiotics were determined by micro-dilution broth method with CCCP and without it. The distribution of efflux systems in genome of A. baumannii W61were characterized by using the TransDB database. A. baumannii ATCC17978and gene recA were selected as reference strain and reference gene. The expression of mRNA of13efflux pumps and2membrane proteins were detected by Realtime quantitative RT-PCR. The controlling gene adeRS and adeL were analyzed and aligned to the references sequences in GenBank.4. Distribution of mobile genetic elements:The distribution and characteristic of mobile genetic elements were analyzed by using the Genbank-nr and IS finder database to explore the mechanism of resistance dissemination.Results:1. Antibiotic susceptibility:A. baumannii clinical isolate W61was resistant to cephalosporin （sensitive to Pseudomonas）, carbopenems, sulbactam compound agents, floquinolones, aminoglycosides, sensitive to polymyxin, was determinded as a pan-drug strain.2. Genome annotation:Genome and2plasmids sizes of A. baumannii W61（GenBank accession no. CP003500） are approximately4million,77,528and110,967bp, respectively. G+C content of genome and plasmids were39.11%,34.05%and41.61%, respectively. The genome and plasmids were predicted to encode3,780,101and123proteins, respectively. The genome and plasmids contain a blaADC gene associated with [3-lactams resistance, aacA4, aadAl,aacCl and arm A genes associated with aminoglycosides resistance, gyrA mutation （Ser83Leu） associated with quinolones resistance, blaoxA-23and blaoxA-66gene associated with carbapenems resistance, sul gene associated with sulfamido resistance, catB8gene associated with chloramphenicol.3. Effect of CCCP and expression of efflux pumps and membrane proteins:The MICs of8antibiotics （Levofloxacin, ciprofloxacin, Ofloxacin, chloramphenicol, gentamycin, amikacin, ceftazidime, cefoperazone/sulbactam） in A. baumannii W61decreased at least4-fold in the presence of CCCP. The A. baumannii W61chromosome encodes8RND,6MFS,1ABC,2MATE and3SMR efflux systems. The difference of relative mRNA expression of adeB, abeM, ABTJ₀0035,ABTJ₀0276and ompA were significant between A. baumannii W61and ATCC17978. The gene adeRS of A. baumannii W61has mutations, however, no point mutation in adeL.4. Distribution of mobile genetic elements:A. baumannii W61has mobile genetic elements including2plasmids,2class1integron,23transposase genes,9kinds of insertion sequences and1AbaR-type resistance island. Class1integron including aminoglycoside degradation enzyme-coding genes, chloramphenicol acetyltransferase gene, and ISCR1binding a16S rRNA methylase gene, insert into3’termination of class1integron, formed approximately10kb-length resistance genes cluster. ISCR2carried efflux gene tetA.Conclusions:1. A. baumannii W61genome analyses （GenBank accession no. CP003500） have identified genes and gene mutation associated with resistance to β-lactams （blaADc）, aminoglycosides （aacA4, aadA1, aacC1,armA,strA,strB）, carbopenems （blaoxA-23, blaoxA-66）, sulfamido （sul）, chloramphenicol （catB8） and quinolones （gyrA mutation）. The genotype was concord with phenotype involved in pan-drug resistance.2. The MICs of antibiotics decreased in the presence of CCCP. The effect of efflux pumps contributes to multidrug resistance. Efflux systems were identified distributing in A. baumannii W61genome generally. The higher expression of adeB, abeM, ABTJ₀0035, ABTJ₀0276in A. baumannii W61may relate to the multi-drug resistance.3. Mobile genetic elements were identified distributing in A. baumannii W61genome generally, including plasmids, class1integron and ISCRs, carried resistance genes and formed a resistance genes cluster. Mobile genetic elements may contribute to the multi-drug resistance and resistance dissemination.