鲍曼不动杆菌IRS-PCR基因分型研究

目的 用低频限制性位点聚合酶链反应(IRS-PCR)对鲍曼不动杆菌进行基因分型,分析基因型与鲍曼不动杆菌耐药谱的关系,并初步探讨其在分子流行病学中的作用.方法 随机收集2008年8月至2009年8月临床分离的73株鲍曼不动杆菌,采用K-B法进行药物敏感试验确定鲍曼不动杆菌耐药谱;同时利用IRS-PCR对此73株鲍曼不动杆菌进行基因分型;并分析IRS-PCR分型与鲍曼不动耐药谱的关系;结合IRS-PCR分型结果与73株鲍曼不动杆菌感染病例的临床资料,分析在此时间段鲍曼不动杆菌在我院流行感染的情况.结果 药物敏感试验将73株鲍曼不动杆菌菌株分为A1(19株全耐药型)和A2 ～ A31(54株耐药谱型)31个药敏谱.IRS-PCR法将其分为A～W共23个基因型,其中A、C、B、D和E型为5种优势菌株,分别为14、11、10、8和6株.对比研究发现A1型菌株(15/19)主要集中在基因型A、C、D内,而基因型B包含A15型耐药菌株9株(69.2％),基因型E包含A3型耐药菌株3株(42.9％).A基因型在院内特别是ICU中心引起2次爆发流行,而C和D型主要在呼吸内科引起感染.结论 IRS-PCR基因分型与药敏分型有较高的一致性,且IRS-PCR基因分型在早期发现和预防感染暴发流行方面优于药敏分型.     

感染耐亚胺培南鲍曼不动杆菌临床分布情况及耐药性分析

目的:探讨感染耐亚胺培南(IPM)鲍曼不动杆菌临床分布情况及耐药性。方法:回顾性分析2013~2015年榆林市中医院患者感染鲍曼不动杆菌的临床分布,并对耐亚胺培南鲍曼不动杆菌进行培养、鉴定及药敏试验,分析其耐药情况。结果:2013~2015年我院共分离培养出鲍曼不动杆菌185株,其中IPM耐药99株(53.51%),IPM敏感86株(46.49%)。绝大部分菌株分离自痰液标本(138株,74.59%),其次是血液标本(14株,7.57%)与尿液标本(12株,6.49%);来自ICU送检标本菌株数量最多(84株,45.41%),其次是呼吸内科标本(53株,28.65%)与神经内科标本(30株,16.22%)。不同标本类型及科室中IPM耐药及IPM敏感鲍曼不动杆菌占比无显著差异(P0.05)。IPM耐药鲍曼不动杆菌对抗菌药物耐药性均较高,其中以头孢哌酮/舒巴坦(CSL)敏感性最高(50.51%);IPM敏感鲍曼不动杆菌对部分抗菌药物耐药性尚可,其中以IPM敏感性最高(93.02%)。结论:耐亚胺培南鲍曼不动杆菌临床分布广泛,多重耐药性严重甚至出现泛耐药,应当对患者进行药敏试验,以药敏结果选择合适的抗菌药物进行使用。     

膜孔蛋白改变在鲍曼不动杆菌对亚胺培南耐药中的作用

目的对鲍曼不动杆菌耐药情况进行分析,探索膜孔蛋白在亚胺培南耐药中的作用,为临床合理用药及控制医院感染提供依据。方法收集非重复亚胺培南耐药鲍曼不动杆菌63株,亚胺培南敏感鲍曼不动杆菌21株,用K-B纸片法检测上述细菌对16种抗菌药物的敏感性,PCR技术检测carO和oprD膜孔蛋白基因携带情况,并采用DNASTAR软件进行序列对比,对CarO蛋白三维结构建模。结果亚胺培南耐药鲍曼不动杆菌除对替加环素(3.2%)、头孢哌酮/舒巴坦(28.6%)和米诺环素(30.2%)耐药率低外,对其他抗菌药物耐药率均较高,而亚胺培南敏感菌对多数抗生素均较敏感。PCR扩增显示所检测菌株carO和oprD基因均阳性。进一步系列比对发现亚胺培南耐药株较敏感株carO基因存在有意义突变,蛋白质分子立体结构有明显差别。结论亚胺培南耐药鲍曼不动杆菌耐药情况严峻,carO膜孔蛋白基因突变发挥重要作用。     

鲍曼不动杆菌的耐药性检测及泛耐药菌株的耐药基因研究

目的考察临床分离的鲍曼不动杆菌的耐药性并对泛耐药菌株的耐药基因进行检测。方法用纸片扩散法对60株临床分离鲍曼不动杆菌进行药物敏感试验,并用PCR法检测8株泛耐药菌株携带7种耐药基因OXA-51、OXA-23、OXA-24、OXA-58、aac(3)-Ⅰ、gyr A、abe M的情况。结果所分离的鲍曼不动杆菌对临床常用的β-内酰胺类抗生素、氨基糖苷类抗生素、四环素类抗生素、磺胺类抗菌药及喹诺酮类抗菌药均产生耐药性;在8株泛耐药鲍曼不动杆菌中OXA-51、OXA-23、OXA-58、gyr A、abe M基因检测均全部呈阳性;OXA-24基因检测均呈阴性;aac(3)-I基因检测有5株呈阳性,3株呈阴性。结论鲍曼不动杆菌耐药情况严重,其耐药机制与多种耐药基因密切相关。     

临床分离鲍曼不动杆菌的外排泵基因检测及同源性分析

目的探讨临床分离鲍曼不动杆菌主动外排基因的分布和菌株克隆相关性,为指导临床合理用药和制定恰当的感染控制措施提供理论依据。方法对临床分离的80株鲍曼不动杆菌采用琼脂稀释法检测对抗菌药物的敏感性,聚合酶链反应(PCR)检测adeB、adeG、adeJ外排泵基因的携带情况,脉冲场凝胶电泳法分析多重耐药鲍曼不动杆菌的同源性。结果 80株临床分离鲍曼不动杆菌中,57株为多重耐药株(MDRAB),且对多粘菌素B、头孢哌酮/舒巴坦和美罗培南较为敏感,对其他临床常用抗生素普遍耐药。adeB、adeG、adeJ外排泵基因分布广泛,在敏感菌株中存在率也很高;PFGE分型结果显示57株MDRAB菌株共分为四大群(A、B、C、D),并以流行克隆A为主。结论临床分离鲍曼不动杆菌耐药形式严峻,其临床分离株普遍存在外排泵编码基因adeB、adeG、adeJ,携带外排泵阳性MDRAB菌株的克隆播散是温州地区鲍曼不动杆菌发生耐药的机制之一。     

鲍曼不动杆菌临床菌株生物膜形成能力的研究

目的对鲍曼不动杆菌临床菌株的生物膜形成能力进行对比研究,并分析生物膜形成的一些可能的影响因素。方法利用在聚苯乙烯板上构建生物膜的技术对51株全耐药的鲍曼不动杆菌的生物膜形成能力进行检测,同时对分离自下呼吸道和无菌体液的各20株鲍曼不动杆菌的生物膜形成能力进行研究,然后对新近报道的鲍曼不动杆菌生物膜形成相关基因abaI在所有菌株中的分布情况进行检测。结果 51株全耐药的鲍曼不动杆菌中35株(68.6%)可以形成生物膜,并且形成生物膜的能力较强。50%(10/20)分离自下呼吸道的鲍曼不动杆菌能够形成生物膜,20株无菌体液中分离的菌株仅有1株可以形成生物膜。78.0%(71/91)鲍曼不动杆菌中abaI基因扩增阳性。结论分离自下呼吸道的鲍曼不动杆菌临床菌株有较强的生物膜形成能力,abaI基因广泛存在于鲍曼不动杆菌临床菌株中。临床在治疗鲍曼不动杆菌感染的同时需要考虑其在感染部位形成生物膜的因素,可能在治疗的同时有必要加入一些对生物膜有穿透性的药物。     

鲍曼不动杆菌耐药程度与其主动外排泵蛋白的相关性研究

目的:探讨鲍曼不动杆菌耐药程度与其主动外排泵蛋白的相关性。方法:首先用纸片扩散法检测64株临床鲍曼不动杆菌对8种抗菌药物的敏感性;将其分为A组(0～2种抗生素耐药)、B组(对3～5种抗生素耐药)和C组(对6～8种抗生素耐药);检测64株临床鲍曼不动杆菌对罗丹明6G的外排情况,筛选出罗丹明6G外排明显增加的菌株;并用逆转录-聚合酶链反应(RT-PCR)方法检测主动外排泵基因AdeABC的表达水平。结果:64株鲍曼不动杆菌中有4株对0～2种抗生素耐药(A组),对3～5种抗生素耐药的有33株(B组),对6～8种抗生素耐药的有27株(C组);多重耐药组鲍曼不动杆菌罗丹6G外排明显增高,外排程度A组相似文献   

医院内鲍曼不动杆菌感染调查及泛耐药情况分析

目的 了解义乌市妇幼保健院近3年来医院内鲍曼不动杆菌感染现状,及泛耐药情况.方法 对医院2009年至2011年分离的鲍曼不动杆菌分布及耐药情况进行回顾性分析.细菌鉴定采用美国德灵细菌鉴定仪.结果 3年间共分离到鲍曼不动杆菌305株,其中2009年72株,2010年91株,2011年142株,泛耐药菌株感染率呈现出逐年上升趋势,2009年5株(占6.94％),2010年9株(占9.89％),2011年有19株(占13.38％).结论 鲍曼不动杆菌感染呈逐年上升趋势,其泛耐药菌株感染率呈上升趋势.应加强抗生素的合理使用,控制鲍曼不动杆菌在医院内的定值和播散,重视泛耐药鲍曼不动杆菌监测.     

从耐药鲍曼不动杆菌中发现β-内酰胺酶ADC基因和膜孔蛋白carO基因新的变异型

目的 调查一组耐药鲍曼不动杆菌中β-内酰胺酶基因和膜孔蛋白基因的存在和变异情况.方法 收集2010年1月至2010年12月浙江某医院ICU患者痰液标本中分离的多耐药和泛耐药鲍曼不动杆菌各10株,用分子鉴定法鉴定菌种,再用聚合酶链反应(PCR)及序列分析的方法分析34种β-内酰胺酶基因与膜孔蛋白carO基因.结果 本组20株耐药鲍曼不动杆菌共检出TEM-1型20株(100％)、OXA-23型10株(50.0％)、ADC-30型12株(60.0％)、ADC基因新的变异型ADC-60型8株(40.0％)(GenBank登录号:JQ692087).10株PDR菌均检出OXA-23型β-内酰胺酶基因,而10株MDR菌则均未检出OXA-23型β-内酰胺酶基因.20株耐药鲍曼不动杆菌膜孔蛋白carO基因均存在有义突变,19株测得序列相同,翻译成氨基酸序列后与鲍曼不动杆菌敏感株(SDF)比较,一致率为76.0％.8号株carO基因序列与其他19株测得DNA序列不同,第351位缺失了12个碱基并导致过早出现终止密码子.结论 本组鲍曼不动杆菌对β-内酰胺类药物耐药主要与产TEM、ADC、OXA-23和carO基因存在有义突变相关.OXA-23型β-内酰胺酶基因阳性是PDR菌耐碳青霉烯类药物的原因.ADC基因存在新变异型:ADC-60是国内外首次报道.     

多重PCR检测鲍曼不动杆菌OXA型碳青酶烯酶基因

目的:利用一种多重PCR方法检测鲍曼不动杆菌OXA型碳青酶烯酶基因。方法:利用耐药性试验筛选出耐药鲍曼不动杆菌菌株;根据OXA型碳青酶烯酶基因设计引物,利用多重PCR方法检测三类OXA基因(OXA-23-like,OXA-58-like andOXA-51-like)。结果:30株鲍曼不动杆菌中有25株同时扩出OXA-51和OXA-23,1株扩出OXA-58条带和OXA-51,4株只扩出OXA-51;结论:实验所的多重PCR方法简单、快速、准确,可广泛应用临床鲍曼不动杆菌OXA型碳青酶烯酶基因的检测。     

Identification of an OprD homologue in Acinetobacter baumannii

With the increased number of resistant Acinetobacter baumannii strains, it is urgently required to decipher the molecular bases of outer membrane permeability. The analyses of the outer membrane from different A. baumannii strains indicated a modification in the expression of two proteins of 29 and 43 kDa, respectively. By electrophoresis and MALDI-MS analyses, the 43 kDa OMP was identified as a protein belonging to the OprD family, a basic amino acid and imipenem porin.     

Proteomic analysis of a fraction enriched in cell envelope proteins of Acinetobacter baumannii

Acinetobacter baumannii is a multiresistant opportunistic nosocomial pathogen. A protein fraction was purified and analyzed by 2-DE. Twenty-nine major protein spots were selected for protein identification using trypsin digestion and MS analysis. As the A. baumannii genome has not yet been described, protein identification was performed by homology with other Acinetobacter species in the NCBi database. We identified ribosomal proteins, chaperones, elongation factors and outer membrane proteins (Omp), such as OmpA and the 33-36-kDa OMP. Proteomic analysis of A. baumannii provides a platform for further studies in antimicrobial resistance.     

鼠伤寒沙门菌外膜蛋白与耐药性关系的分析

目的分析鼠伤寒沙门菌外膜蛋白(OMP)与耐药性的关系。方法用消除剂丫啶橙消除耐药性,盲传测其遗传稳定性,采用超声波物理裂解法制备鼠伤寒沙门菌外膜蛋白标本,用变性聚丙烯酰胺凝胶电泳(SDA-PAGE)检测外膜蛋白,用紫外分光光度计测其吸光值,计算浓度。结果抗性消除表型能稳定遗传,耐药鼠伤寒沙门菌与敏感鼠伤寒沙门菌都含有6条主要的外膜蛋白条带,两者相比,发现耐药菌的外膜蛋白在约57、53、30 kDa处减弱或缺失,总的蛋白浓度也低于后者。结论鼠伤寒沙门菌耐药性与外膜蛋白的减弱或缺失有关。     

Colicinogeny in local isolates of salmonellae and plasmid transfer studies

Colicinogeny was determined in local isolates of S. typhimurium and S. enteritidis. Fourteen out of 35 S. typhimurium isolates of hospital origin were colicin producers whereas only one chicken isolate out of 82 S. enteritidis isolates of various origin (human, chicken or egg) produced colicin. A colicin producing, cephalothin (Cpt)- and piperacillin (Prl)-resistant local isolate of Salmonella havana (H32) harbored 4 plasmids of 54.0, 28.4, 2.7 and 1.9 kb. Upon curing its plasmids, the strain lost the ability to produce colicin and resistance to antibiotics and no longer expressed smooth lipopolysaccharide (LPS). The outer membrane protein (OMP) of 34.6 kDa was also lost. Using nalidixic acid (Nal)-resistant mutant of the cured strain in conjugation experiments, 10 out of 27 transconjugants were found to be resistant to Nal and Prl, 10 were resistant to Nal and Cpt and 7 showed Nal, Prl and Cpt resistance. Cpt and Prl resistance were determined by 54.0 and 28.4 kb plasmids, respectively. There was no direct correlation between plasmid contents and colicinogeny, LPS and OMP profiles.     

Quantitative proteomic analysis of cell wall and plasma membrane fractions from multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and iron-uptake transporters appeared to be dependent on tetracycline conditions, whereas β-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii.     

禽源性大肠杆菌O_1、O_2和O_(78)分离株外膜蛋白型的研究

对江苏省25个禽源性大肠杆菌O_1、O_2和O_(78)分离株的外膜蛋白型进行了测定。用改良的N-十二烷酰肌氨酸法提取其外膜蛋白,经SDS-PAGE电泳后,用考马斯亮蓝法进行染色。结果,8个O_1分离株,9个O_2分离株分属2个OMP型,其中的1个为二者所共有;而8个O_(78)分离株的OMP型也与该型相同。表明从江苏省分离到的禽源性大肠杆菌具有多样性的OMP型,而且这3个血清型的分离株中存在着共同的OMP型。     

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.     

淋病流行株外排系统与外膜通透性和多重耐药性的关系

探讨外排系统、外膜通透性与淋病流行株多重耐药性的关系。应用K—B法和琼脂稀释法从湛江地区分离出62株淋球菌多重耐药株。利用SDS—PAGE测定淋球菌外膜孔蛋白的表达;应用直接荧光法测定能量抑制剂加入前后淋球菌对抗生素的摄入和积累情况,比较耐药菌与敏感菌内膜泵蛋白表达的差异;利用煮沸法提取细菌DNA,PCR扩增mtrR基因,并对扩增产物测序,比较敏感株与多重耐药株的差异。结果5株多重耐药菌均有外膜孔蛋白表达的缺失或下降,同时伴有外排泵蛋白的表达;5株敏感淋球菌无mtrR的突变,10株多重耐药株均有mtrR基因的突变。表明外排系统、外膜通透性与淋病流行株的多重耐药性密切相关。     

我国部分地区禽源性大肠杆菌的外膜蛋白型

测定了从我国１８个省、市、自治区分离到的２０４个禽病原性大肠杆菌优势血清型分离株的外膜蛋白型（ＯｕｔｅｒＭｅｍｂｒａｎｅＰｒｏｔｅｉｎＰａｔｅｒｎｓ,ＯＭＰ型）。这些分离株共产生了４个ＯＭＰ型,５６个Ｏ１８分离株可分为３个ＯＭＰ型,５４个Ｏ７８分离株、２８个Ｏ２分离株、２６个Ｏ８８分离株、２２个Ｏ１１分离株和１８个Ｏ２６分离株,分别出现了４、２、１、３和１个ＯＭＰ型。其中,ＯＭＰ１型为６个血清型所共有,ＯＭＰ３型则同时存在于Ｏ１８、Ｏ７８、Ｏ２和Ｏ１１分离株中。结果表明,优势血清型中,Ｏ１８、Ｏ７８、Ｏ２和Ｏ１１分离株具有多样性的ＯＭＰ型,而Ｏ８８、Ｏ２６分离株的ＯＭＰ型则高度一致,所测６个优势血清型的分离株间存在共同的ＯＭＰ型     

Role of fibronectin in the adhesion of Acinetobacter baumannii to host cells

Adhesion to host cells is an initial and important step in Acinetobacter baumannii pathogenesis. However, there is relatively little information on the mechanisms by which A. baumannii binds to and interacts with host cells. Adherence to extracellular matrix proteins, such as fibronectin, affords pathogens with a mechanism to invade epithelial cells. Here, we found that A. baumannii adheres more avidly to immobilized fibronectin than to control protein. Free fibronectin used as a competitor resulted in dose-dependent decreased binding of A. baumannii to fibronectin. Three outer membrane preparations (OMPs) were identified as fibronectin binding proteins (FBPs): OMPA, TonB-dependent copper receptor, and 34 kDa OMP. Moreover, we demonstrated that fibronectin inhibition and neutralization by specific antibody prevented significantly the adhesion of A. baumannii to human lung epithelial cells (A549 cells). Similarly, A. baumannii OMPA neutralization by specific antibody decreased significantly the adhesion of A. baumannii to A549 cells. These data indicate that FBPs are key adhesins that mediate binding of A. baumannii to human lung epithelial cells through interaction with fibronectin on the surface of these host cells.