鲍曼不动杆菌临床分布、耐药性分析和肺部感染病例的预后研究

目的了解浙江大学医学院附属第一医院临床分离鲍曼不动杆菌的分布情况、耐药状况以及肺部感染病例的临床预后,为临床鲍曼不动杆菌感染的治疗提供参考。方法分析该院2013年1月至2013年6月培养到的非重复鲍曼不动杆菌420株的临床分布和耐药性,回顾性分析鲍曼不动杆菌肺炎并接受头孢哌酮/舒巴坦治疗患者的临床资料。结果 420株鲍曼不动杆菌来源以重症监护室（ICU）为主,占45.96%。其次为外科（神经外科、肝胆外科）,占36.19%。标本构成中以呼吸道标本最高（66.90%）,其次为血培养（8.81%）。共有79例（18.8%）患者≥2个部位分离到鲍曼不动杆菌,其中62例（78.5%）呼吸道是首发的分离部位、之后出现第二个部位培养阳性。体外药敏显示,鲍曼不动杆菌对阿米卡星、替加环素、左氧氟沙星、头孢哌酮/舒巴坦、氨苄西林/舒巴坦、亚胺培南和美罗培南的耐药率分别为5.2%、15.9%、35.6%、64.2%、71.1%、79.1%和88.9%。选取其中诊断为肺炎患者72例。72例患者粗死亡14例（19.4%）。患者病情好转组治疗初C反应蛋白平均水平为69.5 mg/L、死亡组为137.6 mg/L,经比较差异有统计学意义（P〈0.05）;患者病情好转组治疗初APACHE II为16.4、死亡组为19.7,经比较两组差异有统计学意义（P〈0.05）。结论入住重症监护室、外科手术后患者是鲍曼不动杆菌分离的主要人群,18.8%的患者在多个部位培养到鲍曼不动杆菌,鲍曼不动杆菌耐药率高,抗菌药物选择困难。治疗初期C反应蛋白和APACHE II水平对预后有参考价值。     

306株鲍曼不动杆菌的临床分布及耐药性监测

目的调查杭州市某医院鲍曼不动杆菌感染的临床分布及其耐药性现状。方法监测该院2004年1月至2006年12月临床分离的306株鲍曼不动杆菌的临床感染分布及耐药性,药物敏感试验采用琼脂纸片扩散法,耐药性数据分析采用WHONET 5软件。结果2004年至2006年3年间,鲍曼不动杆菌的检出率呈逐年增加趋势(从1.60%增至2.40%);临床分布以重症监护室(ICU)最高(84/306),老年患者多见(131/306);有184株(60.13%)来源于痰液标本。药敏试验结果显示鲍曼不动杆菌对美罗培南最敏感,耐药率为4.6%;其次是亚胺培南/西司他丁和头孢哌酮/舒巴坦,耐药率分别为7.2%及8.5%;对3代头孢菌素及环丙沙星的耐药率均>50.0%;同时发现185株(60.5%)为多重耐药菌株。结论鲍曼不动杆菌对碳青霉烯类抗生素及头孢哌酮/舒巴坦较敏感,临床医师应注意合理使用抗菌药物,以减少多重耐药菌株的产生。     

多重耐药和广泛耐药鲍曼不动杆菌感染治疗的药物选择

鲍曼不动杆菌是医院内感染的重要病原体,其基于内在性和获得性的耐药机制,导致全球抗感染领域面临巨大挑战。目前,针对多重耐药和广泛耐药鲍曼不动杆菌引起的感染尚无有效治疗方案,本文对其可选用的治疗药物及最新进展进行综述。     

鲍曼不动杆菌无痕基因敲除及hcp基因相关功能

【背景】鲍曼不动杆菌耐药严重,基因敲除是研究细菌毒力与耐药的重要方式。但现有的大部分细菌基因敲除方法基于抗生素抗性筛选,导致不适用于多重耐药菌株的基因敲除。【目的】旨在建立一种非依赖于抗生素抗性筛选的方法,用于敲除多重耐药鲍曼不动杆菌基因。【方法】运用同源重组和自杀载体pMo130-TelR对亚碲酸钾的抗性,使用两步筛选法,构建鲍曼不动杆菌VI型分泌系统溶血素共调节蛋白(Hemolysin-Coregulated Protein,Hcp)基因敲除突变体,并对缺失株的生长能力、细菌竞争能力以及血清抵抗能力进行测试。【结果】通过构建含同源片段的重组pMo130-TelR载体,成功敲除了鲍曼不动杆菌标准株ATCC 17978中的hcp基因,获得了ATCC 17978 hcp基因缺失突变体。突变体生长能力没有显著改变,但细菌竞争能力显著下降,血清抵抗能力显著升高。【结论】pMo130-TelR可成功用于鲍曼不动杆菌无痕基因敲除,对于研究鲍曼不动杆菌的耐药机制等相关问题具有深远意义。     

鲍曼不动杆菌耐药性及碳青霉烯酶检测

目的了解临床分离鲍曼不动杆菌对临床常用抗菌药物的耐药性及其产碳青霉烯酶情况,为临床抗感染治疗提供依据。方法采用MicroScan WalkAway-40全自动微生物分析仪对240株临床分离的鲍曼不动杆菌进行鉴定和药敏试验,改良的Hodge试验检测耐碳青霉烯类鲍曼不动杆菌产生的碳青霉烯酶,并采用SPSS 13.0软件进行统计分析。结果 240株鲍曼不动杆菌对亚胺培南的耐药率最低(20.8%),对青霉素类、头孢菌素类、喹诺酮类、复方新诺明的耐药率均大于50%;耐碳青霉烯类鲍曼不动杆菌碳青霉烯酶的检出率为64.0%。结论临床分离鲍曼不动杆菌耐药情况严重,碳青霉烯酶是鲍曼不动杆菌对碳青霉烯类抗菌药物耐药的主要原因。     

鲍曼不动杆菌裂解性噬菌体LZ35的 分离及全基因组分析

摘要：目的 通过分离鉴定鲍曼不动杆菌噬菌体并进行遗传信息分析,为今后噬菌体用于治疗鲍曼不动杆菌引起的感染提供依据。方法 以鲍曼不动杆菌临床分离株为宿主菌,从医院污水中分离鲍曼不动杆菌噬菌体并进行纯化、电镜观察形态特征、提取噬菌体DNA,进行全基因组测序,分析全基因组的结构特征,比较基因组分析其进化关系。结果 分离到鲍曼不动杆菌裂解性噬菌体LZ35,电镜观察显示,该噬菌体属于有尾噬菌体目肌尾病毒科。基因组全长44 885 bp,G＋C含量为37.95%,含有83个开放阅读框,其中22个编码序列可预测其功能,61个编码序列为未知基因。噬菌体LZ35的基因组与鲍曼不动杆菌噬菌体IME-AB2和YMC-13-01-C62具有很高的同源性（分别为97%和99%）,与鲍曼不动杆菌噬菌体YMC11/12/R1215的进化关系最近。结论 以鲍曼不动杆菌临床分离株为宿主菌,分离到鲍曼不动杆菌裂解性噬菌体LZ35,明确了其形态和基因组特征,为防治噬菌体疗法奠定基础。     

Adherence and motility characteristics of clinical Acinetobacter baumannii isolates

Acinetobacter baumannii continues to be a major health problem especially in hospital settings. Herein, features that may play a role in persistence and disease potential were investigated in a collection of clinical A. baumannii strains from Australia. Twitching motility was found to be a common trait in A. baumannii international clone I strains and in abundant biofilm formers, whereas swarming motility was only observed in isolates not classified within the international clone lineages. Bioinformatic analysis of the type IV fimbriae revealed a correlation between PilA sequence homology and motility. A high level of variability in adherence to both abiotic surfaces and epithelial cells was found. We report for the first time the motility characteristics of a large number of A. baumannii isolates and present a direct comparison of A. baumannii binding to nasopharyngeal and lung epithelial cells.     

鲍曼不动杆菌的临床分布及药物敏感性结果分析

目的了解鲍曼不动杆菌的临床分布及药物敏感性分析。方法收集2004年5月至2006年5月抚顺市中心医院住院及门诊患者的临床标本,从中分离鲍曼不动杆菌。质控菌株:大肠埃希菌ATCC 25922,铜绿假单胞菌ATCC 27853,采用美国BD公司生产的凤凰微生物鉴定/药敏分析仪,对62株鲍曼不动杆菌进行鉴定和药物敏感测定并分析其临床分布。结果62株鲍曼不动杆菌感染率高的有呼吸病房22例(35.5%),干部病房14例(22.6%),标本类型以痰为主52例(83.9%)。药敏监测该细菌对17种抗生素的耐药情况为亚胺培南、美洛培南、氨苄西林/舒巴坦、左氧氟沙星的敏感率较高,分别为100%、100%、75.8%、70.9%。有头孢唑林、氨苄西林、奥格门丁、氨曲南耐药率为90.3%、88.7%、80.6%、77.4%。结论鲍曼不动杆菌最有效的抗生素是亚胺培南、美洛培南,一旦临床发生感染,应及时调整和选用合理抗生素治疗,避免滥用抗生素,防止耐药菌株的产生,提高临床治愈率。     

鲍曼不动杆菌体外诱导耐药及其诱导前后交叉耐药和呼吸耗氧率分析

【背景】鲍曼不动杆菌是院内感染的重要病原菌,因其耐药率高、治疗难度大而备受关注。然而,对于该菌的交叉耐药及耐药相关因素尚未完全阐明。【目的】通过体外诱导分别获得耐美罗培南或耐替加环素的鲍曼不动杆菌菌株,并研究其诱导前后的交叉耐药性和细菌呼吸耗氧率差异。【方法】采用多步法对鲍曼不动杆菌ATCC19606进行体外诱导耐药,PCR扩增诱导前后菌株的16S rRNA基因并测序鉴定,微量肉汤稀释法检测诱导前后鲍曼不动杆菌对美罗培南、亚胺培南、替加环素、阿米卡星、头孢吡肟及左氧氟沙星等抗菌药物的最低抑菌浓度变化,Seahorse XF^e96细胞能量代谢实时测定仪对诱导前后菌株的耗氧率进行分析。【结果】通过88d的体外诱导实验,分别获得耐美罗培南或耐替加环素的鲍曼不动杆菌ATCC19606菌株。耐美罗培南鲍曼不动杆菌ATCC19606对替加环素、亚胺培南、阿米卡星、左氧氟沙星仍处于敏感状态,但是对头孢吡肟交叉耐药;耐替加环素鲍曼不动杆菌ATCC19606对美罗培南、亚胺培南、阿米卡星、左氧氟沙星及头孢吡肟仍处于敏感状态。鲍曼不动杆菌ATCC19606被美罗培南或替加环素诱导耐药...     

Differences in Acinetobacter baumannii strains and host innate immune response determine morbidity and mortality in experimental pneumonia

Despite many reports documenting its epidemicity, little is known on the interaction of Acinetobacter baumannii with its host. To deepen our insight into this relationship, we studied persistence of and host response to different A. baumannii strains including representatives of the European (EU) clones I-III in a mouse pneumonia model. Neutropenic mice were inoculated intratracheally with five A. baumannii strains and an A. junii strain and at several days morbidity, mortality, bacterial counts, airway inflammation, and chemo- and cytokine production in lungs and blood were determined. A. baumannii RUH875 and RUH134 (EU clone I and II, respectively) and sporadic strain LUH8326 resulted in high morbidity/mortality, whereas A. baumannii LUH5875 (EU clone III, which is less widespread than clone I and II) caused less symptoms. A. baumannii type strain RUH3023(T) and A. junii LUH5851 did not cause disease. All strains, except A. baumannii RUH3023(T) and A. junii LUH5851, survived and multiplied in the lungs for several days. Morbidity and mortality were associated with the severity of lung pathology and a specific immune response characterized by low levels of anti-inflammatory (IL-10) and specific pro-inflammatory (IL-12p40 and IL-23) cytokines at the first day of infection. Altogether, a striking difference in behaviour among the A. baumannii strains was observed with the clone I and II strains being most virulent, whereas the A. baumannii type strain, which is frequently used in virulence studies appeared harmless.     

Specific and Selective Bacteriophages in the Fight against Multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii causes serious infections especially in immunocompromised and/or hospitalized patients. Several A. baumannii strains are multidrug resistant and infect wounds, bones, and the respiratory tract. Current studies are focused on finding new effective agents against A. baumannii. Phage therapy is a promising means to fight this bacterium and many studies on procuring and applying new phages against A. baumannii are currently being conducted. As shown in animal models, phages against multidrug-resistant A. baumannii may control bacterial infections caused by this pathogen and may be a real hope to solve this dangerous health problem.     

Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial infection. Despite considerable clinical and epidemiological data regarding the role of A. baumannii in nosocomial infection, the specific virulence factor or pathogenic mechanism of this organism has yet to be elucidated. This study investigated the molecular mechanism of apoptosis on the infection of human laryngeal epithelial HEp-2 cells with A. baumannii and examined the contribution of outer membrane protein 38 (Omp38) on the ability of A. baumannii to induce apoptosis of epithelial cells. A. baumannii induced apoptosis of HEp-2 cells through cell surface death receptors and mitochondrial disintegration. The Omp38-deficient mutant was not as able to induce apoptosis as the wild-type A. baumannii strain. Purified Omp38 entered the cells and was localized to the mitochondria, which led to a release of proapoptotic molecules such as cytochrome c and apoptosis-inducing factor (AIF). The activation of caspase-3, which is activated by caspase-9, degraded DNA approximately 180 bp in size, which resulted in the appearance of a characteristic DNA ladder. AIF degraded chromosomal DNA approximately 50 kb in size, which resulted in large-scale DNA fragmentation. These results demonstrate that Omp38 may act as a potential virulence factor to induce apoptosis of epithelial cells in the early stage of A. baumannii infection.     

346株鲍曼不动杆菌临床分布及耐药率分析

目的了解鲍曼不动杆菌引起医院感染的特点以及对抗菌药物的耐药性的变化趋势。方法应用美国BD公司Phoenix^TM100全自动细菌和药敏系统鉴定仪对所分离的364株鲍曼不动杆菌进行鉴定和药敏试验,并进行统计学分析。结果 346株鲍曼不动杆菌来自痰液、伤口分泌物、中段尿、静脉血及大便,分别占58.96%、30.92%、5.49%、4.34%、0.29%。药敏结果显示鲍曼不动杆菌对多粘菌素和米诺环素敏感性最高,耐药率低于10.4%。结论加强鲍曼不动杆菌的耐药监测,了解其耐药性变迁,可合理指导用药,有效控制鲍曼不动杆菌耐药菌株的产生。     

Platelet-activating Factor Receptor Initiates Contact of Acinetobacter baumannii Expressing Phosphorylcholine with Host Cells

Adhesion is an initial and important step in Acinetobacter baumannii causing infections. However, the exact molecular mechanism of such a step between A. baumannii and the host cells remains unclear. Here, we demonstrated that the phosphorylcholine (ChoP)-containing outer membrane protein of A. baumannii binds to A549 cells through platelet-activating factor receptor (PAFR), resulting in activation of G protein and intracellular calcium. Upon A. baumannii expressing ChoP binding to PAFR, clathrin and β-arrestins, proteins involved in the direction of the vacuolar movement, are activated during invasion of A. baumannii. PAFR antagonism restricts the dissemination of A. baumannii in the pneumonia model. These results define a role for PAFR in A. baumannii interaction with host cells and suggest a mechanism for the entry of A. baumannii into the cytoplasm of host cells.     

不同海拔地区同级别医院鲍曼不动杆菌的分布与 耐药性的连续对比性分析

目的连续对比分析不同海拔地区同级别医院非鲍曼不动杆菌的耐药性,寻找不同海拔对鲍曼不动杆菌的耐药性的影响并指导合理应用抗生素。方法回顾分析2011-2013年两家不同海拔地区同级别医院临床分离的鲍曼不动杆菌药敏结果。结果 2011年至2013年,低海拔地区医院鲍曼不动杆菌检出率为12.66%、17.01%、15.33%。高海拔的地区院鲍曼不动杆菌检出率为0.24%、1.50%、1.44%。低海拔地区医院鲍曼不动杆菌仅对美满环素仍保持较高的敏感率;除头孢哌酮/舒巴坦外,对多数常用药物耐药率均高于70%。而且保持稳定。高海拔的地区院鲍曼不动杆菌对常用抗生素的耐药率逐年下降,庆大霉素、左旋氧氟沙星、亚胺培南、头孢哌酮、头孢他啶的敏感率近两年均在60%以上。结论低海拔地区医院鲍曼不动杆菌检出率高,常用抗生素耐药率高。高海拔的地区院鲍曼不动杆菌检出率低,常用抗生素敏感率高。环境因素对微生态具有重要的影响作用。     

694株鲍曼不动杆菌的耐药性分析

目的了解大连市友谊医院临床分离的鲍曼不动杆菌的分布及耐药状况,为临床治疗鲍曼不动杆菌感染提供参考。方法对该院2007年至2009年住院患者送检的标本中分离到的694株鲍曼不动杆菌的分布及药敏结果做回顾性分析。结果鲍曼不动杆菌2007年检出112株（7．1％）,2008年检出210（11．3％）株,2009年检出372（14．7％）株;在所有临床送检的标本中,痰标本中的检出率最高,占88．7％;在对13种抗生素的药敏试验中,对美罗培南最为敏感,其次对头孢哌酮／舒巴坦较为敏感。结论鲍曼不动杆菌的检出率逐年增高,耐药率也逐年增高。     

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.     

鲍曼不动杆菌遗传操作系统研究进展

鲍曼不动杆菌作为一种医院内感染的病原菌,因其易于引起各类感染且耐药性强而受到广泛关注。快速改造鲍曼不动杆菌基因组的工具可有效促进其耐药机制的研究。本文就近些年来适用于鲍曼不动杆菌的遗传操作方法进行了总结,包括各种外源基因转入方法(电转化、自然转化、接合转移)和基因改造技术(等位基因交换、DNA重组工程、转座突变),并对鲍曼不动杆菌的基因组编辑方法的改进作了初步展望。     

Susceptibility, phenotypes of resistance, and extended-spectrum β-lactamases in Acinetobacter baumannii strains

Acinetobacter baumannii plays an increasing role in the pathogenesis of infections in humans. The bacilli are frequently isolated from patients treated in intensive care units. A growing resistance to antibiotics is leading to the emergence of strains that are multidrug-resistant and resistant to all available agents. The objective of this study was to assess susceptibility to antibiotics and to determine the presence and current level of the extended-spectrum β-lactamases (ESBLs) and attempt to isolate the Acinetobacter baumannii strain carrying the blaPER gene. A total of 51 strains of A. baumannii identified by phenotypic features were examined. That the strains belonged to the species was confirmed by the presence of the blaOXA-51-like; gene. A broth microdilution method was used for antibacterial susceptibility testing. The occurrence of ESBLs was determined using phenotypic double-disk synergy tests. The PCR technique was used to confirm the presence of the blaPER-1; gene encoding ESBL. The most active antibiotics were meropenem, cefepime and ampicillin/sulbactam, with susceptibility shown by 76.5%, 60.8% and 56.9% of the strains, respectively. The strains exhibited the highest resistance (> 75%) to piperacillin, tetracycline, ciprofloxacin and cefotaxime. Phenotypic tests revealed ESBL mechanism of resistance in approximately 20% of Acinetobacter baumannii isolates. However, the PCR technique did not confirm the presence of the blaPER-1; gene in any of the Acinetobacter baumannii strains examined in our hospital. Acinetobacter baumannii strains demonstrate considerable resistance to many groups of antibiotics. Our findings indicate the involvement of enzymes belonging to families other than PER β-lactamase in resistance to β-lactams in A. baumannii.     

Crude oil degradation efficiency of a recombinant Acinetobacter baumannii strain and its survival in crude oil-contaminated soil microcosm

A hydrocarbon degrading Acinetobacter baumannii S30 strain, isolated from crude oil-contaminated soil, was inserted with the lux gene from the luciferase gene cassette luxCDABE. Soil microcosms were designed to study the degradation efficacy for total petroleum hydrocarbon (TPH) of crude oil by lux-tagged A. baumannii S30 pJES. Bioaugmentation of a TPH-contaminated microcosm with A baumannii S30 pJES showed that TPH levels were reduced from 89.3 to 53.9 g/kg soil in 90 days. Biodegradation of TPH by A baumannii S30 pJES was also monitored in shake flask conditions, which showed a reduction of initial TPH levels by over 50% at the end of 120 h. A lux-PCR-based approach along with the standard dilution plating with selective antibiotics was successfully utilized to monitor the survivability of the lux-tagged strain A. baumannii S30 pJES in soil microcosms and stability of the lux insert in the host strain A. baumannii S30. The selective plating technique indicated the population of A. baumannii S30 pJES to be 6.5+/-0.13 x 10(8) CFU/g at day zero (just after bioaugmentation) and 2.09+/-0.08 x 10(8) CFU/g of soil after 90 days of incubation. lux-PCR confirmed the stability of the insert in all the randomly selected colonies of A. baumannii strains from the antibiotic plates. The lux insert was stable after 50 generations in Luria Bertini broth and storage at -70 degrees C as glycerol stocks for over a year. These results revealed that the lux insert was stable and lux-tagged A. baumannii S30 strain could survive in a TPH-contaminated soil microcosm and could degrade TPH in the soil microcosm conditions. It can be used as an effective marker to monitor the survival of augmented strains at a bioremediation site.