Colistin stimulates the activity of neutrophil elastase and Pseudomonas aeruginosa elastase.

Nonantimicrobial effects of antibiotics may contribute to their activity in the treatment of infective airway disease. The aim of this study was to identify antibiotics used for the treatment of infection in cystic fibrosis that may alter the activity of human neutrophil elastase (HNE) and Pseudomonas aeruginosa elastase (PE). The effect of antibiotics on the activity of purified HNE and PE, and HNE in sputum was assessed using colourimetric and fluorescent substrate assays by kinetic measurements, and by examining the interaction of HNE with inhibitors. Ceftazidime, tobramycin, and gentamycin slightly inhibited purified HNE activity whereas erythromycin and colistin significantly stimulated purified HNE and PE (395 and 557%, respectively). However, only colistin increased HNE activity in sputum (+102%) and was therefore studied in more detail. This increase in activity was not due an interference with the specific inhibition of HNE by alpha1-antitrypsin but colistin was found to reverse the inhibitory effects of small molecular weight molecules like heparin. Colistin increases the activity of human neutrophil elastase and Pseudomonas aeruginosa elastase, two proteases that contribute to the pathogenesis of cystic fibrosis airway disease.     

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。     

Early pulmonary granulocyte recruitment in response to Streptococcus pneumoniae

Although polymorphonuclear leukocytes (PMN) are a conspicuous histologic feature of clinical and experimental pneumococcal pneumonia, neither the mechanism nor the magnitude of recruitment of these cells to the lung following lesser pneumococcal challenge is known. We have, therefore, investigated the early process of recruitment of PMN to alveolar spaces after pulmonary inoculation of Streptococcus pneumoniae in doses less than those causing pneumonia. We injected Balb/c mice with water and varying inoculums of pneumococci via an endobronchial catheter. Bronchoalveolar lavage (BAL) was performed on the inoculated lung at 0, 2, or 4 h after injection. Cellular response was measured and chemotactic activity was assayed on BAL supernatants at each time interval using the migration of human PMN through 3-micron filters in modified Boyden chambers by the leading front techniques. The BAL of normal and control animals (inoculum of sterile water only used for the control animals) yielded 5.03 +/- 1.51 X 10(2) and 0.17 +/- 0.04 X 10(5) PMN, respectively. The PMN recruitment at 4 h as a function of pneumococcal inoculum was described by the following equation: log PMN = 0.751 log Pn + 1.119 (r2 = 0.82, p less than 0.001). The PMN were, therefore, recruited in a dose-dependent manner. That recruitment may be caused by chemotactic substance(s) was suggested by the significant correlation between the PMN response and the distance of in vitro migration: log PMN = 0.057 micron + 0.52 (r = 0.77, p less than 0.005). We have defined quantitatively the recruitment of PMN to the lung after pneumococcal challenge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute lung injury induced by Pseudomonas aeruginosa elastase in hamsters.

Human neutrophil elastase (HNE) is the predominant elastolytic enzyme in the sputum of cystic fibrosis (CF) patients. However, a variably small portion of the activity can be ascribed to Pseudomonas aeruginosa elastase (PaE). The purpose of these studies was to evaluate the activities of the two elastases in an in vivo model of acute lung injury (ALI). The elastolytic activity of Pseudomonas aeruginosa elastase (MW = 39K) and human neutrophil elastase (MW = 33K) were also examined using insoluble bovine neck and lung elastin. The ability of hamster serum to inhibit elastinolysis by the two elastases was also examined. On a per milligram protein basis, PaE was the more potent elastase, regardless of substrate, and it preferentially hydrolyzed lung relative to neck elastin. PaE is poorly inhibited by hamster serum compared to HNE. In vivo, PaE is much more efficient than HNE in inducing an acute lung injury in hamsters. The duration of effects induced by doses of the two proteases that produce similar acute biological effects are essentially identical. The increases of lung weight and total lavagable WBCs persist for at least 7 days. All other parameters return to baseline between 3 and 5 days. The predominant cells in the lavage 1 and 2 days post insult are PMNs. By day 7, the predominant cell is the macrophage. These data suggest that even though PaE is a minor component of the elastolytic activity in CF patients, it may still contribute significantly to the pathology of the disease.     

Fast solubilization of human lung elastin by Pseudomonas aeruginosa elastase

Pseudomonas aeruginosa may cause severe lung infections in humans. This bacteria secretes an elastase that might degrade lung elastin. We have studied the solubilization of human lung elastin by P. aeruginosa elastase in an attempt to delineate the pathogenic role of this proteinase in P. aeruginosa lung infections. We also used bovine ligamentum nuchae elastin and human leukocyte elastase for comparative purposes. With an elastin concentration of 5 mg X ml-1 and at physiologic ionic strength, P. aeruginosa elastase is about 50 times more active on human lung elastin than on bovine elastin. In contrast, human leukocyte elastase has similar specific activities on the 2 substrates. In addition, the bacterial enzyme is about 10 times more active on human elastin than the neutrophil elastase but the latter is about 5 times more active on bovine elastin than the former. In order to better quantitate these enzyme-substrate interactions, we have measured initial rates of elastolysis, derived from product versus time curves, as a function of elastin concentration. The substrate-velocity curves, analyzed using an equation similar to the classic Michaelis-Menten one, yielded 2 empirical kinetic parameters: [S50]-1, the apparent elastase-elastin affinity and Vm, the apparent catalytic efficiency of elastase. This analysis shows that human leukocyte elastase exhibits similar [S50]-1 and Vm values for the 2 elastins. The low activity of P. aeruginosa elastase on bovine elastin is due to the combined effects of low S50(-1) and Vm values, which could not be measured separately.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corneal infections in mice with toxin A and elastase mutants of Pseudomonas aeruginosa

The data presented indicate that in experimental infections of the mouse cornea, toxin A of Pseudomonas aeruginosa contributes to the organism's pathogenicity, whereas active elastase may not be required. After traumatization, corneas were infected with wild-type parental toxin A-producing strains, two toxin A-deficient mutants (Tox-), or an elastase mutant. The infections produced by both Tox- mutants were less severe than infections produced by their parental strains. Furthermore, the Tox- mutants were not able to persist in the eyes as long as their parental strains. Addition of subdamaging doses of exogenous toxin A to eyes infected with the Tox- mutant PA103-29 significantly increased is virulence. The course of infection and the resulting corneal damage produced by the elastase mutant were indistinguishable from those of its parental strain.     

Antibody response to Pseudomonas aeruginosa in children with cystic fibrosis

Cystic fibrosis (CF) is the most frequent life threatening autosomal recessive disease in white subjects. The primary cause of morbidity and mortality in children with CF is chronic pulmonary infection, mainly caused by Pseudomonas aeruginosa. The purpose of this study was to assess the value of the measurement of antibodies to P. aeruginosa in diagnosing lung infection by the bacteria in CF patients. We assessed P. aeruginosa antibody titers in CF patients from Rio de Janeiro, Brazil, using cell lysate antigens as well as recombinant PcrV, a Type III Secretion System protein. Sputum (more than 70% of the specimens) or oropharyngeal swabs were obtained whenever patients were regularly followed for their pulmonary disease. Blood samples were obtained with an average interval of 6 months for a period of 2 years. The ELISA cut‐offs were assigned as the positive 95% confidence interval of the mean antibody levels from non‐fibrocystic controls. Our data showed that most CF patients (81%) of whom were not chronically infected by P. aeruginosa (Groups I and II), had their first serology positive for rPcrV. Cell‐lysate ELISA was able to detect P. aeruginosa antibodies before positive culture in the first serum sample of 44% of the patients from Groups I and II. When serum reactivity to rPcrV and cell lysate were combined, 94% of CF patients from Groups I and II (n = 16) had the first serology positive for P. aeruginosa over a mean time of 20 months before the first isolation of P. aeruginosa. In conclusion, longitudinal P. aeruginosa serology should become part of respiratory care follow‐up, in conjunction with other lung parameter functions. Pediatr Pulmonol. 2009; 44:392–401. © 2009 Wiley‐Liss, Inc.     

Moxifloxacin and ciprofloxacin protect human respiratory epithelial cells against Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, and Haemophilus influenzae in vitro

The fluoroquinolones moxifloxacin and ciprofloxacin display excellent in vitro activities against many respiratory tract pathogens. Here we show that moxifloxacin and ciprofloxacin accumulate approximately 7- to 10-fold in primary human respiratory epithelial cells, derived from nasal polyps and grown in 3-dimensional vesicles. Furthermore, using these vesicles, we assessed the bactericidal effect of moxifloxacin on Staphylococcus aureus and Streptococcus pneumoniae and that of ciprofloxacin on Pseudomonas aeruginosa and Haemophilus influenzae. Finally, we determined the protective effect of the fluoroquinolones on vesicles infected with these pathogens. All four bacterial strains were highly toxic for vesicles. S. aureus and S. pneumoniae were readily killed by moxifloxacin regardless whether the antibiotics were present intra/extracellularly or only intracellularly in vesicles. Similar results were obtained for the killing of H. influenzae and P. aeruginosa. Exclusively intracellularly located fluoroquinolones rescued 42% to 76% of the cells after bacterial challenge compared to the rescue of 48% to 94% cells when the fluoroquinolones were present intra/ extracellularly. Without addition of fluoroquinolones cell survival in vesicles was 0% to 38%. The results suggest that intracellular accumulation of moxifloxacin and ciprofloxacin is important for the protection of respiratory epithelial cells from the cytotoxic effects of major respiratory tract pathogens.     

Antibacterial activity of beta-lactam antibiotics in experimental meningitis due to Streptococcus pneumoniae

In order to define the characteristics of the antibacterial activity of beta-lactam antibiotics in the treatment of bacterial meningitis, the relationship between cerebrospinal fluid (CSF) drug concentrations and the rate of bacterial killing was investigated for penicillin G and four new cephalosporins in an animal model of meningitis due to Streptococcus pneumoniae. All five drugs showed a significant correlation between increasing drug concentrations in CSF and increasing bactericidal rates. Minimal activity was observed in CSF at drug concentrations of approximately the broth minimal bactericidal concentration (MBC). Maximal activity occurred with CSF concentrations 10-30 times higher. In vitro tests did not reproduce the unique correlation of increasing drug concentrations and killing activity found in vivo. When evaluating new beta-lactam antibiotics for the treatment of bacterial meningitis, it is reasonable to establish a minimum standard of CSF drug concentrations of greater than or equal to 30 times the MBC against the infecting organism.     

Heteroresistance to penicillin in Streptococcus pneumoniae

Heteroresistance to beta-lactam antibiotics has been mainly described for staphylococci, for which it complicates diagnostic procedures and therapeutic success. This study investigated whether heteroresistance to penicillin exists in Streptococcus pneumoniae. Population analysis profile (PAP) showed the presence of subpopulations with higher penicillin resistance in four of nine clinical pneumococcal strains obtained from a local surveillance program (representing the multiresistant clones ST179, ST276, and ST344) and in seven of 16 reference strains (representing the international clones Spain(23F)-1, Spain(9V)-3, Spain(14)-5, Hungary(19A)-6, South Africa(19A)-13, Taiwan(23F)-15, and Finland(6B)-12). Heteroresistant strains had penicillin minimal inhibitory concentrations (MICs) (for the majority of cells) in the intermediate- to high-level range (0.19-2.0 mug/ml). PAP curves suggested the presence of subpopulations also for the highly penicillin-resistant strains Taiwan(19F)-14, Poland(23F)-16, CSR(19A)-11, and CSR(14)-10. PAP of bacterial subpopulations with higher penicillin resistance showed a shift toward higher penicillin-resistance levels, which reverted upon multiple passages on antibiotic-free media. Convergence to a homotypic resistance phenotype did not occur. Comparison of two strains of clone ST179 showed a correlation between the heteroresistant phenotype and a higher-penicillin MIC and a greater number of altered penicillin-binding proteins (PBP1a, -2b, and -2x), respectively. Therefore, heteroresistance to penicillin occurs in international multiresistant clones of S. pneumoniae. Pneumococci may use heteroresistance to penicillin as a tool during their evolution to high penicillin resistance, because it gives the bacteria an opportunity to explore growth in the presence of antibiotics before acquisition of resistance genes.     

Immunization with a Pseudomonas aeruginosa elastase peptide reduces severity of experimental lung infections due to P. aeruginosa Or Burkholderia cepacia

Pseudomonas aeruginosa and Burkholderia cepacia produce metalloproteases that effect lung injury. Two epitopes (peptides 15 and 42) previously identified on P. aeruginosa elastase induce the production of antibodies that neutralize protease activity. The effects of immunization with synthetic peptides based on these epitopes on experimental lung infections due to P. aeruginosa or B. cepacia were examined. Rats were immunized with peptides conjugated to keyhole limpet hemocyanin or tetanus toxoid before infection. Immunization with peptide 15 (pep15) resulted in a decrease in total cells and polymorphonuclear leukocytes in bronchoalveolar lavage (BAL) fluid and a 50%-70% decrease in lung histopathologic changes, compared with findings in controls. Immunization with peptide 42 decreased cells in BAL fluid but did not decrease lung pathologic changes. Immunization with pep15 alone was just as effective in protecting against lung injury as immunization with a combination of both peptides. These studies suggest that immunization with pep15 can reduce the severity of lung infections due to P. aeruginosa or B. cepacia.     

Effects of antibiotic class on the macrophage inflammatory response to Streptococcus pneumoniae

Antibiotic choice can alter host inflammation during invasive bacterial infections. Previous studies of gram-negative organisms concluded that antibiotic-mediated release of bacterial cell wall components amplifies inflammation. Less has been reported about antibiotic effect on gram-positive organisms. This study explored the hypothesis that Streptococcus pneumoniae would induce greater macrophage inflammatory mediator production when killed with cell wall active antibiotics rather than protein synthesis inhibitors. Stimulation of RAW 264.7 murine macrophages with pneumococci and oxacillin led to significantly higher inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation than did the same concentrations of pneumococci and clindamycin. Neither antibiotic alone or in combination with lipopolysaccharide acted directly on macrophages to modify the immune response. Endotoxin contamination did not confound the results, as preincubation with polymyxin B did not change iNOS or TNF protein levels. Thus, the antimicrobial mechanism of action affects macrophage inflammatory mediator production after stimulation with pneumococci.     

IgG proteolytic activity of Pseudomonas aeruginosa in cystic fibrosis

To study how fragmented IgG antibodies might arise within the respiratory secretions of individuals with cystic fibrosis (CF), we screened protease extracts from CF polymorphonuclear leukocytes and mucoid and nonmucoid transformants of Pseudomonas aeruginosa from patients with CF for IgG proteolytic activity. All strains of P. aeruginosa tested exhibited IgG proteolytic activity. Incubation for 7 hr at 37 C was required to demonstrate generation of free Fc gamma immunoreactivity. Further analysis of these cleavage products of CF IgG demonstrated generation of Fc gamma polypeptides with 4S sedimentation coefficients and F(ab')2 fragments with 5S coefficients. Bacterial IgG proteolytic activity was inhibited by EDTA and was associated with levels of bacterial elastase exceeding 5 micrograms/mg of total protein. Pseudomonas elastase was significantly more active on IgG1 and IgG3; IgG2 and IgG4 were more resistant. This bacterial exoproduct appears to digest IgG molecules into Fab gamma, F(ab')2 fragments, and a free Fc gamma piece with a molecular weight of 40,000.     

采用Red重组系统敲除铜绿假单胞菌弹性蛋白酶基因

目的敲除铜绿假单胞菌弹性蛋白酶基因,获得无弹性蛋白酶活性的铜绿假单胞菌菌株。方法采用PCR从pKD46质粒上扩增λ噬菌体的Red重组酶基因,并将其克隆到大肠杆菌和铜绿假单胞菌穿梭质粒pUCP多克隆位点上,电击转化铜绿假单胞菌PAO1感受态细胞,构建PAO1/pUCP-Red基因敲除体系。常规基因操作构建两端与弹性蛋白酶基因上、下游同源,中间为庆大霉素抗性基因的线性打靶片段;并将其电击转化pUCP-Red/PAO1感受态;采用庆大霉素和羧苄青霉素抗性平板初步筛选阳性重组菌;通过PCR、RT-PCR及弹性蛋白酶活性检测方法,鉴定菌株弹性蛋白酶基因的敲除情况。结果本研究通过构建Red重组系统,获得了无弹性蛋白酶活性的铜绿假单胞菌菌株。结论本研究成功敲除了铜绿假单胞菌弹性蛋白酶基因,所获无弹性蛋白酶活性的菌株将为系统深入研究弹性蛋白酶在铜绿假单胞菌致病性中的详细作用机理提供材料和基础。     

Immunoglobulin-G subclasses in cystic fibrosis. IgG2 response to Pseudomonas aeruginosa lipopolysaccharide

Pulmonary macrophage phagocytosis of Pseudomonas aeruginosa is defective when this pathogen is opsonized with IgG antibodies isolated from serum samples from patients with cystic fibrosis (CF). To evaluate this defect further, IgG subclasses in the serum and lung fluids of patients with CF were quantitated. The pattern of IgG subclasses in serum specimens from patients with CF (n = 15) and in patients without CF but with chronic obstructive airway disease and recurrent P. aeruginosa infection (n = 4) was significantly altered from that found in normal subjects (n = 31). Immunoglobulin-G2 and IgG3 expressed as percentages of total IgG subclasses or in micrograms per milliliter of serum were significantly elevated in the serum specimens of these patients (p less than 0.05), and IgG1 was significantly decreased (p less than 0.01). It appears that the increase in IgG2 in the serum of patients with CF and those without CF but with chronic P. aeruginosa infection may be in response to chronic antigenic stimulation by P. aeruginosa lipopolysaccharide. Evidence presented to support this includes: (1) IgG2 is not increased in CF serum if a history of P. aeruginosa infection is absent, (2) IgG2 levels expressed as percentages of total IgG subclasses in CF lung fluids were positively correlated (r = 0.73) with the number of colony-forming units of P. aeruginosa present in CF sputum specimens, and (3) IgG antibodies specifically eluted from P. aeruginosa lipopolysaccharide ligands on affinity gels were largely restricted to IgG2. The opsonic index, ([IgG3] + [IgG1]) divided by ([IgG2] + [IgG4]), is inverted in CF lung fluids (0.73:1; normal, 2:1). Because pulmonary macrophages show surface receptors binding primarily with IgG3 and IgG1, it may be that such an alteration in IgG subclasses in the respiratory secretions of patients with CF further inhibits opsonin-mediated clearance of P. aeruginosa.     

Natural and vaccine-related immunity to Streptococcus pneumoniae

To investigate the protective effects of pneumococcal vaccine, we assayed serum from healthy adults and from elderly bronchitics for antibody and opsonic activity against nine serotypes of S. pneumoniae. Before vaccination, there was no relation between opsonization and the level of antibody measured by RIA. Some serotypes were well opsonized in the absence of detectable antibody to capsular polysaccharide; others were not, despite modest levels of antibody. These in vitro studies did not support the concept that a certain level of antibody (e.g., greater than or equal to 250 ng of antibody nitrogen/ml) was specifically associated with the capacity to opsonize pneumococci. Nearly all postvaccination sera had increased antibody and opsonic activity against all serotypes, but the lack of correlation in any individual serum persisted. RIA showed that pre- and postvaccination levels of antibody in elderly adults with chronic lung disease were similar to those of younger adults. In elderly bronchitics, opsonizing activity for six of the nine serotypes was lower after vaccination, a result of suggesting a possible explanation for the failure of pneumococcal vaccine to be fully protective in these subjects. Elderly subjects had higher levels of antibody to phosphocholine, but when isolated, this antibody did not opsonize any of the vaccine strains of pneumococci. These results suggest that alternative strategies are needed to maximize the protective effect of pneumococcal vaccine in the population at greatest risk.