泛耐药鲍曼不动杆菌临床株W61耐药与播散机制的基因组学研究
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摘要
鲍曼不动杆菌(Acinetobacter baumannii, Ab)是医院内感染中常见的一种病原菌,其耐药问题日趋严重,中国CHINET细菌耐药性监测报告,2010年鲍曼不动杆菌泛耐药株分离率高达21.4%,较2009年17.0%又有所增加,且均高于同年铜绿假单胞菌、肺炎克雷伯菌、肠杆菌科细菌的泛耐药株分离率。多重耐药和泛耐药的鲍曼不动杆菌,已成为临床亟待解决的棘手难题。近年来,随着高通量测序技术的快速发展,基因组学和比较基因组学的研究方法为细菌学领域引入新的研究平台。本实验采用微生物学、分子生物学和生物信息学分析方法揭示泛耐药鲍曼不动杆菌临床株W61的多重耐药和耐药播散的机制,为指导临床合理使用抗菌药物,避免或减少细菌耐药性的出现,防控细菌耐药播散,研发新型抗菌药物和新作用靶位提供依据。
研究目的
1、了解泛耐药鲍曼不动杆菌临床株W61的基因组基本特征,分析其多重耐药表型与耐药基因型。
2、了解泛耐药鲍曼不动杆菌临床株W61基因组中外排泵系统的分布,分析外排泵(AdeABC、AdeIJK、AdeFGH、AbeM、AbeS、CraA、MdtL、AmvA及ABTJ_00034/35/36、ABTJ_00275/276/277等5个功能未知的外排泵)和外膜蛋白(OmpA、OmpW)的表达,探索外排泵和外膜蛋白在菌株多重耐药中的作用。
3、了解泛耐药鲍曼不动杆菌临床株W61基因组中可移动元件(整合子、转座子、插入序列、质粒)的分布,探讨耐药播散机制。
研究方法
1、菌株收集与药敏试验：收集自2009年天津市某三级甲等医院临床分离的鲍曼不动杆菌W61,经常规、生化、API20NE纸条鉴定。采用微量肉汤稀释法和琼脂纸片扩散法测定鲍曼不动杆菌W61对16种常用抗菌药物(哌拉西林他唑巴坦、头孢氨噻肟、头孢他啶、头孢曲松、头孢哌酮、头孢吡肟、头孢哌酮／舒巴坦、阿米卡星、庆大霉素、左氧氟沙星、环丙沙星、氧氟沙星、莫西沙星、亚胺培南、美罗培南、多粘菌素)的MIC值(或抑菌圈直径),确定其耐药表型。
2、全基因组高通量测序与序列分析：应用Roche454FLX测序平台,对鲍曼不动杆菌临床株W61进行全基因组测序,利用相关软件和聚合酶链式反应(PCR)填补空缺片段,拼接完整的基因组序列,提交Genbank。应用BASys细菌注释系统进行基因组和质粒的基因注释,分析菌株耐药表型与耐药基因型的关系。
3、泵抑制剂干预试验与外排泵及外膜蛋白基因表达检测：采用微量肉汤稀释法测定未加用和加用泵抑制剂CCCP时鲍曼不动杆菌临床株W61对8种抗菌药物(左氧氟沙星、环丙沙星、氧氟沙星、氯霉素、庆大霉素、阿米卡星、头孢他定、头孢哌酮/舒巴坦)的MIC值,了解泵抑制剂对菌株耐药表型的影响。应用TransDB数据库分析多重耐药株W61基因组中外排泵系统的分布情况。选取鲍曼不动杆菌ATCC17978为对照株,以recA为内参基因,采用实时荧光定量RT-PCR方法对鲍曼不动杆菌W61和ATCC17978的13种外排泵基因(adeB、 adeJ、adeG、abeM、abeS、craA、mdtL、amvA及ABTJ_00035、ABTJ_00276等5个功能未知的泵基因)和2种外膜蛋白基因(ompA、ompW) mRNA的表达进行相对定量分析。泛耐药株W61外排泵AdeABC的调控系统AdeRS、外排泵ABTJ_00034/35/36上游调控序列ABTJ_00033和外排泵AdeFGH的调控系统AdeL的结构基因,与GenBank公布的ATCC17978相应序列进行比对分析。
4、可移动元件分布：应用Genbank-nr和IS finder数据库,经序列比对分析鲍曼不动杆菌临床株W61基因组中可移动元件(整合子、转座子、插入序列、质粒)的分布及其携带的耐药基因,分析可移动元件在菌株耐药性的水平播散中的作用。
研究结果
1、药敏试验结果：鲍曼不动杆菌临床株W61对抗假单胞菌头孢菌素、碳青霉烯类抗菌药物、含舒巴坦的复合制剂、氟喹诺酮类抗菌药物和氨基糖苷类抗菌药物均耐药,对多粘菌素敏感,为泛耐药株。
2、基因组序列分析：泛耐药株W61基因组(Genbank登录号CP003500)全长3964912bp,平均GC含量为39.11%,包含3731个蛋白质编码基因,6个rRNA编码基因,74个tRNA编码基因；泛耐药株W61携带2个质粒,plasmidA全长77528bp,平均GC含量34.05%,含有109个蛋白质编码基因,plasmidB全长110967bp,平均GC含量41.61%,含有123个蛋白质编码基因。基因组携带：β-内酰胺类耐药基因ADC型ampC,氨基糖苷类耐药基因(盒)aacA4、aadA1、 aacC1、armA、strA、strB,氟喹诺酮类作用位点gyrA突变,碳氢霉烯类耐药基因oxa-23、oxa-66,磺胺类耐药基因sul1、sul2,氯霉素耐药基因盒catB8。
3、泵抑制剂对耐药表型的影响及外排泵与外膜蛋白基因表达情况：泛耐药株W61在使用CCCP后对8种抗菌药物(左氧氟沙星、环丙沙星、氧氟沙星、氯霉素、庆大霉素、阿米卡星、头孢他定、头孢哌酮／舒巴坦)的MIC值均较前下降4倍或4倍以上。泛耐药株W61基因组共包含RND家族外排泵8个,MFS家族外排泵6个,ABC家族外排泵1个,MATE家族外排泵2个,SMR家族外排泵3个。泛耐药株W61外排泵基因adeB、abeM、ABTJ_00035、ABTJ_00276和外膜蛋白基因ompA的mRNA相对表达水平高于对照株ATCC17978,其差异有统计学意义。调控基因adeRS、ABTJ_00033出现基因突变及缺失,而调控基因adeL序列无基因突变。外膜蛋白基因carO出现基因突变。
4、可移动元件分布情况：泛耐药株W61基因组携带可移动原件：质粒2个、Ⅰ类整合子2个、插入序列片段9类(包括ISCRs2个)、转座酶基因23个、AbaR型耐药岛1个。其中质粒、Ⅰ类整合子和ⅠSCRs均携带耐药基因,并构成耐药基因簇-复合Ⅰ类整合子和AbaR型耐药岛。
研究结论
1、泛耐药鲍曼不动杆菌W61基因组(Genbank登录号CP003500)全长3.96Mbp,包含2个质粒。基因组和质粒中携带β-内酰胺类耐药基因ampC、氨基糖苷类耐药基因(盒)aacA4、aadA1、aacC1、armA、strA、strB、碳氢霉烯类耐药基因oxa-23、oxa-66、磺胺类耐药基因sul1、sul2、氯霉素耐药基因盒catB8,存在氟喹诺酮类作用位点gyrA突变,与菌株泛耐药表型相一致。
2、泵抑制剂CCCP的应用,可使泛耐药鲍曼不动杆菌W61对多种抗菌药物MIC值较前降低,这表明外排泵功能对菌株W61多重耐药有一定贡献。外排泵系统在泛耐药鲍曼不动杆菌W61的基因组中广泛分布,外排泵AdeABC、AbeM、 ABTJ_00034/35/36、ABTJ_00275/276/277的高表达可能与菌株W61的多重耐药有关,调控基因adeRS和ABTJ_00033基因突变和氨基酸替换可能与外排泵AdeABC和ABTJ_00034/35/36表达上调有关。
3、可移动元件在泛耐药鲍曼不动杆菌W61的基因组中广泛分布,其中质粒、Ⅰ类整合子和ISCRs均携带耐药基因,并构成耐药基因簇-复合Ⅰ类整合子和AbaR型耐药岛,对泛耐药株W61多重耐药性的形成及水平传播可能有一定作用。
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, ABTJ_00035,ABTJ_00276and 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, ABTJ_00035, ABTJ_00276in 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.
研究目的
1、了解泛耐药鲍曼不动杆菌临床株W61的基因组基本特征,分析其多重耐药表型与耐药基因型。
2、了解泛耐药鲍曼不动杆菌临床株W61基因组中外排泵系统的分布,分析外排泵(AdeABC、AdeIJK、AdeFGH、AbeM、AbeS、CraA、MdtL、AmvA及ABTJ_00034/35/36、ABTJ_00275/276/277等5个功能未知的外排泵)和外膜蛋白(OmpA、OmpW)的表达,探索外排泵和外膜蛋白在菌株多重耐药中的作用。
3、了解泛耐药鲍曼不动杆菌临床株W61基因组中可移动元件(整合子、转座子、插入序列、质粒)的分布,探讨耐药播散机制。
研究方法
1、菌株收集与药敏试验：收集自2009年天津市某三级甲等医院临床分离的鲍曼不动杆菌W61,经常规、生化、API20NE纸条鉴定。采用微量肉汤稀释法和琼脂纸片扩散法测定鲍曼不动杆菌W61对16种常用抗菌药物(哌拉西林他唑巴坦、头孢氨噻肟、头孢他啶、头孢曲松、头孢哌酮、头孢吡肟、头孢哌酮／舒巴坦、阿米卡星、庆大霉素、左氧氟沙星、环丙沙星、氧氟沙星、莫西沙星、亚胺培南、美罗培南、多粘菌素)的MIC值(或抑菌圈直径),确定其耐药表型。
2、全基因组高通量测序与序列分析：应用Roche454FLX测序平台,对鲍曼不动杆菌临床株W61进行全基因组测序,利用相关软件和聚合酶链式反应(PCR)填补空缺片段,拼接完整的基因组序列,提交Genbank。应用BASys细菌注释系统进行基因组和质粒的基因注释,分析菌株耐药表型与耐药基因型的关系。
3、泵抑制剂干预试验与外排泵及外膜蛋白基因表达检测：采用微量肉汤稀释法测定未加用和加用泵抑制剂CCCP时鲍曼不动杆菌临床株W61对8种抗菌药物(左氧氟沙星、环丙沙星、氧氟沙星、氯霉素、庆大霉素、阿米卡星、头孢他定、头孢哌酮/舒巴坦)的MIC值,了解泵抑制剂对菌株耐药表型的影响。应用TransDB数据库分析多重耐药株W61基因组中外排泵系统的分布情况。选取鲍曼不动杆菌ATCC17978为对照株,以recA为内参基因,采用实时荧光定量RT-PCR方法对鲍曼不动杆菌W61和ATCC17978的13种外排泵基因(adeB、 adeJ、adeG、abeM、abeS、craA、mdtL、amvA及ABTJ_00035、ABTJ_00276等5个功能未知的泵基因)和2种外膜蛋白基因(ompA、ompW) mRNA的表达进行相对定量分析。泛耐药株W61外排泵AdeABC的调控系统AdeRS、外排泵ABTJ_00034/35/36上游调控序列ABTJ_00033和外排泵AdeFGH的调控系统AdeL的结构基因,与GenBank公布的ATCC17978相应序列进行比对分析。
4、可移动元件分布：应用Genbank-nr和IS finder数据库,经序列比对分析鲍曼不动杆菌临床株W61基因组中可移动元件(整合子、转座子、插入序列、质粒)的分布及其携带的耐药基因,分析可移动元件在菌株耐药性的水平播散中的作用。
研究结果
1、药敏试验结果：鲍曼不动杆菌临床株W61对抗假单胞菌头孢菌素、碳青霉烯类抗菌药物、含舒巴坦的复合制剂、氟喹诺酮类抗菌药物和氨基糖苷类抗菌药物均耐药,对多粘菌素敏感,为泛耐药株。
2、基因组序列分析：泛耐药株W61基因组(Genbank登录号CP003500)全长3964912bp,平均GC含量为39.11%,包含3731个蛋白质编码基因,6个rRNA编码基因,74个tRNA编码基因；泛耐药株W61携带2个质粒,plasmidA全长77528bp,平均GC含量34.05%,含有109个蛋白质编码基因,plasmidB全长110967bp,平均GC含量41.61%,含有123个蛋白质编码基因。基因组携带：β-内酰胺类耐药基因ADC型ampC,氨基糖苷类耐药基因(盒)aacA4、aadA1、 aacC1、armA、strA、strB,氟喹诺酮类作用位点gyrA突变,碳氢霉烯类耐药基因oxa-23、oxa-66,磺胺类耐药基因sul1、sul2,氯霉素耐药基因盒catB8。
3、泵抑制剂对耐药表型的影响及外排泵与外膜蛋白基因表达情况：泛耐药株W61在使用CCCP后对8种抗菌药物(左氧氟沙星、环丙沙星、氧氟沙星、氯霉素、庆大霉素、阿米卡星、头孢他定、头孢哌酮／舒巴坦)的MIC值均较前下降4倍或4倍以上。泛耐药株W61基因组共包含RND家族外排泵8个,MFS家族外排泵6个,ABC家族外排泵1个,MATE家族外排泵2个,SMR家族外排泵3个。泛耐药株W61外排泵基因adeB、abeM、ABTJ_00035、ABTJ_00276和外膜蛋白基因ompA的mRNA相对表达水平高于对照株ATCC17978,其差异有统计学意义。调控基因adeRS、ABTJ_00033出现基因突变及缺失,而调控基因adeL序列无基因突变。外膜蛋白基因carO出现基因突变。
4、可移动元件分布情况：泛耐药株W61基因组携带可移动原件：质粒2个、Ⅰ类整合子2个、插入序列片段9类(包括ISCRs2个)、转座酶基因23个、AbaR型耐药岛1个。其中质粒、Ⅰ类整合子和ⅠSCRs均携带耐药基因,并构成耐药基因簇-复合Ⅰ类整合子和AbaR型耐药岛。
研究结论
1、泛耐药鲍曼不动杆菌W61基因组(Genbank登录号CP003500)全长3.96Mbp,包含2个质粒。基因组和质粒中携带β-内酰胺类耐药基因ampC、氨基糖苷类耐药基因(盒)aacA4、aadA1、aacC1、armA、strA、strB、碳氢霉烯类耐药基因oxa-23、oxa-66、磺胺类耐药基因sul1、sul2、氯霉素耐药基因盒catB8,存在氟喹诺酮类作用位点gyrA突变,与菌株泛耐药表型相一致。
2、泵抑制剂CCCP的应用,可使泛耐药鲍曼不动杆菌W61对多种抗菌药物MIC值较前降低,这表明外排泵功能对菌株W61多重耐药有一定贡献。外排泵系统在泛耐药鲍曼不动杆菌W61的基因组中广泛分布,外排泵AdeABC、AbeM、 ABTJ_00034/35/36、ABTJ_00275/276/277的高表达可能与菌株W61的多重耐药有关,调控基因adeRS和ABTJ_00033基因突变和氨基酸替换可能与外排泵AdeABC和ABTJ_00034/35/36表达上调有关。
3、可移动元件在泛耐药鲍曼不动杆菌W61的基因组中广泛分布,其中质粒、Ⅰ类整合子和ISCRs均携带耐药基因,并构成耐药基因簇-复合Ⅰ类整合子和AbaR型耐药岛,对泛耐药株W61多重耐药性的形成及水平传播可能有一定作用。
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, ABTJ_00035,ABTJ_00276and 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, ABTJ_00035, ABTJ_00276in 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.
引文
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