基因组学研究多药耐药鲍曼不动杆菌生物被膜的基因特征

目的 基因组学研究细菌生物被膜独特的存在方式和独特的基因表达特征.方法 选取多药耐药鲍曼不动杆菌菌株,研究同一菌株在不同生长状态、浮游细菌状态和生物被膜状态下,两个样本间基因组的表达差异;分析细菌生物被膜状态下独特的基因表达.结果 我们发现,不同生长状态下两个样本间存在基因表达差异.相对于浮游状态菌,生物被膜菌株有198个基因出现表达差异;其中106个基因表达上调,92个基因表达下调.结论 细菌生物被膜状态下的菌体,有着自己独特的基因和蛋白质表达.菌体生理生化特性、致病力和对药物敏感性都与浮游菌状态显著不同.这些表达差异的基因中,主要集中于外膜蛋白和调控因子相关基因的表达.     

鲍曼不动杆菌耐药性与生物被膜及外膜蛋白基因ompA相关性研究

目的 对鲍曼不动杆菌耐药性与生物被膜及外膜蛋白基因ompA的相关性进行研究.方法 收集非重复鲍曼不动杆菌126株,采用琼脂扩散(K-B)法进行体外药敏实验;改良平板法测定生物被膜形成能力;采用聚合酶链反应(PCR)法扩增外膜蛋白基因ompA;采用反转录聚合酶链反应(RT-PCR)半定量法测定生物被膜形成的不同阶段外膜蛋白基因ompA表达水平的变化.结果 126株鲍曼不动杆菌中,多重耐药菌分离率为62.70％ (79/126);PCR检测到多重耐药鲍曼不动杆菌中ompA基因的检出率为87.34％ (69/79);RT-PCR检测到浮游菌、生物被膜3d、生物被膜5d外膜蛋白基因表达的相对灰度值分别为0.557 3±0.305 3、0.820 8±0.095 7、1.097 5±0.208 6,统计学分析显示,外膜蛋白基因ompA在生物被膜生长的不同时期其表达水平的差异有统计学意义.结论 鲍曼不动杆菌耐药情况严峻,多重耐药菌分离率呈升高趋势;多重耐药性可能与生物被膜形成及外膜蛋白基因ompA高表达有关.     

鲍曼不动杆菌临床分离株生物被膜形成能力与耐药性的研究

目的研究鲍曼不动杆菌生物被膜形成能力与耐药性之间的相关性,为生物膜耐药机制的研究提供依据。方法平板结合结晶紫染色法建立鲍曼不动杆菌生物被膜模型,定量分析66株临床分离的鲍曼不动杆菌生物被膜形成能力,采用K-B纸片法测定菌株的耐药性,概率法检验生物被膜形成能力与耐药性的相关性。结果所测66株鲍曼不动杆菌中,60株(90.9%)具有生物被膜形成能力,其中形成试验弱阳性19株(28.8%),阳性19株(28.8%),强阳性22株(33.3%),阴性6株(9.1%)。鲍曼不动杆菌对头孢吡肟、头孢他啶、头孢噻肟、亚胺培南、美洛培南、哌拉西林、复方新诺明等的耐药性随着生物被膜的形成能力增强而降低(P0.05)。结论绝大多数临床分离株鲍曼不动杆菌具有较强的生物被膜形成能力;鲍曼不动杆菌的生物被膜形成能力与耐药存在负相关性。     

多重耐药鲍曼不动杆菌主动外排泵基因abeM的测定及耐药性分析

目的了解临床分离株鲍曼不动杆菌对16种抗菌药物的耐药性,为抗耐药菌药物选择提供依据,并扩增外排泵蛋白编码基因abeM,以探讨鲍曼不动杆菌多重耐药与主动外排作用的关系。方法采用纸片扩散法(Kirby-Bauer)对收集的65株不重复鲍曼不动杆菌进行药物敏感性试验。应用聚合酶链反应测定多重耐药鲍曼不动杆菌主动外排系统abeM基因,并进行序列分析。结果收集的鲍曼不动杆菌对头孢哌酮/舒巴坦敏感,耐药率15.38%,对亚胺培南耐药率为52.31%,对其余抗菌药物的耐药率在60.00%~73.85%之间;经PCR扩增,65株鲍曼不动杆菌中adeB基因阳性60株(92.31%),序列分析显示与Genbank登录的鲍曼不动杆菌ATCC 19606药物外排蛋白编码基因abeM(AB204810)的同源性为98.00%。结论 abeM主动外排作用可能是多重耐药鲍曼不动杆菌多重耐药的重要机制之一。     

鲍曼不动杆菌多重耐药及其播散机制的研究进展

近20年来,鲍曼不动杆菌已经成为医院感染中重要的病原菌[1].由于鲍曼不动杆菌具有快速获得对多种抗菌药物耐药的能力,多重耐药、泛耐药的鲍曼不动杆菌分离株不断在医院中出现,已成为威胁公众健康的重要问题,而对其多重耐药及播散机制的研究也日趋深入[2].鲍曼不动杆菌的固有耐药途径包括:酶的降解作用,靶位修饰或保护,降低对抗菌药物的通透或增加对抗菌药物的主动泵出;获得性耐药的途径为携带耐药决定簇的遗传元件的水平传播,或是由于结构基因的突变导致细菌细胞功能失活或修饰改变.其中多重耐药性的形成主要通过:携带多种耐药基因的可移动元件的水平传播;染色体基因编码的外排泵系统的高表达[3].外排泵也可以在质粒、整合子等遗传元件上存在,即可移动的遗传元件可以携带多种耐药基因(包括外排泵编码基因),使多重耐药性快速传递.现将鲍曼不动杆菌多重耐药及播散机制研究进展情况综述如下.     

连翘对多药耐药鲍曼不动杆菌主动外排泵编码基因adeB的影响

目的研究中药连翘对多药耐药鲍曼不动杆菌主动外排泵编码基因adeB的影响,从分子水平探讨其主动外排耐药机制。方法从临床分离的30株多药耐药鲍曼不动杆菌中筛选出对环丙沙星（CIP）外排表型和外排泵编码基因均阳性的9株菌作为实验菌株,应用琼脂对倍稀释法检测连翘水煎剂作用前后CIP对上述9株菌的最小抑菌浓度（MIC）,选取作用前后MIC变化较大的代表菌株进行基因测序,并将作用前后的测序结果进行比对分析。结果连翘水煎剂可降低CIP对多药耐药鲍曼不动杆菌的MIC;PCR扩增目的基因片段为2 242 bp,与预期结果一致;测序分析表明,连翘作用后鲍曼不动杆菌adeB基因序列共有12个碱基突变;突变发生氨基酸替换的有E140V、K145I、A147V、K150N、A151V、K153I,并有3个未发生氨基酸替换的突变。结论连翘水煎剂可抑制鲍曼不动杆菌的主动外排泵,并可引起主动外排泵编码基因adeB序列发生变异。     

鲍曼不动杆菌喹诺酮类耐药基因突变分析

目的调查鲍曼不动杆菌对喹诺酮类药物的耐药情况,并对喹诺酮类耐药基因突变进行分析。方法对中南大学湘雅附二医院2002~2008年间鲍曼不动杆菌临床分离株的耐药情况进行统计。同时于2008年7~12月随机收集50株鲍曼不动杆菌,PCR扩增gyrA基因和parC基因,并对扩增产物进行限制性片段长度多态性分析(PCR-RFLP),选取部分PCR扩增产物进行测序和分析。结果鲍曼不动杆菌对喹诺酮类药物的耐药率呈逐年上升的趋势。50株鲍曼不动杆菌对环丙沙星耐药率为62%,对左氧氟沙星耐药率为46%。gyrA基因及parC基因扩增分别获得305 bp和400 bp的基因产物。PCR-RFLP结果显示,对gyrA基因,耐药株100%不能被HinfⅠ酶切,敏感株53%能被HinfⅠ酶切;对parC基因,耐药株29%能被HinfⅠ酶切,敏感株有63%能被HinfⅠ酶切。分析测序结果,耐药株中的gyrA基因和parC基因存在突变,未被酶切的敏感株也存在突变,使HinfⅠ的识别序列GACTC变为GACTT,酶切位点GANTC消失。被酶切耐药株中发现parC基因新的突变点,第89碱基密码子GAA中的A突变成G,被酶切敏感株的parC基因出现多个位点的同义突变。结论鲍曼不动杆菌临床分离株对喹诺酮类药物耐药情况严重,呈逐年上升的趋势。鲍曼不动杆菌对喹诺酮类药物产生耐药与gyrA基因突变和parC基因突变有关。     

60株鲍曼不动杆菌喹诺酮类耐药基因分析

目的探究鲍曼不动杆菌喹诺酮类耐药的机制。方法收集2009年1-12月天津市三所三甲医院各类标本分离的鲍曼不动杆菌60株。采用聚合酶链反应（PCR）扩增技术对质粒介导喹诺酮类耐药基因qnrA、qnrB、qnrS、acc（6’）．Ib．cr、qepA及染色体基因gyrA和parC进行基因检测,并对阳性结果进行酶切和测序鉴定。结果60株鲍曼不动杆菌中qnrA、qnrB、qmS和qepA基因均为阴性,acc（6’）-Ib基因12株阳性,经测序证实均未出现变异。37株环丙沙星耐药的鲍曼不动杆菌中30株（81．0％）gyrA基因不被HinfI酶切,26株（70％）parc基因不被Hinf I酶切,证实有基因突变存在。结论天津地区尚未发现鲍曼不动杆菌中存在质粒介导的喹诺酮耐药机制,gyrA和parC基因变异仍为鲍曼不动杆菌喹诺酮耐药的主要原因,但同时亦有其他喹诺酮耐药机制的存在。     

呼吸重症监护病房多重耐药鲍曼不动杆菌耐药基因研究

目的 观察我院呼吸重症监护病房(RICU)临床分离多重耐药鲍曼不动杆菌β-内酰胺酶基因和消毒剂耐药基因qacE△1-sul1存在状况.方法 收集RICU分离多重耐药鲍曼不动杆菌16株,采用PCR方法检测8种β-内酰胺酶基因(TEM、SHV、PER、DHA、IMP、VIM、OXA-23、OXA-24)和消毒剂耐药基因(qacE△1-sul1)共9种基因.采用多基因聚类分析方法进行多重耐药菌株亲缘性分析.结果 16株多重耐药鲍曼不动杆菌中blaOXA-23阳性5株(31.25%),blaTEM阳性2株(12.50%),blaDHA 2株(12.50%),blaOXA-24 1株(6.25%),blaPER 1株(6.25%).blaSHV、blaIMP、blaVIM均未检出,消毒剂耐药基因检测qacE△1-sull阳性16株(100.00%).多基因聚类分析发现存在克隆传播现象.结论 我院RICU多重耐药鲍曼不动杆菌携带多种β-内酰胺酶基因,消毒剂耐药基因携带率高,聚类分析显示存在克隆传播现象,应引起临床重视.     

部分非鲍曼不动杆菌耐药性及相关基因分析

目的了解目前非鲍曼不动杆菌临床分离株的耐药现状及耐药基因携带情况。方法随机抽取100株不动杆菌临床分离株,采用分子生物学方法筛选非鲍曼不动杆菌,用E-test方法检测非鲍曼不动杆菌对12种抗生素的耐药性,用PCR方法检测16种相关耐药基因的分布。结果共筛选出17株非鲍曼不动杆菌,7株为多重耐药(41.2%),其中对头孢噻肟的耐药率为100%,对氯霉素、哌拉西林、氨苄西林和氨曲南的耐药率分别为76.5%、76.5%、64.7%和52.9%,对多粘菌素B普遍敏感;检出6种耐药基因分别为ampC、blaTEM、blaPER-1、blaOXA-23-like、blaOXA-58和Int1。结论携带超广谱β-内酰胺酶(ESBLs)基因和blaOXA-23-like基因为非鲍曼不动杆菌对β-内酰胺类和碳青霉烯类抗生素耐药的主要原因。非鲍曼不动杆菌的耐药形势严峻,应加强监测。     

鲍曼不动杆菌临床株中可移动遗传元件ISCR1的作用

目的了解鲍曼不动杆菌临床株中新型可移动遗传元件ISCR1的携带情况及其下游基因环境,探讨鲍曼不动杆菌耐药播散机理。方法收集多重耐药鲍曼不动杆菌临床株34株和敏感株21株,PCR扩增ISCR1基因及其下游的基因环境,扩增产物测序分析。结果 34株多重耐药鲍曼不动杆菌临床株中,携带ISCR1基因14株,其中11株ISCR1阳性多重耐药株,在ISCR1基因下游携带氨基糖甙类耐药基因armA基因;21株敏感株中,ISCR1基因扩增结果均为阴性。结论 ISCR1元件可能参与了armA耐药基因的水平播散。     

Antimicrobial susceptibility patterns and distribution of blaOXA genes among Acinetobacter spp. Isolated from patients at Tehran hospitals

Multiple drug-resistant strains of Acinetobacter have created therapeutic problems worldwide. This study was conducted to determine the antimicrobial susceptibility patterns and prevalence of bla(OXA-type) carbapenemases among isolates of Acinetobacter spp. obtained from Iranian patients. Here, 128 Acinetobacter isolates were identified at the species level, and their susceptibilities to different antibiotics were determined using disk agar diffusion testing. Isolates were then subjected to multiplex-PCR targeting bla(OXA) genes. More than 50% of the isolates showed multidrug resistance to different antibiotics. The rates of susceptibility to imipenem, meropenem, piperacillin-tazobactam, and amikacin were 50.7, 50, 42.1, and 38.2%, respectively. The MICs of carbapenems for the resistant isolates ranged from 64 to > or = 256 microg/ml. All strains of Acinetobacter baumannii possessed a bla(OXA-51-like) gene. The co-existence of bla(OXA-51-like)/bla(OXA-23-like) and bla(OXA-51-like)/bla(OXA-24-like) was detected in 25% (n=32) and 17.9% (n=23) of the isolates, respectively. Over 70% of carbapenem-resistant strains contained at least two genes encoding OXA-type carbapenemase. Resistance to carbapenems in the population of Acinetobacter strains in Iran is high, with the majority of isolates showing multidrug resistance. A wide diversity of OXA genes exists among the strains of A. baumannii in Iran. Detection of bla(OXA-51-like) can be used as a simple and reliable method to differentiate A. baumannii strains from other species.     

High prevalence of metallo-beta-lactamase-producing acinetobacter baumannii in a teaching hospital in Tabriz, Iran

Metallo-beta-lactamase (MBL)-producing Acinetobacter baumannii has become a growing therapeutic concern worldwide. The aims of this study were to evaluate the antimicrobial susceptibility of A. baumannii isolates and to determine the prevalence of MBL genes among carbapenem non-susceptible isolates. During a period of 16 months (March 2008-June 2009), 100 isolates of A. baumannii were collected from different clinical specimens of inpatients admitted to the largest teaching hospital in the northwest of Iran. All isolates were tested for antimicrobial susceptibility by Kirby-Bauer disk diffusion method. Carbapenem non-susceptible isolates were further screened for production of MBL by Etest and were then subjected to PCR for detection of MBL genes of types bla(IMP) and bla(VIM). Among 63 carbapenem (imipenem and meropenem) non-susceptible isolates of A. baumannii, 31 (49%) were found to be MBL producers. Of 31 MBL-producing isolates, 19 (61%) carried the bla(IMP) gene and 9 (29%) carried the bla(VIM) gene. All MBL-producing isolates were multidrug resistant. This is the first report of IMP and VIM types among MBL-producing A. baumannii in Iran.     

Role of integrons in antimicrobial susceptibility patterns of Acinetobacter baumannii

The relationship between the presence and types of integrons and the antimicrobial susceptibility patterns of Acinetobacter baumannii was investigated. A total of 134 non-duplicated A. baumannii isolates, 54.5% (n=73) of which were subsequently found to carry class 1 integrons, were collected from a regional hospital in Taiwan between March and September 2007. Only two types of gene cassette array, aacA4-catB8-aadA1 and aacC1-orfP-orfP-orfQ-aadA1, were identified. Susceptibility data showed that those strains carrying integrons were significantly more resistant to all antibiotics tested except ampicillin/sulbactam and imipenem. An epidemiological study revealed that the same integron could be found in different unrelated strains. These findings suggest that the presence of integrons in A. baumannii is responsible for both the horizontal transfer of antibiotic-resistance genes related to aminoglycosides and chloramphenicol and also represents a marker of multidrug resistance and epidemic potential.     

Outbreak of Acinetobacter spp septicemia in a neonatal ICU

An outbreak of Acinetobacter spp infection in the neonatal unit at Lok Nayak Hospital, New Delhi, India, is described. During a 6-month period, 68 strains of Acinetobacter baumannii were isolated from the blood and CSF of 47 neonates admitted to the intensive care unit. Diagnosis of clinically significant bacteremia was made in 36 patients. On environmental/personnel sampling, Acinetobacter spp isolates with similar antibiogram were recovered from intravenous catheter and washbasin. Control of the outbreak was possible only after strict infection control practices in the unit. It was concluded that any clinical multidrug resistant A. baumannii isolate can be a potential nosocomial outbreak strain.     

cDNA芯片检测耐药急淋患者细胞凋亡调控相关基因的差异表达

目的通过观察耐药／非耐药急性淋巴细胞性白血病（急淋）患者细胞凋亡调控相关基因的差异表达,探索多药耐药的分子病理机制。方法用TRIzol抽提外周血标本总RNA，逆转录生成cDNA并标记后，与含有4096个人类已知基因的cDNA表达谱芯片杂交，检测细胞凋亡调控相关基因在两者间的差异表达。结果耐药组与非耐药组有150个基因表达显著差异，包括耐药组83个基因低表达，67个基因高表达，其中凋亡调控相关基因有6个低表达，8个高表达。结论细胞凋亡调控相关基因表达异常可能是白血病多药耐药的分子病理机制之一。     

48株鲍曼不动杆菌氨基糖苷类抗生素耐药基因检测

目的了解鲍曼不动杆菌氨基糖苷类抗生素耐药基因分布情况。方法收集蚌埠医学院第一附属医院2015年1月至12月临床分离的48株泛耐药鲍曼不动杆菌,VITEK 2Compact进行鉴定和药敏实验。PCR法检测12个氨基糖苷类修饰酶基因和3个甲基化酶基因以及外排泵基因adeB。结果在实验的16个基因中,共检出4种氨基糖苷类抗生素耐药基因aac(6′)-Ib、armA、adeB和ant(3″)-Ia,其中aac(6′)-Ib检出率为39.6%(19/48),armA基因检出率为89.6%(43/48),adeB检出率89.6%(43/48),ant(3″)-Ia检出率为10.4%(5/48),其余基因均未检出;存在两种以上耐药基因的共39株,检出率为81.3%(38/48)。结论 aac(6′)-Ib、armA基因以及外排泵adeB是介导我院鲍曼不动杆菌氨基糖苷类抗生素耐药的主要基因。