OmpA is the principal nonspecific slow porin of Acinetobacter baumannii

Acinetobacter species show high levels of intrinsic resistance to many antibiotics. The major protein species in the outer membrane of Acinetobacter baumannii does not belong to the high-permeability trimeric porin family, which includes Escherichia coli OmpF/OmpC, and instead is a close homolog of E. coli OmpA and Pseudomonas aeruginosa OprF. We characterized the pore-forming function of this OmpA homolog, OmpA(Ab), by a reconstitution assay. OmpA(Ab) produced very low pore-forming activity, about 70-fold lower than that of OmpF and an activity similar to that of E. coli OmpA and P. aeruginosa OprF. The pore size of the OmpA(Ab) channel was similar to that of OprF, i.e., about 2 nm in diameter. The low permeability of OmpA(Ab) is not due to the inactivation of this protein during purification, because the permeability of the whole A. baumannii outer membrane was also very low. Furthermore, the outer membrane permeability to cephalothin and cephaloridine, measured in intact cells, was about 100-fold lower than that of E. coli K-12. The permeability of cephalothin and cephaloridine in A. baumannii was decreased 2- to 3-fold when the ompA(Ab) gene was deleted. These results show that OmpA(Ab) is the major nonspecific channel in A. baumannii. The low permeability of this porin, together with the presence of constitutive β-lactamases and multidrug efflux pumps, such as AdeABC and AdeIJK, appears to be essential for the high levels of intrinsic resistance to a number of antibiotics.     

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。     

Structural and binding studies of phosphopantetheine adenylyl transferase from Acinetobacter baumannii

Phosphopantetheine adenylyltransferase (PPAT, EC. 2.7.7.3) catalyzes an essential step in the reaction that transfers an adenylyl group from adenosine tri phosphate (ATP) to 4′-phosphopantetheine (pPant) yielding 3′- dephospho-coenzyme A (dPCoA) and pyrophosphate (PP) in the coenzyme A (CoA) biosynthesis pathway_. The enzyme PPAT from Acinetobacter baumannii (AbPPAT) was cloned, expressed and purified. The binding studies of AbPPAT were carried out with two compounds, tri‑sodium citrate (TSC) and l-ascorbic acid (LAA, vitamin-C) using fluorescence spectroscopic (FS) and surface Plasmon resonance (SPR) methods. Both methods provided similar values of dissociation constants for TSC and LAA which were of the order of 10⁻⁸ M and 10⁻⁵ M respectively. The computer aided docking studies indicated fewer interactions of LAA with AbPPAT as compared to those of TSC. The freshly purified samples of AbPPAT were crystallized. The crystals of AbPPAT were soaked in the solutions containing TSC and LAA. However, the crystals of the complex of AbPPAT with LAA did not diffract well and hence the structure of the complex of AbPPAT with LAA could not be determined. On the other hand, the crystals of the complex of AbPPAT with TSC diffracted well and the structure was determined at 1.76 Å resolution. It showed that TSC bound to AbPPAT at the ATP binding site and formed several intermolecular contacts including 12 hydrogen bonds. The results of binding studies for both TSC and LAA and the structure of the complex of AbPPAT with TSC clearly indicated a potential role of TSC and LAA as antibacterial agents.     

Structural and Functional Characterization of Acinetobacter baumannii Nucleoside Diphosphate Kinase

近年来,鲍曼不动杆菌(Acinetobacter baumannii)在医院里越来越受到人们的关注,尤其是在重症监护病房(ICUs)．它以强大的多重耐药性(multiresistance)而闻名．核苷二磷酸激酶(nucleoside diphosphate kinase,NDK)是一种进化上非常保守的酶,它能催化核苷之间磷酸基团的转移．我们解析了鲍曼不动杆菌NDK野生型和C端氨基酸残基Arg141-Thr142-Arg143(RTR)截短突变体的结构．通过和黄色黏菌(Myxococcus xanthus)NDK的三维结构进行比较,推断鲍曼不动杆菌NDK的催化机制和黄色黏菌类似．通过激酶活性实验和圆二色谱实验,发现鲍曼不动杆菌NDK E28A突变体二级结构发生了改变,从而导致蛋白催化活性降低,说明Glu28是鲍曼不动杆菌NDK结构中非常关键的氨基酸残基．鲍曼不动杆菌NDK C端RTR截短突变体显示出催化活性极大的降低,这可能与C端RTR残基介导的二体间相互作用有关．虽然RTR截短突变体中的Lys33伸向了和野生型中不同的方向,和Val15产生相互作用弥补了一部分因为RTR截短丢失的相互作用,维持了RTR截短突变体和野生型类似的结构．但是,Lys33产生的相互作用依然太弱,不足以维持蛋白在催化的动态过程中整体结构的高效转换．我们解析的鲍曼不动杆菌NDK晶体高分辨率结构将有助于科学家设计针对鲍曼不动杆菌的药物．     

Isolation and structure elucidation of acinetobactin., a novel siderophore from Acinetobacter baumannii

A novel siderophore, called acinetobactin, with both catecholate and hydroxamate functional groups was isolated from low-iron cultures of Acinetobacter baumannii ATCC 19606. The structure was elucidated by chemical degradation, fast-atom bombardment mass spectrometry and ¹H and ¹³C NMR spectroscopy. Acinetobactin was composed of -N-hydroxyhistamine, threonine and 2,3-dihydroxybenzoic acid, the last two components forming an oxazoline ring. Acinetobactin was structurally related to anguibactin, a plasmid-encoded siderophore of Vibrio anguillarum. The only difference was that acinetobactin possessed an oxazoline ring instead of a thiazoline ring. Four of 12 other clinical A. baumannii strains examined produced acinetobactin, indicative of strain-to-strain variation in the ability to produce acinetobactin. In addition, a relatively small amount of acinetobactin was also detected in A. haemolyticus ATCC 17906.Abbreviations COSY chemical shift correlation spectroscopy - DHBA 2,3-dihydroxybenzoic acid - EDDA ethylenediamine-di(o-hydroxyphenylacetic acid) - FAB fast-atom bombardment - GC-MS gas chromatography-mass spectrometry     

鲍曼不动杆菌感染的呼吸机相关肺炎20例

目的 了解我院NICU内鲍曼氏不动杆菌感染呼吸机相关肺炎的情况,探讨有效预防和控制的该类感染的措施。方法 收集我院NICU内2006.1-2007.2之间发生呼吸相关肺炎病通过细菌培养确定为鲍曼氏不动杆菌感染的患儿的临床资料以及该菌的药敏试验结果进行分析。结果 鲍曼氏不动杆菌是呼吸机相关肺炎的主要致病菌之一,其流行情况复杂,并且出现了耐碳青酶烯类的菌株感染,可造成严重的不良后果。结论 预防该菌在NICU内导致感染需要引起临床工作者更多的重视,加强对其定植情况的监测是预防其感染和流行的重要措施,对该菌开展及分子流行病学的深入研究将有助于指导NICU内NI防治。     

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter baumannii strain 24

Extraction of dry bacteria of Acinetobacter baumannii strain 24 by phenol-water yielded a lipopolysaccharide (LPS) that was studied by serological methods and fatty acid analysis. After immunisation of BALB/c mice with this strain, monoclonal antibody S48-3-13 (IgG(3) isotype) was obtained, which reacted with the LPS in western blot and characterized it as S-form LPS. Degradation of the LPS in aqueous 1% acetic acid followed by GPC gave the O-antigenic polysaccharide, whose structure was determined by compositional analyses and NMR spectroscopy of the polysaccharide and O-deacylated polysaccharide as [carbohydrate structure: see text] where QuiN4N is 2,4-diamino-2,4,6-trideoxyglucose and GalNAcA 2-acetamido-2-deoxygalacturonic acid. The amino group at C-4 of the QuipN4N residues is acetylated in about 2/3 of LPS molecules and (S)-3-hydroxybutyrylated in the rest.     

Purification and characterization of OXA-23 from Acinetobacter baumannii

Although the existence of bla_OXA-23 is reported in various parts of the world, the product of bla_OXA-23 gene, OXA-23, has not been purified and its kinetic properties are not known. In this study, OXA-23 of Acinetobacter baumannii isolated from Kocaeli University intensive care unit was characterized after purification using recombinant methods. Preliminary results showed that conventional protein purification methods were not effective for purification of OXA-23. Therefore, OXA-23 was fused to maltose-binding protein of Escherichia coli, the fused protein was expressed and purified to homogeneity. Kinetic properties of the pure protein were then studied with substrates e.g., imipenem, meropenem, cefepime, ceftazidime, ampicilline, piperacillin, penicillin G, and nitrocefin. Also clavulanic acid, tazobactam, and sulbactam concentrations that inhibit 50% of OXA-23 enzyme activity were calculated. Modelling of OXA-23 revealed its ionic surface structure, conformation in the fused form and its topology allowing us to make predictions for OXA-23 substrate specificity.     

Two methionine aminopeptidases from Acinetobacter baumannii are functional enzymes

Drug resistance in Gram-negative bacteria, such as Acinetobacter baumannii, is emerging as a significant healthcare problem. New antibiotics with a novel mechanism of action are urgently needed to overcome the drug resistance. Methionine aminopeptidase (MetAP) carries out an essential cotranslational methionine excision in many bacteria and is a potential target to develop such novel antibiotics. Two putative MetAP genes were identified in A. baumannii genome, but whether they actually function as MetAP enzymes was not known. Therefore, we established an efficient E. coli expression system for their production as soluble and metal-free proteins for biochemical characterization. We demonstrated that both could carry out the metal-dependent catalysis and could be activated by divalent metal ions with the order Fe(II) ≈ Ni(II) > Co(II) > Mn(II) for both. By using a set of metalloform-selective inhibitors discovered on other MetAP enzymes, potency and metalloform selectivity on the A. baumannii MetAP proteins were observed. The similarity of their catalysis and inhibition to other MetAP enzymes confirmed that both may function as competent MetAP enzymes in A. baumannii and either or both may serve as the potential drug target.     

鲍曼不动杆菌疫苗的研究进展

鲍曼不动杆菌为不动杆菌属中最常见的一种革兰阴性杆菌,同时也是医院获得性感染的主要致病菌之一。由于其广泛的耐药性,普通抗生素对其治疗效果已不明显。近年来,国内外学者尝试通过研制抗菌疫苗的手段来防治鲍曼不动杆菌感染,主要有灭活全菌体疫苗、外膜囊泡、重组蛋白亚单位疫苗、荚膜多糖候选疫苗、联合疫苗等。现就鲍曼不动杆菌疫苗的研究进展进行了综述。     

Carbapenem-Resistant Acinetobacter baumannii from Serbia: Revision of CarO Classification

Carbapenem-resistant A. baumannii present a significant therapeutic challenge for the treatment of nosocomial infections in many European countries. Although it is known that the gradient of A. baumannii prevalence increases from northern to southern Europe, this study provides the first data from Serbia. Twenty-eight carbapenem-resistant A. baumannii clinical isolates were collected at a Serbian pediatric hospital during a 2-year period. The majority of isolates (67.68%) belonged to the sequence type Group 1, European clonal complex II. All isolates harbored intrinsic OXA-51 and AmpC cephalosporinase. OXA-23 was detected in 16 isolates (57.14%), OXA-24 in 23 isolates (82.14%) and OXA-58 in 11 isolates (39.29%). Six of the isolates (21.43%) harbored all of the analyzed oxacillinases, except OXA-143 and OXA-235 that were not detected in this study. Production of oxacillinases was detected in different pulsotypes indicating the presence of horizontal gene transfer. NDM-1, VIM and IMP were not detected in analyzed clinical A. baumannii isolates. ISAba1 insertion sequence was present upstream of OXA-51 in one isolate, upstream of AmpC in 13 isolates and upstream of OXA-23 in 10 isolates. In silico analysis of carO sequences from analyzed A. baumannii isolates revealed the existence of two out of six highly polymorphic CarO variants. The phylogenetic analysis of CarO protein among Acinetobacter species revised the previous classification CarO variants into three groups based on strong bootstraps scores in the tree analysis. Group I comprises four variants (I-IV) while Groups II and III contain only one variant each. One half of the Serbian clinical isolates belong to Group I variant I, while the other half belongs to Group I variant III.     

外排泵介导鲍曼不动杆菌耐药性的研究进展

目的鲍曼不动杆菌的多重耐药性问题日趋严重,该菌外膜上外排泵过表达是导致其耐药性的重要机制。详尽地研究多药外排泵的机制以及寻找阻断其功能的外排泵抑制剂,将为多耐药鲍曼不动杆菌的治疗开辟新的路径。本文就近年来鲍曼不动杆菌外排泵的研究现状进行综述,着重描述多药外排泵RND家族的耐药谱特征及其表达调控机制,同时,还阐述了MFS和MATE家族外排泵的研究进展。     

鲍曼不动杆菌基因分型技术的研究进展

鲍曼不动杆菌为非发酵糖类革兰阴性杆菌,具有强大的获得耐药性和克隆传播的能力,广泛分布于医院环境,是医院内感染的重要病原菌之一。因此,建立精准、高效的鲍曼不动杆菌基因分型技术,是开展其医院内感染流行病学研究和临床防控的重要工具。本文就近年来鲍曼不动杆菌基因分型技术的研究进展进行综述,并分析比较多种基因分型技术的原理、优点、局限性及应用范围。     

鲍曼不动杆菌噬菌体的研究进展

鲍曼不动杆菌能引起严重的院内感染,近年来鲍曼不动杆菌的耐药性越来越强,给治疗和控制感染带来极大困难。噬菌体可在宿主菌内复制并可破坏宿主菌,而对动植物没有毒性。因此,噬菌体以其特有的自然特征有望成为较好的抗菌制剂,作为一种新兴的治疗方法,已经越来越受到人们的关注,对噬菌体展开广泛和深入的研究具有重要的基础意义和潜在的临床应用价值。     

鲍曼不动杆菌血流感染的研究进展

近年来随着抗生素的广泛应用、免疫抑制剂的使用以及侵入性操作的增加,多重耐药、泛耐药甚至全耐药鲍曼不动杆菌日益增多,鲍曼不动杆菌引起的血流感染也呈不断上升趋势,给临床抗感染治疗带来极大的困难,导致临床预后差、病死率高,大大延长了患者的住院时间,造成医疗资源的浪费。了解鲍曼不动杆菌血流感染的危险因素、研究其耐药机制、尽早采取有效的干预措施、合理的应用抗生素、研究新型的治疗策略对预防和治疗鲍曼不动杆菌血流感染至关重要。本文就鲍曼不动杆菌血流感染的危险因素、耐药性、治疗、预后及预防进行综述,为临床预防和治疗提供一定的参考依据。     

黄酮化合物对耐药性鲍曼不动杆菌抑菌作用研究

为探索中药黄酮化合物对耐药性鲍曼不动杆菌的抑菌效果。研究通过微量肉汤稀释法,考察40种中药及天然来源的黄酮化合物对三株耐药性鲍曼不动杆菌菌株的体外抑菌作用,测试其最低抑菌浓度与最低杀菌浓度;采用结晶紫染色法进一步测定目标化合物对鲍曼不动杆菌生物被膜形成的能力;探索黄酮联合头孢哌酮/舒巴坦对鲍曼不动杆菌的抑菌效果。研究表明,黄酮单体对于鲍曼不动杆菌具有不同程度的体外抑菌作用。其中部分具有轻微作用,其最小抑菌浓度仅为0.5 mg/mL;黄芩素和汉黄芩苷具有显著的抑菌作用,其最小抑菌浓度分别为0.062 5和0.125 mg/mL;同时,黄芩素对鲍曼不动杆菌生物被膜形成具有明显的抑制作用。此外,黄芩素和头孢哌酮/舒巴坦联合作用于耐药鲍曼不动杆菌与单用黄芩素或头孢哌酮/舒巴坦比较,浓度有所降低。综上,黄芩素对耐药性鲍曼不动杆菌有较好的体外抑菌效果,这为治疗耐药性鲍曼不动杆菌引发的感染的治疗提供新思路和方向。