Infektionen der Atemwege mit Pseudomonas aeruginosa bei der Cystischen Fibrose

Summary The main cause of death in cystic fibrosis (CF) patients is progressive pulmonary insufficiency frequently associated with chronic infections of the respiratory tract by Pseudomonas aeruginosa. Bacteria of this species synthesize numerous extracellular products contributing to its pathogenicity. An alginate-like exopolysaccharide is characteristic for mucoid mutants predominating among P. aeruginosa isolates from CF patients. It interferes with immune defense mechanisms of the host and probably protects the bacteria against certain antibiotics. Furthermore, it is involved in the formation of bacterial microcolonies that resist mucociliary clearance, opsonisation, and phagocytosis. Exotoxin A and elastase are regarded as the most important among various extracellular enzymes involved in pulmonary injury in CF patients. Exotoxin A inhibits eukaryotic protein synthesis leading to necrosis; elastase, together with other Pseudomonas-proteases, induces hemorrhagic lesions and necrosis and seems to inactivate immunoglobulins and complement factors. Phospholipase C and glycolipid represent two hemolysins of P. aeruginosa that may contribute to cytopathogenic effects in infected lungs. No primary defect in the immunological defense mechanisms of CF patients has been described so far. Antibodies against various P. aeruginosa antigens including those mentioned above have been demonstrated, but a complete elimination of the bacteria from infected lungs has not been observed. Therapy of pulmonary P. aeruginosa infections in CF patients usually includes combinations of antibiotics of the-lactam and aminoglycoside type. Difficulties arise from an unusually high intrinsic resistance of P. aeruginosa as well as from poor penetration of many antibiotics into the sputum of CF patients. Therefore, future efforts to manage the Pseudomonas problem in CF will probably concentrate on prophylactic therapy, e.g. childhood vaccination of CF patients in order to prevent bacterial colonization of the respiratory tract.

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Abkürzungen ADP Adenosindiphosphat - CF Cystische Fibrose - GLP Glycolipoprotein - LPS Lipopolysaccharid - NAD Nicotinamidadenindinukleotid Dieser Aufsatz ist die erweiterte Fassung eines Vortrages, der bei der Jahrestagung der Deutschen Gesellschaft zur Bekämpfung der Mucoviscidose e.V. am 1.6.1984 in Bonn gehalten wurde.     

Src kinase Lyn is crucial for Pseudomonas aeruginosa internalization into lung cells

Lyn is an important B cell signaling kinase of the Src tyrosine kinase family with a broad range of functions from cytoskeletal changes to induction of apoptosis. However, the role of Lyn in infectious diseases is not clear. Here, we demonstrate that Lyn activation by phosphorylation significantly impacted invasion of an alveolar epithelial cell line, primary lung cells, and rat lungs by Pseudomonas aeruginosa (PA), a common opportunistic lung pathogen affecting individuals with deficient lung immunity. Our results indicate that activation of Lyn and its interaction with rafts and TLR2, played an important role in the initial stages of PA interaction with host cells. The role of Lyn was further evaluated using the pharmacologic Src-specific inhibitor PP2, a dominant negative mutant, and finally confirmed with Lyn-deficient (Lyn(-/-)) bone marrow-derived mast cells. Inhibition of Lyn's function by above approaches prevented PA internalization. Moreover, blocking of Lyn also affected downstream events: induction of inflammatory cytokines and apoptosis. This report brings out a new role of Lyn in infectious diseases and indicates potential new targets for prevention and treatment of infections.     

The immune response to chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is predominantly of the Th2 type

Most cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa lung infection have a persistent acute type lung inflammation dominated by polymorphonuclear neutrophils (PMN) and a pronounced antibody response against P. aeruginosa. We speculated whether this immune response in CF is of the Th2 type and whether a change to a Th1 type immune response could improve the prognosis. Therefore, we studied 14 CF patients with (CF +P) and 14 CF patients without (CF -P) chronic P. aeruginosa lung infection. The specific production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) by peripheral blood mononuclear cells was determined. Cells from CF +P patients had lower IFN-gamma (p<0.05) and higher IL-4 (p<0.005) production as compared to cells from CF -P patients. Furthermore, a positive correlation between IFN-gamma production and lung function was found (FVC: Rho = 0.637; p<0.03; FEV1: Rho=0.524; p<0.07). We conclude that a Th2 type immune response is most frequent in CF patients with chronic P. aeruginosa lung infection, and the patients with a Th1-dominated immune response had the best lung function. The clinical implication is that a change to a Th1 type immune response might improve the prognosis in these patients.     

Cellular immunity in healthy volunteers treated with an octavalent conjugate Pseudomonas aeruginosa vaccine

Humoral immunity in response to an octavalent O-polysaccharide-toxin A conjugate Pseudomonas aeruginosa vaccine is well studied, and a phase III clinical study in cystic fibrosis (CF) patients is currently ongoing. In contrast, little is known about cellular immunity induced by this vaccine. Fifteen healthy volunteers were immunized on days 1 and 60. Parameters of cellular immunity were studied before vaccination on day 1, and on day 74. Analyses included flow cytometry of whole blood and antigen-induced proliferation of and cytokine production by lymphocyte cultures. The effects of immunization on the composition of peripheral blood lymphocytes as determined by flow cytometry were minor. In contrast, after immunization a highly significant increase of proliferation in response to stimulation with detoxified toxin A was noted: the stimulation index rose from 1.4 on day 1 to 42.2 on day 74 (restimulation with 0.4 microg/ml; P = 0.003). Immunization led to significant production of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha by antigen-stimulated lymphocytes. In contrast, no significant induction of interleukin (IL)-4 or IL-10 was observed. In conclusion, immunization of healthy volunteers led to activation of cellular immunity including strong antigen-specific proliferation and cytokine production. In CF patients priming of the cellular immune system towards a Th1-like pattern would be of potential advantage. Therefore, confirmatory analyses in immunized CF patients with and without chronic infection with P. aeruginosa are foreseen.     

Cellular immunity in healthy volunteers treated with an octavalent conjugate Pseudomonas aeruginosa vaccine

Humoral immunity in response to an octavalent O-polysaccharide-toxin A conjugate Pseudomonas aeruginosa vaccine is well studied, and a Phase III clinical study in cystic fibrosis (CF) patients is currently ongoing. In contrast, little is known about cellular immunity induced by this vaccine. Fifteen healthy volunteers were immunized on days 1 and 60. Parameters of cellular immunity were studied before vaccination on day 1, and on day 74. Analyses included flow cytometry of whole blood, and antigen-induced proliferation of and cytokine production by lymphocyte cultures. The effects of immunization on the composition of peripheral blood lymphocytes as determined by flow cytometry were minor. In contrast, after immunization a highly significant increase of proliferation in response to stimulation with detoxified toxin A was noted: the stimulation index rose from 1.4 on day 1 to 42.2 on day 74 (restimulation with 0.4 microg/ml; P = 0.003). Immunization led to significant production of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha by antigen-stimulated lymphocytes. In contrast, no significant induction of interleukin (IL)-4 or IL-10 was observed. In conclusion, immunization of healthy volunteers led to activation of cellular immunity including strong antigen-specific proliferation and cytokine production. In CF patients priming of the cellular immune system towards a Th1-like pattern would be of potential advantage. Therefore, confirmatory analyses in immunized CF patients with and without chronic infection with P. aeruginosa are foreseen.     

耐亚胺培南铜绿假单胞菌的分布及耐药性分析

目的:了解耐亚胺培南铜绿假单胞菌的感染情况及其耐药特性,为临床合理使用抗生素及防治耐药菌感染提供参考。方法:收集汕头大学医学院第一附属医院2005年4月到2006年4月间分离的92株耐亚胺培南的铜绿假单胞菌,VITEK-60全自动微生物鉴定仪及其配套试剂(GNS-506、GNS-120、GNS-114)进行细菌鉴定及测定其对多种抗生素的耐药性,肉汤二倍稀释法测定其对亚胺培南的MIC值。结果与结论:耐亚胺培南的铜绿假单胞菌感染主要发生神经外科、外科ICU、呼吸科及ICU和神经内科,对亚胺培南的耐药多为中低度耐药(MIC≤32 mg.L-1),对其它多种抗生素的耐药率均达到100%,仅头孢哌酮/舒巴坦复合制剂保持了较高的敏感率,作为经验性用药可考虑选用。     

抗生素单一或联合用药诱导铜绿假单胞菌释放内毒素的比较性研究

目的 观察不同种类抗生素单独或联合应用对铜绿假单胞菌（Pseudomonas aeruginosa，Pa）释放内毒素（lipopolysaccharide，LPS）的影响，探讨其规律、特点及可能机制，为临床抗生素的合理选择提供参考。方法 选择临床上常用的6种抗生素：哌拉西林/他唑巴坦钠（PIP／TAZ），头孢哌酮（CPZ），亚胺培南/西司他丁钠（IPM／CIL），美罗培南（MEM），环丙沙星（CIP），阿米卡星（AMK），以0．5倍最小抑菌浓度（0．5mc）单一或联合体外作用于Pa，观察不同时相点抗生素的杀菌作用和细菌形态学改变，检测培养上清液中LPS水平。结果 6种抗生素单独应用时杀菌效应差异无统计学意义，联合用药组优于单一用药组。各种抗生素在引起细菌形态学改变的同时，诱导LPS释放程度不一。PIP／TAZ、CIP、MEM组作用后导致细菌成丝状变并伴随大量LPS释放，IMP／CIL组则引起细菌球状变，释放LPS相对较少。联合用药组及CPZ组大部分裂解，释放LPS明显减少。随作用时间延长，抗生素诱导LPS释放水平增加。结论 抗生素单独或联合体外作用于铜绿假单胞菌均可诱导LPS的释放，其释放水平与抗生素的种类、作用时间及细菌形态改变有关。临床抗感染治疗时在考虑抗菌效应同时应尽量选用低LPS诱导量的抗生素。     

三种检测铜绿假单胞菌AmpC酶方法的评价

目的探讨一种简便易行，适用于临床微生物实验室常规开展检测AmpCβ内酰胺酶（简称AmpC酶）的方法。方法对临床分离的150株铜绿假单胞菌用表型筛选试验作AmpC酶测定，对AmpC酶阳性菌株同时进行氯唑西林双纸片协同试验、氟氯西林（FCC）双抑制剂扩散协同试验、PCR基因检测。用基因检测来评价比较三种测定方法的检出率及差异。结果表型筛选试验AmpC酶阳性37株同时进行氯唑西林双纸片协同试验、氟氯西林双抑制剂扩散协同试验、基因检测，检测出阳性株分别为15、14、11株，阳性率分别是40．54％（15／37）、37．84％（14／37）、29．73％（11／37）。表型筛选试验与后三种方法比较差异有统计学意义（P〈0．01），后三种方法结果比较差异无统计学意义（P〉0．05）。结论氯唑西林双纸片协同试验、氟氯西林双抑制剂扩散协同试验检测AmpC酶特异性较表型筛选试验高，且操作简便、结果可靠，适合临床实验室常规检测应用。     

Bacterial RNA is recognized by different sets of immunoreceptors

Innate immunity recognizes microbial nucleic acids by endosomal TLR and cytosolic recognition systems. Despite increasing knowledge on the properties of nucleic acid recognition for viral RNA and bacterial DNA, little is known about the immunogenicity of prokaryotic RNA. Here we show that bacterial RNA is a potent trigger for type‐I IFN secretion in human PBMC. Activation of human plasmacytoid dendritic cells was dependent on endosomal maturation and could be blocked by a TLR7‐specific inhibitor. Murine plasmacytoid dendritic cells from TLR7‐deficient mice were unresponsive to bacterial RNA. Surprisingly, in myeloid DC, TLR were dispensable for TNF‐α and IL‐12 induction by bacterial RNA. Even non‐immune stroma cells were able to mount a NF‐κB response upon triggering with bacterial RNA. Retinoic‐acid inducible gene I and melanoma‐differentiation‐associated gene 5 could be ruled out to be responsible for this reactivity. Although the inflammasome adaptor protein, apoptosis‐associated speck‐like protein, and a functional type‐I IFN receptor were necessary for IL‐1β secretion in myeloid DC, these proteins were dispensable for TNF‐α and IL‐12 induction by cytosolic bacterial RNA. Our results show that besides of activation of TLR7 and inflammasomes, bacterial RNA activates additional cytosolic receptors similarly as has been reported for recognition of bacterial DNA.     

利用Au蛋白芯片技术快速鉴定铜绿假单胞菌的研究

目的 利用Au蛋白芯片建立铜绿假单胞菌蛋白指纹图谱诊断模型,为铜绿假单胞菌感染快速鉴定奠定基础.方法 收集临床分离鉴定的铜绿假单胞菌64株及对照菌199株,随机分成训练组和验证组.运用表面增强激光解析电离飞行时问质谱技术及Au蛋白芯片(proteinchip golden array)检测铜绿假单胞菌及对照菌的蛋白表达,用Ciphergen Proteinchip和BioMarker Wizard软件自动采集数据,筛选铜绿假单胞菌特征性蛋白.结果 经 BioMarker Patterns 软件建立分类树模型.利用该模型对29株铜绿假单胞菌和64株对照菌进行盲法验证.结果 在相对分子质量3000～20 000范围内共检测到80个蛋白峰,其中具有统计学差异的蛋白峰58个(P＜0.01).BioMarker Patterns软件判别分析,择优选出质荷比(M/Z)为14 045.2的蛋白质峰建立铜绿假单胞菌分类树模型.验证结果表明其对铜绿假单胞菌诊断的灵敏度和特异度分别为96.55%(28/29)、100%(64/64).结论 利用Au蛋白芯片技术可快速鉴定铜绿假单胞菌.

Abstract：

Objective To establish protein fingerprinting identification model of Pseudomonas aeruginosa (P. aeruginosa) and to lay a foundation for rapid identification of P. aeruginosa by proteinchip golden array. Methods Sixty-four P. aeruginosa and one hundred and ninety-nine control bacteria identified in our laboratory were collected and divided into training and testing group. Surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) and proteinchip golden array were used to detect the protein profiling of the bacteria. Data were automatically collected by Ciphergen Proteinchip Software and protein markers of P. aeruginosa were screened by BioMarker Wizard Software. Classification tree model was developed and validated by BioMarker Patterns Software. The model was blindly tested with twenty-nine P. aeruginosa and sixty-four control bacteria. Results Eighty protein peaks were detected between 3000 and 20 000, among which fifty-eight ones showed significantly difference between P. aeruginosa and the control bacteria (P＜0.01). By BioMarker Patterns Software, one protein peak ( M/Z at 14 045.2) was chosen to develop a classification tree model. The results exhibited with sensitivity of 96. 55% and specificity of 100%. Conclusion Proteinchip golden array has the potential for rapid identification of P. aeruginosa.     

铜绿假单胞菌随机扩增多态性DNA指纹法基因分型研究

建立了铜绿假单胞菌（ＰＡ）随机扩增多态性ＤＮＡ（ＲＡＰＤ）指纹图基因分型方法，并应用于流行病学相关或不相关的７５个ＰＡ菌株的基因分型。分型率为１００％。３２株新生儿暴发感染菌株，可分成４个血清型和５个ＲＡＰＤ谱型，其中２５株属于同一谱型。在８例患者的垂直追踪菌株中，除有１例其第１和第３个分离株的血清型和ＲＡＰＤ谱型均相同，但与第２个分离株的血清型和ＲＡＰＤ谱型不同外，其余７例前后菌株的谱型相同。１３株散发菌株可分成３个血清型和１０个ＲＡＰＤ谱型；１２株流行病学无关菌株的ＲＡＰＤ谱型均不相同。本研究结果表明，ＲＡＰＤ指纹图基因分型法具有分型率高、分辨力强、比较快速简便等优点，并且不需要已知核酸序列，是在分子水平上对微生物感染的病原学、发病机理及流行病学研究的较理想的方法。     

耐喹诺酮类铜绿假单胞菌的耐药机制研究

目的探讨广州地区铜绿假单胞菌对喹诺酮类药物的耐药机制。方法对喹诺酮耐药株的gyrA和parC基因进行限制性片段长度多态性分析（PCR-RFLP）,并对其中的高水平耐药株gyrB和parE基因进行测序;用琼脂稀释法测定加入碳酰氰基-对-氯苯腙（CCCP）前后环丙沙星的最小抑菌浓度（MIC）;同时用SDS-PAGE对高水平耐药株的外膜蛋白进行电泳分析。结果有72.7%（72/99）的菌株发生gyrA突变,主要为Thr-83-Ile;25.3%（25/99）的菌株发生parC突变,主要为Ser-87-Leu,且均是gyrA和parC双基因突变;gyrB和parE突变较少见。53.3%（53/99）的菌株的MIC可被CCCP逆转,其MIC能明显降低;7%（7/10）的高水平耐药株的外膜蛋白在43～67kDa间条带增多,其蛋白含量有差异。结论抗菌药物作用靶位的改变和外排泵机制是本地区铜绿假单胞菌对喹诺酮类耐药的重要机制。     

临床分离的铜绿假单胞菌对头孢噻肟的耐药性分析

目的：测定铜绿假单胞菌对头孢噻肟的耐药性。方法：采用琼脂二倍稀释法测定铜铝假单胞菌对头孢噻肟的MIC值。用铜绿假单胞菌ATCC27853标准菌株作为质控标准。按照美国临床实验标准委员会（NCCLS）2004年颁布的标准操作和判定结果。结果：铜绿假单胞菌对头孢噻肟的耐药率为59．18％。结论：临床分离的铜绿假单胞菌对头孢噻肟高度耐药，应考虑通过进行细菌培养和药敏试验，合理选用抗生素，或选用有协同作用的二联疗法来提高抗铜绿假单胞菌感染的疗效。     

碳青霉烯类治疗铜绿假单胞菌肺炎的临床效果分析

目的 探究碳青霉烯类治疗铜绿假单胞菌肺炎的效果。方法 选取2017年1月~2018年1月收治的86例PA肺炎患者临床资料进行分析,随机分为对照组和研究组,每组43例。对照组行头孢哌酮钠舒巴坦钠治疗,研究组行碳青霉烯类双联法治疗,比较两组肺功能（FEV1/FVC、FEV1）、实验室指标（CRP、PCT、WBC）及生活质量（SGRQ评分）。结果 两组患者治疗后FEV1/FVC、FEV1水平均优于治疗前,且研究组改善幅度大于对照组,统计学意义显著（P＜0.01）。研究组治疗后实验室各指标水平均优于治疗前,且优于对照组,统计学意义显著（P＜0.01）。治疗后,研究组SGRQ评分为（30.87±3.55）分,低于对照组的（40.54±4.36）分,统计学意义显著（P＜0.01）。结论 碳青霉烯类治疗铜绿假单胞菌肺炎,可有效改善患者肺功能,减轻炎症反应,提高生活质量。     

铜绿假单胞菌感染豚鼠后生物被膜形成的研究

目的 建立体内铜绿假单胞菌生物被膜模型，研究体内细菌生物被膜的组织学及细菌学特征。方法 通过吸入法使铜绿假单胞菌以气雾剂的形式吸入豚鼠肺内并生长定植，分别观察不同时期肺组织内细菌生物被膜的特征。结果 定植在肺内的铜绿假单胞菌以肉芽肿结节的形式存在，外周包绕类上皮细胞和成纤维细胞。结节内细菌被被膜基质所包绕并彼此连结，中间镶嵌宿主炎性细胞。接种后3周，肺内仍可见结节并培养出铜绿假单胞菌。结论 用吸入法可建立较稳定的铜绿假单胞菌肺感染生物被膜，其以肉芽肿结节的形式存在，宿主的反应细胞参与了生物被膜的形成。     

The antimicrobial peptide LL‐37 modulates the inflammatory and host defense response of human neutrophils

The human cathelicidin antimicrobial peptide acts as an effector molecule of the innate immune system with direct antimicrobial and immunomodulatory effects. The aim of this study was to test whether the cathelicidin LL‐37 modulates the response of neutrophils to microbial stimulation. Human neutrophils were exposed to LPS, Staphylococcus aureus and Pseudomonas aeruginosa subsequent to incubation with LL‐37 and cytokine release was measured by ELISA. The incubation with LL‐37 significantly decreased the release of proinflammatory cytokines from stimulated human neutrophils. ROS production of neutrophils was determined by a luminometric and a flow cytometry method. The peptide induced the production of ROS and the engulfment of bacteria into neutrophils. Peritoneal mouse neutrophils isolated from CRAMP‐deficient and WT animals were treated with LPS and TNF‐α in the supernatant was measured by ELISA. Antimicrobial activity of neutrophils was detected by incubating neutrophils isolated from CRAMP‐knockout and WT mice with bacteria. Neutrophils from CRAMP‐deficient mice released significantly more TNF‐α after bacterial stimulation and showed decreased antimicrobial activity as compared to cells from WT animals. In conclusion, LL‐37 modulates the response of neutrophils to bacterial activation. Cathelicidin controls the release of inflammatory mediators while increasing antimicrobial activity of neutrophils.     

An outbreak of carbapenem-resistant Pseudomonas aeruginosa in a urology ward

Objective  To investigate an outbreak of carbapenem-resistant Pseudomonas aeruginosa (CRPA) in a urology ward.
Methods  Patients infected or colonized with CRPA were prospectively identified by daily laboratory surveillance. Routine infection-control measures were reinforced, disinfection protocols were revised, and a surveillance program was set up, analyzing cross-transmission in the nursing ward and environment cultures from urology wards and the operating theater. CRPA isolates from clinical and environment samples were studied by pulsed-field gel electrophoresis (PFGE), following Xba I and Spe I restriction.
Results  From February 1998 to September 2000, 59 adult urology patients were colonized or infected by CRPA. All patients had been operated on prior to identification of the CRPA isolate and 79% of these procedures were performed in the same cystoscopy room. No patients had received prior carbapenem therapy. No cross-transmission was detected, and environment cultures from the urology ward and theater were negative except for five samples collected in the cystoscopy room. PFGE identified a single clone in the isolates from different patients and the environment samples.
Conclusions  The PFGE analysis indicated that the CRPA outbreak resulted from the contamination of the cystoscopy room via an unsealed drain. The outbreak ended when the drain was sealed.     

铜绿假单胞菌生物被膜形成过程中多糖合成相关基因表达的研究

目的 了解铜绿假单胞菌(Pseudomonas aerugirtosa,Pa)生物被膜形成过程中多糖生物合成相关基因在生物被膜形成中的表达,探讨其在生物被膜形成中的调控作用.方法 分别收集非黏液型铜绿假单胞菌PAO1的浮游菌及生物被膜菌,用实时荧光定量RT-PCR的方法对基因的表达进行相对定量分析.结果 多糖合成相关基因pslA、algD.pelA的mRNA在生物被膜菌中的相对表达量均高于浮游菌.结论 .pslA、algD,pelA的表达与铜绿假单胞菌生物被膜形成密切相关,在生物被膜形成中具有重要作用.