生物膜铜绿假单胞菌对小白鼠的致病作用

目前生物膜菌感染的治疗是临床亟待解决的课题 ,因此对生物膜菌的致病机理需要深入的研究 ,才能有效的治疗生物膜菌引起的感染。对 6周龄、雄性清洁级小鼠 ,分别用铜绿假单胞菌悬液和生物膜状态的铜绿假单胞菌在小鼠气管内接种 ,进行 3项试验 :①以细菌浓度为 5× 10 6ＣＦＵ ｍｌ接种 2 0 μｌ 只测定鼠肺泡灌洗液 (ＢＡＬＦ)中白细胞数 ,结果见表 1。 2种细菌接种后ＢＡＬＦ中的白细胞数差异有显著性 (Ｐ <0 .0 1)。②测定 2组感染小鼠的存活率每组 30只小鼠分别将 10 6ＣＦＵ ｍｌ的 2组菌液 2 0 μｌ 只接种到鼠气管内 ,观察 10ｄ小鼠存…     

用表面增强激光解析/电离飞行时间质谱蛋白指纹快速鉴定4种临床常见菌

目的 建立临床常见细菌蛋白指纹数据模型,为细菌的快速鉴定奠定基础.方法 利用表面增强激光解析/电离飞行时间质谱检测大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌和金黄色葡萄球菌参考株的细菌蛋白,筛选每种细菌稳定表达的蛋白峰,建立细菌的蛋白指纹数据模型,将其数据导人自建Fingerwave软件.收集临床分离的大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌和金黄色葡萄球菌共256株并运用质谱仪检测其细菌蛋白峰,蛋白峰数据与Fingerwave软件中蛋白指纹数据模型进行相似度比较,以评价其鉴定符合率.结果 初步建立了4种临床常见细菌的蛋白指纹数据模型,利用其对临床菌株进行鉴定,鉴定结果与传统微生物学鉴定及分子生物学鉴定结果的符合率分别为大肠埃希菌93.1％ (54/58)、肺炎克雷伯菌87.2％( 75/86)、铜绿假单胞菌95.2％( 60/63)和金黄色葡萄球菌96.2％ (51/53).结论 通过蛋白指纹数据的相似度比较,可同时对4种常见临床细菌进行鉴定,为细菌感染的快速诊断提供了可能性.     

ESBL阳性肺炎克雷伯菌所致肺炎临床特点和耐药性分析

目的 研究产超广谱β内酰胺酶(ESBL)的肺炎克雷伯菌的耐药特性及其所致肺炎的临床特点及临床危险因素,为预防ESBL阳性肺炎克雷伯菌所致肺炎的发生和临床治疗中抗生素的合理使用提供参考依据.方法 回顾性收集首都医科大学附属北京同仁医院93例(其中包括感染ESBL阳性肺炎克雷伯菌48例,非ESBL阳性肺炎克雷伯菌45例)住院患者的病历资料包括患者性别、年龄、治疗所用抗生素、住院时长、基础疾病以及侵入性操作6方面的信息,以及培养肺炎克雷伯菌的临床药敏结果.并对其危险因素进行单因素和多因素的分析.结果 ESBL阳性肺炎克雷伯菌菌株对阿米卡星的耐药率最低为41.6％,其次为复方新诺明和亚胺培南分别为56.3％和58.3％;对头孢菌素类药物(除头孢替坦外)耐药率为100％.临床治疗用药中单独用药最多见的是哌拉西林、美罗培南;联合用药最常见的是哌拉西林联合舒巴坦、美罗培南联合莫西沙星.感染ESBLs阳性肺炎克雷伯菌的肺炎患者,60岁以上占79.2％,平均住院天数为28.5 ±11.6天;死亡率为31％;合并高血压的有18例,合并糖尿病的有9例,合并脑梗的有3例,合并呼吸系统疾病的有10例;治疗过程中采用侵入性操作的有35例;感染ESBLs阴性肺炎克雷伯菌的肺炎患者,60岁以上33.4％,平均住住院天数为15.5 ±5.0天;死亡率6％;合并高血压的有18例,合并糖尿病的有9例,合并脑梗的有3例,合并呼吸系统疾病的有10例;治疗过程中采用侵入性操作的有16例.结论 住院时长(≥20天)、侵入性操作(包括气管插管、鼻导管吸氧、鼻饲等)、治疗过程中阿米卡星的使用,都是造成感染ESBL阳性肺炎克雷伯菌肺炎的临床感染危险因素(P＜0.05).在治疗感染ESBL阳性肺炎克雷伯菌的肺炎患者时应根据药敏结果调整临床用药.控制和预防感染ESBL阳性肺炎克雷伯菌的临床高危因素,可减少感染的可能性.     

大鼠慢性铜绿假单胞菌生物膜肺部感染IL-4的变化

铜绿假单胞菌（Pseudomonas aeruginosa，Pa）是慢性呼吸道感染主要致病菌之一，它可以产生藻酸盐形成生物膜。本研究通过建立慢性铜绿假单胞菌生物膜肺部感染动物模型，观察大鼠肺部的细菌学、病理学特点及IL-4的表达，了解铜绿假单胞菌生物膜肺部感染的免疫应答情况。     

肺炎克雷伯杆菌在T24细胞内存活的动态观察

目的：了解肺炎克雷伯杆菌与膀胱上皮细胞的相互关系，观察肺炎克雷伯杆菌在人膀胱上皮细胞抹T24中生存的动态变化。方法：采用肺炎克雷伯杆菌临床分离抹03138侵袭T24细胞，并用庆大霉素杀死细胞外的细菌，分别于细菌进入细胞后的4、24、48及72h裂解细胞，释放出细胞内的活细菌，用平板菌落计数法计数胞内活菌数。结果：T24细胞内的肺炎克雷伯杆菌03138抹在实验48h内有一定生长，试验72h细胞内活菌数量明显减少。加入细胞因子（TNF-αd和INF-γ）可以促进上皮细胞清除胞内细菌。结论：膀胱上皮细胞清除进入细胞内的肺炎克雷伯杆菌，可能是泌尿道天然免疫的一种防御机制，而细胞因子可以调控上皮细胞的抗菌作用。     

产超广谱β内酰胺酶肺炎克雷伯菌医院感染肺炎临床危险因素的病例对照研究

目的探讨产超广谱β内酰胺酶(ESBLs)肺炎克雷伯菌医院感染肺炎的临床危险因素。方法用SPSS11.5软件进行统计,对101例肺炎克雷伯菌医院感染肺炎的临床危险因素进行病例对照研究。结果单因素分析发现住院时间大于20天、入住重症监护病房、气管插管或切开、留置导管、机械通气、头孢噻肟的使用是产ESBLs肺炎克雷伯菌医院感染肺炎的主要危险因素。多因素非条件logistic回归分析表明,头孢噻肟的使用是产ESBLs肺炎克雷伯菌医院感染肺炎的独立危险因素。结论合理使用头孢噻肟、采用替换性抗生素治疗策略是防止产ESBLs肺炎克雷伯菌感染肺炎流行的重要措施。     

矿井瓦斯爆炸伤创面病原菌的分离及其药敏分析

目的：寻找瓦斯爆炸伤创面感染的主要病原菌及其临床使用抗生素的抗菌谱，为临床治疗瓦斯爆炸伤伤员提供微生物学依据。 方法：将所取创面分泌物直接接种于绵羊血平板和巧壳力琼脂平板上，35℃培养18～24h，参照伯杰氏细菌学手册(第九版)进行分离、鉴定，采用K-B法进行药敏试验。结果：瓦斯爆炸伤剖面感染的主要病原菌以肺炎克雷伯氏菌肺炎亚种占首位，铜绿假单胞菌和类产碱假单胞菌依序次之，这3种菌种对环丙沙星、多粘菌索B敏感。 结论：瓦斯爆炸伤伤员不仅易受铜绿假单胞菌、类产碱假单胞菌医源性感染，更易受矿井内的肺炎克雷伯氏菌肺炎亚种感染，这与地面一般烧伤感染情况不同。     

心脏外科术后多重耐药细菌感染的病原学特征与耐药性分析

目的了解心脏外科术后患者多重耐药菌(multidrug-resistant organism, MDRO)感染的病原学特征以及耐药模式, 指导临床医生合理应用指南推荐的抗菌药物, 提高经验性抗菌治疗的成功率、改善住院患者预后。方法收集2018年1月—2021年10月心脏外科术后409例确诊为医院感染患者的完整病历资料, 根据合格的临床标本阳性培养结果分为多重耐药细菌感染组(MDR=176例)和非多重耐药细菌感染组(non-MDR=233例), 分析MDRO感染的病原学特征以及对临床常用抗菌药物的耐药模式, 同时对不同的外科手术类型和临床感染类型相对应的病原学分布、耐药情况以及临床结局进行统计分析。结果合格的临床标本检出多重耐药细菌306株, 97例(97/176, 55.1%)患者同时存在两种及两种以上多重耐药细菌感染, 主要表现为肺炎克雷伯菌、鲍曼不动杆菌和(或)铜绿假单胞菌混合感染, 其中包括碳青霉烯类耐药细菌213株(213/306, 69.6%)。无论外科手术类型和临床感染类型如何, 病原学分布均以鲍曼不动杆菌(114/306, 37.3%)最常见, 其次是肺炎克雷伯菌(72/...     

肺炎克雷伯菌感染中的宿主细胞程序性死亡

肺炎克雷伯菌可以引起多种感染性疾病, 在免疫功能低下的人群中尤其严重。耐多药高毒力肺炎克雷伯菌的出现大大缩小了治疗肺炎克雷伯菌感染的选择, 对新的治疗策略的探索迫在眉睫。在感染进程中, 宿主细胞程序性死亡与肺炎克雷伯菌侵袭之间有着复杂的相互作用, 本文主要从肺炎克雷伯菌引起的宿主细胞焦亡、凋亡、坏死性凋亡、自噬等几种程序性细胞死亡机制的研究进展进行综述。     

生物材料植入感染与表皮葡萄球菌生物膜形成的相关基因调控

表皮葡萄球菌是寄居于人体皮肤和黏膜表面的共生菌群,现已发现其是引发临床生物材料相关感染的主要条件致病菌,在生物材料植入感染中占有重要地位.医用生物材料表面细菌生物膜的形成是其主要致病因素,细菌生物膜可有效抵御机体的防御反应和抗生素治疗,导致生物材料植入感染难以彻底治愈,使感染呈慢性、持续性和反复性特点,从而在临床上造成了极高的死亡率.就表皮葡萄球菌生物膜的形成、胞间黏附素基因(ica)操纵子和附属基因调节子(agr)基因对表皮葡萄球菌生物膜的调控及其在临床生物材料植入感染中的作用等方面作一综述.     

Type 3 fimbrial shaft (MrkA) of Klebsiella pneumoniae, but not the fimbrial adhesin (MrkD), facilitates biofilm formation

Isolates of Klebsiella pneumoniae are responsible for opportunistic infections, particularly of the urinary tract and respiratory tract, in humans. These bacteria express type 3 fimbriae that have been implicated in binding to eucaryotic cells and matrix proteins. The type 3 fimbriae mediate binding to target tissue using the MrkD adhesin that is associated with the fimbrial shaft comprised of the MrkA protein. The formation of biofilms in vitro by strains of K. pneumoniae was shown to be affected by the production of fimbriae on the bacterial surface. However, a functional MrkD adhesin was not necessary for efficient biofilm formation. Nonfimbriate strains were impaired in their ability to form biofilms. Using isogenic fimbriate and nonfimbriate strains of K. pneumoniae expressing green fluorescent protein it was possible to demonstrate that the presence of type 3 fimbriae facilitated the formation of dense biofilms in a continuous-flowthrough chamber. Transformation of nonfimbriate mutants with a plasmid possessing an intact mrk gene cluster restored the fimbrial phenotype and the rapid ability to form biofilms.     

Comparative antibody-mediated phagocytosis of Staphylococcus epidermidis cells grown in a biofilm or in the planktonic state

Staphylococcus epidermidis is an important cause of nosocomial infections. Virulence is attributable to elaboration of biofilms on medical surfaces that protect the organisms from immune system clearance. Even though leukocytes can penetrate biofilms, they fail to phagocytose and kill bacteria. The properties that make biofilm bacteria resistant to the immune system are not well characterized. In order to better understand the mechanisms of resistance of bacteria in biofilms to the immune system, we evaluated antibody penetration throughout the biofilm and antibody-mediated phagocytic killing of planktonic versus biofilm cells of S. epidermidis by using a rabbit antibody to poly-N-acetylglucosamine (PNAG). These antibodies are opsonic and protect against infection with planktonic cells of PNAG-positive Staphylococcus aureus and S. epidermidis. Antibody to PNAG readily penetrated the biofilm and bound to the same areas in the biofilm as did wheat germ agglutinin, a lectin known to bind to components of staphylococcal biofilms. However, biofilm cells were more resistant to opsonic killing than their planktonic counterparts in spite of producing more PNAG per cell than planktonic cells. Biofilm extracts inhibited opsonic killing mediated by antibody to PNAG, suggesting that the PNAG antigen within the biofilm matrix prevents antibody binding close to the bacterial cell surface, which is needed for efficient opsonic killing. Increased resistance of biofilm cells to opsonic killing mediated by an otherwise protective antibody was due not to a biofilm-specific phenotype but rather to high levels of antigen within the biofilm that prevented bacterial opsonization by the antibody.     

Biofilm formation in vitro and virulence in vivo of mutants of Klebsiella pneumoniae

One of the early stages of Klebsiella pneumoniae airway infections may involve biofilm formation. Bacterial biofilm formation is frequently investigated using in vitro techniques that facilitate identification and analysis of individual genes. We investigated the correlation between K. pneumoniae biofilm formation in vitro and ability to cause infection in vivo following construction of a bank of mini-Tn5 mutants.     

Antimicrobial susceptibility testing in biofilm-growing bacteria

Biofilms are organized bacterial communities embedded in an extracellular polymeric matrix attached to living or abiotic surfaces. The development of biofilms is currently recognized as one of the most relevant drivers of persistent infections. Among them, chronic respiratory infection by Pseudomonas aeruginosa in cystic fibrosis patients is probably the most intensively studied. The lack of correlation between conventional susceptibility test results and therapeutic success in chronic infections is probably a consequence of the use of planktonically growing instead of biofilm-growing bacteria. Therefore, several in vitro models to evaluate antimicrobial activity on biofilms have been implemented over the last decade. Microtitre plate-based assays, the Calgary device, substratum suspending reactors and the flow cell system are some of the most used in vitro biofilm models for susceptibility studies. Likewise, new pharmacodynamic parameters, including minimal biofilm inhibitory concentration, minimal biofilm-eradication concentration, biofilm bactericidal concentration, and biofilm-prevention concentration, have been defined in recent years to quantify antibiotic activity in biofilms. Using these parameters, several studies have shown very significant quantitative and qualitative differences for the effects of most antibiotics when acting on planktonic or biofilm bacteria. Nevertheless, standardization of the procedures, parameters and breakpoints, by official agencies, is needed before they are implemented in clinical microbiology laboratories for routine susceptibility testing. Research efforts should also be directed to obtaining a deeper understanding of biofilm resistance mechanisms, the evaluation of optimal pharmacokinetic/pharmacodynamic models for biofilm growth, and correlation with clinical outcome.     

Impact of polymicrobial biofilms in catheter-associated urinary tract infections

Recent reports have demonstrated that most biofilms involved in catheter-associated urinary tract infections are polymicrobial communities, with pathogenic microorganisms (e.g. Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) and uncommon microorganisms (e.g. Delftia tsuruhatensis, Achromobacter xylosoxidans) frequently co-inhabiting the same urinary catheter. However, little is known about the interactions that occur between different microorganisms and how they impact biofilm formation and infection outcome. This lack of knowledge affects CAUTIs management as uncommon bacteria action can, for instance, influence the rate at which pathogens adhere and grow, as well as affect the overall biofilm resistance to antibiotics. Another relevant aspect is the understanding of factors that drive a single pathogenic bacterium to become prevalent in a polymicrobial community and subsequently cause infection. In this review, a general overview about the IMDs-associated biofilm infections is provided, with an emphasis on the pathophysiology and the microbiome composition of CAUTIs. Based on the available literature, it is clear that more research about the microbiome interaction, mechanisms of biofilm formation and of antimicrobial tolerance of the polymicrobial consortium are required to better understand and treat these infections.     

Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.