Dispersion by Pseudomonas aeruginosa requires an unusual posttranslational modification of BdlA

Dispersion enables biofilm bacteria to transit from the biofilm to the planktonic growth state and to spawn novel communities in new locales. Although the chemotaxis protein BdlA plays a role in the dispersion of Pseudomonas aeruginosa biofilms in response to environmental cues, little is known about regulation of BdlA activity or how BdlA modulates the dispersion response. Here, we demonstrate that BdlA in its native form is inactive and is activated upon nonprocessive proteolysis at a ClpP-protease-like cleavage site located between the Per Arnt Sim (PAS) sensory domains PASa and PASb. Activation of BdlA to enable biofilm dispersion requires phosphorylation at tyrosine-238 as a signal, elevated c-di-GMP levels, the chaperone ClpD, and the protease ClpP. The resulting truncated BdlA polypeptide chains directly interact and are required for P. aeruginosa biofilms to disperse. Our results provide a basis for understanding the mechanism of biofilm dispersion that may be applicable to a large number of biofilm-forming pathogenic species. Insights into the mechanism of BdlA function have implications for the control of biofilm-related infections.     

Pseudomonas aeruginosa. Vaccines and immunotherapy

Among opportunistic infections with gram-negative bacilli, those caused by Pseudomonas aeruginosa are associated with particularly high mortalities. Accordingly, considerable interest exists to develop immunotherapeutic or immunoprophylactic agents for this pathogen. In vitro as well as in vivo studies in animal models have demonstrated that LPS serotype-specific antibodies against P. aeruginosa confer protection. Thus, cell wall-derived LPS P. aeruginosa vaccines have been developed for active immunization. Toxic side effects from LPS and relatively slow immune response to active immunization in patients needing rapid protection have led to the development of high-titered anti-P. aeruginosa immunoglobulin G preparations. Passive immunotherapy with these polyclonal antibody preparations has shown promising results in animal models and in clinical pilot studies. More recently, murine and human monoclonal antibodies against P. aeruginosa have been developed. These preparations offer the potential advantage over polyclonal globulin preparations of low protein dosages and virtually unlimited supply.     

Active immunization with Pseudomonas aeruginosa vaccine protects mice from secondary Pseudomonas aeruginosa challenge post-influenza virus infection

BackgroundInfluenza virus infection complicated by secondary bacterial pneumonia contributes significantly to death during seasonal or pandemic influenza. Secondary infection of Pseudomonas aeruginosa (P. aeruginosa) in influenza virus-infected patients contributes to morbidity and mortality.MethodsMice were first infected with PR8 influenza virus, followed by a secondary infection of P. aeruginosa. Body weights and survival rate of mice was monitored daily over 20 days. Bronchoalveolar lavage fluids (BALFs) and lung homogenates were harvested for measuring bacterial titers. Lung tissue section slides were stained with hematoxylin and eosin for microscopic observation. After vaccination with inactivated P. aeruginosa cells or recombinant PcrV protein, the mice were subjected to PR8 influenza virus infection followed by a secondary infection of a P. aeruginosa. The inhibition against P. aeruginosa of serum was evaluated by detecting the growth of P. aeruginosa in broth containing diluted sera.ResultsThe prior influenza infection greatly enhanced the susceptibility to secondary infection of P. aeruginosa and increased morbidity and mortality in mice. Active immunization with inactivated P. aeruginosa cells could protect mice from secondary P. aeruginosa challenge in influenza virus infected mice.ConclusionsTo develop an effective P. aeruginosa vaccine might be a promising strategy to decrease the threat of secondary P. aeruginosa infection in influenza patients.     

Pseudomonas aeruginosa pneumonia

PURPOSE OF REVIEW: Recent articles of clinical interest on Pseudomonas aeruginosa respiratory tract infections including CAP, nosocomially-acquired pneumonia, particularly in the ventilated patient, and chronic infections in cystic fibrosis patients are reviewed. RECENT FINDINGS: The growing importance of P. aeruginosa as an etiologic agent of CAP, the occurrence of CAP in previously healthy adults and its high prevalence as an etiologic agent of late VAP are stressed in recent studies. The effect of antibiotics on the recovery of bacteria in respiratory samples of patients with VAP can be marked and as early as 12 h after administration of antimicrobials certain organisms are no longer cultivable; in contrast, P. aeruginosa can still be recovered even after 48 h of adequate therapy. Type III secretory proteins are recognized as important virulent factors in P. aeruginosa. This phenotype predicts a worse outcome in patients with VAP. Fluoroquinolones have a major role in the emergence of multiply resistant P. aeruginosa in patients with VAP. Pharmacokinetic/pharmacodynamic parameters of antimicrobials with antipseudomonal activity are gaining importance as a means of optimization of antibiotic therapy. In CF, the knowledge of the pharmacokinetics and bioavailability of inhaled tobramycin and its long term beneficial effect in lung function are important developments in this area. SUMMARY: P. aeruginosa continues to be a serious problem worldwide as a cause of respiratory tract infections in selected populations. Microbiologic diagnosis remains difficult and plagued with pitfalls. The application of modern PK/PD concepts should help to optimize antibiotic therapy of this increasingly difficult to treat infection, particularly at the respiratory tract level and with an increasing prevalence of resistance to all antipseudomonal agents. Inhaled antibiotics, particularly tobramycin, are increasingly used for the prevention and treatment of P. aeruginosa infection in CF patients.     

铜绿假单胞菌弹性蛋白酶的高效表创

目的 提取具有弹性蛋白酶活性的铜绿假单胞菌(绿脓杆菌)临床分离株的染色体DNA，PCR扩增弹性蛋白酶基因成熟蛋白编码区，A—T克隆于质粒pMD 18-T载体中，阳性重组子经酶切后连接至相应线性化的表达型载体pQE一31中，转化大肠杆菌JM109感受态，筛选高效表达株，表达产物经SDS—PAGE初步鉴定。所克隆的基因在大肠杆菌中获得高效表达，表达产物经重组质粒测序初步确定为铜绿假单胞菌弹性蛋白酶。本研究为该表达蛋白的纯化、复性和酶动力学研究奠定了基础。     

健康肉鸡高比率携有多重耐药铜绿假单胞菌

摘要： 目的 为了解健康成年肉鸡中铜绿假单胞菌的流行情况及其抗生素抗菌谱。方法 2011年1月~3月在河南省洛阳市两个菜市场随机抽取市售健康肉鸡鸡肠内容物,经SCDLP液体培养基增菌,菌液十六烷基三甲基溴化铵琼脂培养基培养,分离细菌经氧化酶试验、绿脓菌素测定、明胶液化试验、42℃生长试验和硝酸盐还原产气试验等一系列试验鉴定,鉴定的铜绿假单胞菌采用K-B法测定对头孢他啶、头孢吡肟、头孢哌酮、羧苄西林、氨曲南、庆大霉素、阿米卡星、妥布霉素、氯霉素、四环素、诺氟沙星、环丙沙星的耐药性。结果 20个样品9个分离出11株铜绿假单胞菌。11株细菌具有7种耐药模式;3种模式为多重耐药模式,共6株细菌,来源6只肉鸡。结论 45%肉鸡携带铜绿假单胞菌,30%肉鸡携有多重耐药铜绿假单胞菌。     

铜绿假单胞菌Ⅲ型分泌系统的研究进展

铜绿假单胞菌是临床常见的重要条件致病菌,具有多种毒力因子,能引起各种急慢性感染。其中最重要的毒力因子是Ⅲ型分泌系统,主动向宿主细胞靶向输送效应蛋白,引起宿主细胞的病理变化,并逃避免疫细胞的降解。研究Ⅲ型分泌系统不仅有助于明确铜绿假单胞菌的致病机制,更重要的是为临床治疗及新药研发提供新思路。     

Pseudomonas aeruginosa Strains from Nosocomial Pneumonia Are More Serum Resistant than P. aeruginosa Strains from Noninfectious Respiratory Colonization Processes

Abstract Background: Serum resistance is regarded as a major virulence factor of bacteria and is thought to be mediated by O side chains of the lipopolysaccharides (LPS). We investigated the serum–resistance properties and O serogroups of Pseudomonas aeruginosa strains isolated from intensive careunit (ICU) patients with pneumonia and from the respiratory tract of ICU patients without respiratory tract infections. Materials and Methods: 171 P. aeruginosa strains were consecutively isolated from bronchoalveolar lavage fluid or transtracheal aspirates of ICU patients with monobacterial nosocomial pneumonia and 49 strains were isolated from the respiratory tract of ICU patients without respiratory tract infections. All strains were O serogrouped using Oantigen– specific sera for 14 O serogroups and tested for their sensitivity to the serum’s bactericidal effect. Results: Using two different analyses, the frequency of serum–sensitive isolates was significantly lower in strains from patients with pneumonia (56.1%; n = 96/171 and 22.8%, n = 39/171, respectively) than in strains from asymptomatically colonized patients (73.46%; 36/49 and 38.8%, n = 19/49, respectively) (p = 0.03; OR = 2.163; 95% CI = 1.072–4.368 and p = 0.0289; OR = 2.144; 95% CI = 1.089–4.368, respectively). O serogrouping revealed higher frequency of the serogroups A (11.9% and 16.3%, respectively), B (14.3% and 21%), E (26.5% and 24.6%), and I (28.6% and 28%) in both strain collections. The frequency of serum–sensitive strains (13/28 and 3/45, respectively) was significantly lower among strains expressing the A and B serogroups, than for all other serogroups (p < 0.05). Conclusion: Strains isolated from patients with pneumonia and strains possessing O–A or O–B serogroups appear to have greater pathogenic potential by virtue of their ability to resist serum–mediated killing. The linkage, however, between the O serogroups, serum resistance, and a strain's virulence remains unclear at this stage. This paper is dedicated to the founders of the Walter Marget Foundation, D. Adam and F. Daschner, in gratitude for their support of the training in infectious diseases.     

Cloned avirulence genes from the tomato pathogen Pseudomonas syringae pv. tomato confer cultivar specificity on soybean

Three different cosmid clones were isolated from a genomic library of the tomato pathogen Pseudomonas syringae pv. tomato, which, when introduced into the soybean pathogen P. syringae pv. glycinea, caused a defensive hypersensitive response (HR) in certain soybean cultivars. Each clone was distinguished by the specific cultivars that reacted hypersensitively and by the intensity of the HR elicited. Unlike wild-type P. syringae pv. tomato isolates, which elicit the HR on all soybean cultivars, all three clones exhibited cultivar specificities analogous to avirulence genes previously cloned from P. syringae pv. glycinea. However, the collective phenotypes of the three clones accounted for HRs on all tested soybean cultivars. One of the three P. syringae pv. tomato clones contained an avirulence gene homologous to avrA, which was previously cloned from P. syringae pv. glycinea race 6. The other two P. syringae pv. tomato clones expressed unique HR patterns on various soybean cultivars, which were unlike those caused by any known P. syringae pv. glycinea race or previously cloned P. syringae pv. glycinea avr gene. Further characterization of the second P. syringae pv. tomato clone indicated that the avirulence phenotype resided on a 5.6-kilobase HindIII fragment that, in Southern blot analyses, hybridized to an identical-size fragment in various P. syringae pathovars, including all tested glycinea races. These results demonstrate that avirulence genes may be distributed among several P. syringae pathovars but may be modified so that the HR is not elicited in a particular host plant. Furthermore, the data raise the possibility that avirulence genes may function in host-range determination at levels above race—cultivar specificity.     

Ventilator-associated Pseudomonas aeruginosa pneumonia

Summary Pseudomonas aeruginosa is the causative organism in 16% to 31% of reported cases of ventilator-associated pneumonia (VAP). Despite the availability of potent antimicrobials and the facilities offered by the intensive care unit, the associated overall mortality is approximately 70%, with an excess mortality of more than 40%. By these observation alone, P. aeruginosa pneumonia represents one of the most serious clinical problems facing the intensive care clinician today. This article includes a review of epidemiology, risk factors for infection, diagnosis and treatment, together with a discussion of predictors of mortality. Summary data from a study of 35 patients with P. aeruginosa VAP in the Oxford ICU are presented. In the search for more effective combinations of available antibiotics we have treated 12 patients with nebulised colistin in conjunction with standard anti-pseudomonal parenteral agents. We present data from this pilot study, including outcome compared to a historical control group. Major improvements in outcome may not be realised until novel therapeutic strategies are developed. The forerunner to this is the acquisition of an indepth understanding of bacterial mechanisms in disease. This overview, therefore, also includes data from some areas of research which shed light on bacterial pathogenesis as a cellular and molecular level. Received: 26 January 1998 Accepted: 10 February 1998     

Mutasynthesis of siderophore analogues by Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa produces the phenolic siderophore pyochelin. Salicylic acid is an intermediate in the pyochelin biosynthetic pathway, and mutants blocked in salicylic acid biosynthesis (Sal-) are able to incorporate exogenously supplied salicylic acid into pyochelin. A P. aeruginosa Sal- mutant was incubated with 13 salicylic acid analogues and was found to incorporate three (5-fluorosalicylic acid, 4-methylsalicylic acid, and 3-hydroxypicolinic acid) into pyochelin analogues, trivially designated as 5-fluoropyochelin, 4-methylpyochelin, and 6-azapyochelin. The structures of the mutasynthetic products were confirmed by 1H and 13C NMR and high-resolution fast atom bombardment mass spectrometry as being identical to pyochelin except for the expected changes in the aromatic ring. The biological activity of the three pyochelin analogues was determined in iron transport assays. In comparison to pyochelin, 4-methylpyochelin was more active in the assays whereas the activities of 5-fluoropyochelin and 6-azapyochelin were markedly decreased. In coincubation assays, 5-fluoropyochelin substantially inhibited iron transport by pyochelin; 4-methylpyochelin and 6-azapyochelin did not demonstrate this inhibitory effect.     

Pathogenesis of Pseudomonas aeruginosa pneumonia during immunosuppression.

A guinea pig model of immunosuppression was utilized to study the effects of immunosuppressive chemotherapy on lung response to challenge with Pseudomonas aeruginosa. Study groups included normal guinea pigs, as well as guinea pigs that received a one-week course of cortisone acetate (CA, 100 mg/kg per day) plus 15 mg of cyclophosphamide (CTX)/kg per day (CA + LoCTX group) or 30 mg of cyclophosphamide/kg per day (CA + HiCTX group). Separate groups received CA or HiCTX alone. Intratracheal instillation of P. aeruginosa resulted in bilateral hemorrhagic pneumonia in both normal and immunosuppressed animals. Survival was 100% for normal animals and for those given CA alone, 67% in the CA + LoCTX and the HiCTX groups, and 0 in the CA + HiCTX group. Increased mortality correlated with a diminished polymorphonuclear leukocyte inflammatory response in infected lung tissues and also with the addition of CA to CTX. Clearance of viable P. aeruginosa from lung tissue was significantly reduced in animals receiving the combination CA + HiCTX. Thus, decreased lung inflammation and the addition of CA appeared to be important determinants for fatal pseudomonas pneumonia.