Induction of phagocytic inhibitory activity in cats with chronic Pseudomonas aeruginosa pulmonary infection.

Chronic pulmonary infection has been established in cats by repeated intrapulmonary inoculation of viable Pseudomonas aeruginosa enmeshed in agarose beads. In the serum of all chronically infected animals, a substance(s) developed which inhibited phagocytosis of P. aeruginosa by normal cat alveolar macrophages. Phagocytosis was measured by incubating macrophage monolayers (5 X 10(5) alveolar macrophages) for 20 min in the presence of 3H-labeled bacteria and 5% serum from control or infected animals. Inhibitory activity developed 4 to 16 weeks after initial infection, and inhibition of phagocytosis of P. aeruginosa in the presence of infected cat serum ranged from 30 to 79%. After inhibitory activity developed, it persisted throughout the remainder of the experiment in each animal. The activity was specific for P. aeruginosa of the infecting serotype and did not affect phagocytosis of gram-positive organisms. Inhibitory activity was unchanged by heating serum at 56 degrees C for 30 min. We have previously described a P. aeruginosa-specific, heat-stable, phagocytosis-inhibitory activity in the serum of patients with cystic fibrosis. Since inhibitory activity also develops in cats with chronic P. aeruginosa pulmonary infection, such activity may not be a primary intrinsic abnormality in patients with cystic fibrosis. The animal model described here offers a system for following the development of and for characterization of the P. aeruginosa-specific phagocytosis-inhibitory activity.     

Plasma serine protease activity during healing of experimental aseptic and infected wounds

I. M. Sechenov First Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR T. T. Berezov.) Translated from Byuelleten' Éksperimental'noi Biologii i Meditsiny, Vol. 106, No. 7, pp. 99–101, July, 1988.     

Protective immunization against chronic Pseudomonas aeruginosa pulmonary infection in rats.

Rats were immunized systemically with various doses of the polyvalent Pseudomonas aeruginosa vaccine PEV-01. After a series of two or three doses (25 to 50 micrograms each) at 8- to 11-day intervals, animals were challenged intratracheally by the agarose bead technique with a serotype 5 P. aeruginosa strain at periods of 9 to 42 days. Immunized animals developed circulating antibodies (primarily immunoglobulin M) against vaccine components at levels significantly higher than challenged, nonimmunized controls (P less than 0.005). Eight to ten days postinfection, histological sections of lungs from immunized animals showed only minimal inflammation associated with infectious foci (agarose beads) as compared with the extensive pathological changes of airways and parenchyma seen in infected nonimmunized control animals. However, no significant reduction in bacterial numbers was observed. Such protection lasted at least 6 weeks after the final immunization. It is speculated that the vaccine may contain components of cell surface proteins and virulence exoproducts.     

Effect of experimental Pseudomonas aeruginosa infection on the activity of mouse phagocytes

It has been stated that experimental infection with 0.01 LD50 Pseudomonas aeruginosa 74 decreases the phagocytic activity of mouse neutrophiles and peritoneal macrophages in relation to latex particles. In the course of infection suppression of phagocytic activity of peritoneal macrophages was delayed for two days as compared with that of neutrophiles. During the dying out of infection phagocytic activity of neutrophils and macrophages returned to the control values. Possible mechanisms of these events were discussed.     

Direct evaluation of Pseudomonas aeruginosa biofilm mediators in a chronic infection model

Biofilms contribute to Pseudomonas aeruginosa persistence in a variety of diseases, including cystic fibrosis, burn wounds, and chronic suppurative otitis media. However, few studies have directly addressed P. aeruginosa biofilms in vivo. We used a chinchilla model of otitis media, which has previously been used to study persistent Streptococcus pneumoniae and Haemophilus influenzae infections, to show that structures formed in vivo are biofilms of bacterial and host origin within a matrix that includes Psl, a P. aeruginosa biofilm polysaccharide. We evaluated three biofilm and/or virulence mediators of P. aeruginosa known to affect biofilm formation in vitro and pathogenesis in vivo--bis-(3',5')-cyclic dimeric GMP (c-di-GMP), flagella, and quorum sensing--in a chinchilla model. We show that c-di-GMP overproduction has a positive impact on bacterial persistence, while quorum sensing increases virulence. We found no difference in persistence attributed to flagella. We conclude from these studies that a chinchilla otitis media model provides a means to evaluate pathogenic mediators of P. aeruginosa and that in vitro phenotypes should be examined in multiple infection systems to fully understand their role in disease.     

Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres

Alginate-producing, mucoid P. aeruginosa is frequently found in the lungs of patients with cystic fibrosis (CF), where it causes a chronic infection. The importance of alginate in the pathogenesis was demonstrated by the ability to establish chronic P. aeruginosa lung infection in rats if P. aeruginosa entrapped in minute alginate-beads were inoculated transtracheally. Alginate beads containing P. aeruginosa were formed by nebulizing a suspension of seaweed sodium-alginate and P. aeruginosa into a calcium solution. The alginate bead method of establishing infection was compared to an agar-bead method and proved to be quantitatively similar after 4 weeks. The ability of the two methods to induce formation of precipitins, IgA and IgG antibodies against P. aeruginosa antigens, including outer membrane proteins, flagella, exoenzymes and alginate, was assessed by crossed immunoelectrophoresis, enzyme-linked immunosorbent assay and immunoblotting. The two methods of inducing infection were comparable and infected rats had significantly higher antibody response than rats inoculated with sterile beads. We suggest that the alginate bead model closely resembles the later stages of CF-lung infection and that it offers the theoretical advantage of using a substance which is chemically similar to the alginate produced in vivo by P. aeruginosa.     

Antibiotic inhibition of protease production by Pseudomonas aeruginosa

The effects of tetracycline, chloramphenicol and polymyxin B on growth and protease production by Pseudomonas aeruginosa were studied. Tetracycline inhibited protease production at concentrations much lower than those required to cause growth inhibition; the effect was not due to inhibition of protease activity by the antibiotic. In contrast, chloramphenicol and polymyxin B inhibted protease production in direct proportion to the inhibition of growth. Lysozyme-release experiments with washed tetracycline-treated cells indicated that the protease did not accumulate intracellularly. The protease-inhibiting effect of tetracycline might have therapeutic significance if it were found to occur in vivo.     

Production of alkaline protease by Pseudomonas aeruginosa.

A highly sensitive and specific radioimmunoassay has been developed for Pseudomonas aeruginosa alkaline protease. Production of alkaline protease was found to be strain variable and medium dependent.     

Pseudomonas aeruginosa infection and vaccination in the rat

A large molecular-weight fraction of Pseudomonas aeruginosa culture filtrate protected rats and mice against a lethal infection with a heterologous serotype, and to some extent against Escherichia coli and Listeria monocytogenes. The active components were obtained from cultures grown for several days in a simple synthetic medium. Infection and vaccination with P. aeruginosa serotype 16 induced agglutinating and precipitating antibodies to the components of this serotype only; in rats infected or vaccinated with serotype 1, low titres of agglutinating antibody against type 16 were found. Vaccine prepared from type 1 or 16 increased, within 3 days of infection, the resistance of rats to type-16 organisms; within the same period agglutinins or precipitins were not produced. It is possible that the protection was based on opsonic and antitoxic activities.     

铜绿假单胞菌lasR rhlR基因缺陷对大鼠慢性肺部感染的影响

目的观察铜绿假单胞菌（Pseudomonas aeruginosa，Pa）PAO1野生型及群体感应（quorum sensing，QS）系统的lasR rhlR基因缺陷型Pα菌株人工生物被膜致病性的差异，及QS系统的lasR rhlR基因在Pα生物被膜感染致病过程中的地位。方法从大鼠支气管内直接予以Pα（菌种为PAO1野生型和PAO1 lasR rhlR基因缺陷型）海藻酸盐微菌粒悬液（10^9CFU／ml）攻击，建立PAO1野生型及QS系统的lasR rhlR基因缺陷型菌株人工生物被膜肺部感染动物模型。分别于感染1周和2周后评估各组大鼠的肺部病理学、细菌学及细胞因子（IL-4、IL-10）反应的变化。结果感染1周和2周后，PAO1野生型组的肺部病理学改变和细菌学改变均明显重于PAO1 lasR rhlR基因缺陷组（P〈0．001）；感染1周后PAO1野生型组肺部的IL-4、IL-10水平都高于PAO1 lasR rhlR基因缺陷组（P〈0．01，P〈0．05）；2周后PAO1野生型组肺部IL-10水平进一步升高（P〈0．01），明显高于PAO1 lasR rhlR基因缺陷组（P〈0．001）。结论PAO1野生型组在病程中引起持久和严重的肺部感染，激发了TH2样免疫反应，而PAO1 lasR rhlR基因缺陷组的肺部病变明显轻于PAO1组，说明QS系统的lasR rhlR基因在Pα肺部感染的建立及慢性化发展过程中发挥着重要作用。     

Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis

Pseudomonas aeruginosa causes a chronic infection in the lungs of cystic fibrosis (CF) patients by establishing an alginate-containing biofilm. The infection has been studied in several animal models; however, most of the models required artificial embedding of the bacteria. We present here a new pulmonary mouse model without artificial embedding. The model is based on a stable mucoid CF sputum isolate (NH57388A) with hyperproduction of alginate due to a deletion in mucA and functional N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Chronic lung infection could be established in both CF mice (Cftr(tmlUnc-/-)) and BALB/c mice, as reflected by the detection of a high number of P. aeruginosa organisms in the lung homogenates at 7 days postinfection and alginate biofilms, surrounded by polymorphonuclear leukocytes in the alveoli. In comparison, both an AHL-producing nonmucoid revertant (NH57388C) from the mucoid isolate (NH57388A) and a nonmucoid isolate (NH57388B) deficient in AHL were almost cleared from the lungs of the mice. This model, in which P. aeruginosa is protected against the defense system of the lung by alginate, is similar to the clinical situation. Therefore, the mouse model provides an improved method for evaluating the interaction between mucoid P. aeruginosa, the host, and antibacterial therapy.     

Effect of chronic Pseudomonas aeruginosa infection on the development of atherosclerosis in a rat model

In order to investigate the possible relationship between atherosclerosis and chronic Pseudomonas aeruginosa infection, 66 Wistar rats were given five separate intratracheal inoculations of either P. aeruginosa or sterile saline at 4-week intervals. The rats were divided into four groups: group 1 was infected with P. aeruginosa and fed a diet containing cholesterol 1% w/v; group 2 was infected with P. aeruginosa and fed a normal diet; group 3 was not infected and was fed a diet containing cholesterol 1% w/v; and group 4 (the control group) was not infected and was fed a normal diet. One month after the final inoculation, the rats were killed humanely; computerised image analysis was used to evaluate sections of the aorta and heart, and the maximal wall thickness of the aorta and coronary artery. The aortic wall thickness was significantly greater for group 1 (329.53 +/- 58.06 microm) compared to groups 2 (190.59 +/- 27.81 microm; p < 0.0001), 3 (262.90 +/- 61.12 microm; p < 0.0004) and 4 (158.00 +/- 30.30 microm; p < 0.0001). Similarly, the coronary artery wall thickness was significantly greater for group 1 (72.96 +/- 10.67 microm) compared to groups 2 (35.07 +/- 8.53 microm; p < 0.0001), 3 (41.45 +/- 10.22 microm; p < 0.0001) and 4 (32.30 +/- 5.27 microm; p < 0.0001). These findings strengthen the hypothesis that chronic infection plays a role in the pathogenesis of atherosclerosis.     

Phospholipase A activity in Pseudomonas aeruginosa

Our study describes the production, purification and properties of an enzyme from Pseudomonas aeruginosa displaying the properties of phospholipase A. Maximal amounts of enzyme could be detected in the culture supernatant when the bacterium was grown for 3 to 5 days at 37°C in stirred flask cultures containing brain heart infusion. The enzyme was purified by polyethylenimine precipitation and ammonium sulfate precipitation followed by gel filtration. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme preparation exhibited two bands with molecular weights of 13.5 and 60 kD, respectively. Correspondingly, two peaks of the same molecular weight could be demonstrated by high performance size exclusion chromatography. The activity toward the sn-2 ester binding of phospholipids was characterized and found to be highest towards phosphatidylcholine. Enzymatic activity was not influenced by the addition of calcium or EDTA while magnesium and strontium caused a decrease of activity. The lyophilized enzyme was found to be stable when stored at −70°C and most active at pH 8.0.     

大鼠铜绿假单胞菌慢性肺部感染生物被膜的致病作用研究

目的 建立慢性铜绿假单胞菌（P.aeruginosa,Pa）生物被膜（biofilm,BF）肺部感染的大鼠模型,探讨Pa生物被膜的致病作用特点.方法 体外测定左旋氧氟沙星（levofloxacin,LFX）及头孢他啶（ceftazidime,CAZ）对藻酸盐包裹的PAO579及浮游PAO579的最低抑菌浓度（MIC）、最低杀菌浓度（MBC）.从大鼠切开的气管内注入0.1ml 10^9CFU/ml藻酸盐包裹的PAO579或浮游PAO579,术后第3、7、14天观察大鼠肺部的细菌学、病理学特点及TNF-α反应.结果 体外LFX、CAZ对藻酸盐包裹的PAO579的MIC、MBC均高于浮游PAO579.体内藻酸盐珠组的细菌形成单位（CFU）显著高于浮游菌组（P=0.002,P=0.004,P=0.002）;肺大体病理及炎症反应程度均较浮游菌组严重;肺TNF-α水平均高于浮游菌组（P=0.002,P=0.002,P=0.022）.相关分析表明,TNF-α水平与大体病理有明显相关性.结论 Pa生物被膜有保护细菌抵御抗菌药物杀伤的作用,同时有介导肺免疫损伤及保护细菌逃避宿主免疫防御作用.     

Diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis by enzyme-linked immunosorbent assay.

An easily applicable test for diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis by enzyme-linked immunosorbent assay (ELISA) for determination of serum immunoglobulin G to P. aeruginosa was developed. Soluble antigens obtained by ultrasonication of P. aeruginosa, serotypes O:1 to O:17, were used as antigens immobilized to polystyrene microtiter plates. The intraplate, plate-to-plate, and day-to-day variations were 14, 19, and 20%, respectively. Plates coated with the antigens could be stored for at least 64 days at +4 and +22 degrees C without any significant change in activity. Normal values were determined in sera from 164 controls (100 children and 64 adults). The sensitivity and specificity of the ELISA was determined by using serum samples from 243 cystic fibrosis patients and were compared to results with crossed immunoelectrophoresis (CIE). The ELISA could diagnose chronic P. aeruginosa infection with a diagnostic sensitivity of 93% and specificity of 92%. The sensitivity and specificity for the diagnosis of the early stages of chronic P. aeruginosa infection by a single sample were 90 and 100%, respectively, and by using an increased antibody response in paired samples, the sensitivity was 93% and specificity was 87%. There was a statistically significant correlation between antibody levels obtained by ELISA and those obtained by CIE. The sensitivity and specificity of the ELISA were equal to those of CIE, and because of its simplicity, the ELISA is recommended as a routine test in patients with cystic fibrosis.     

Antilipopolysaccharide antibodies and differential diagnosis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis

Chronic lung infection in cystic fibrosis is characteristically associated with polyagglutinable, serum-sensitive, mucoid strains of Pseudomonas aeruginosa. Enzyme-linked immunosorbent assay (ELISA) methods for standard-free quantitation of immunoglobulin G (IgG) and IgM antibodies to P. aeruginosa lipopolysaccharides (LPSs) have been developed. We now report the development of assays for quantitation of monomer and dimer total IgA and IgA anti-LPS antibodies. Use of these methods in diagnosis of early chronic P. aeruginosa lung infection was assessed. IgG and IgA anti-LPS levels increased significantly at the onset of chronic infection and continued to increase to very high levels in the later stages of infection. IgM anti-LPS levels also rose at the onset of chronic infection but did not increase further. The function of true- and false-positive rates was illustrated by using various concentrations of IgG, IgA, and IgM anti-LPS for discrimination of patients. Values that gave optimum separations were used for statistical evaluation of the diagnostic sensitivities and specificities of anti-LPS antibody concentrations. The results obtained in these assays were compared with a diagnosis, based on the number of precipitins in crossed immunoelectrophoresis, of serum samples from cystic fibrosis patients. In 64 paired serum samples taken before and immediately after the onset of chronic infection, as defined by crossed immunoelectrophoresis precipitins, the predictive values of a positive ELISA were 86% for IgG and 89% for IgA. The predictive values for a negative ELISA were 98% for IgG and 97% for IgA. Results of the IgM anti-LPS ELISA had a lower predictive value. Immunoblotting and absorption studies showed that IgG anti-LPS antibodies were directed specifically against LPS of P. aeuruginosa. ELISAs were developed to determine the specific IgG sublclasses involved. The increase in IgG anti-LPS involved all four subclasses. Highest anti-LPS titers were seen with IgG1 and IgG4, but the largest relative increases were seen with IgG2 and IgG3.     

Characterization of chronic bronchopulmonary Pseudomonas aeruginosa infection in resistant and susceptible inbred mouse strains

Chronic bronchopulmonary Pseudomonas aeruginosa infection, initiated by intratracheal instillation of 1 to 2 x 10(5) colony-forming units of a mucoid strain of bacteria trapped in agar beads, was characterized in resistant BALB/c mice and susceptible C57BL/6 (B6) mice through 28 d postinfection. B6 mice experienced a more severe infection than BALB/c mice as evidenced by significantly higher mortality and significantly greater weight loss during the first 14 d. Furthermore, B6 mice had significantly higher numbers of bacteria in the lungs through 21 d after infection. Overall, only 22% of these hosts cleared the infection. In contrast, 67% of BALB/c mice cleared the infection. These differences between resistant and susceptible mice were found to correlate with histopathologic differences in the type of inflammation and the extent of tissue damage. An acute, predominantly neutrophilic inflammation and extensive tissue damage were apparent in the lungs of susceptible B6 mice, whereas chronic, granulomatous inflammation and little or no tissue damage were visible in resistant BALB/c mice. The finding of acute inflammation in the lungs of infected B6 mice was confirmed by fluorescence-activated cell sorter (FACS) analyses, which demonstrated that these mice had significantly greater proportions of polymorphonuclear neutrophils in the lungs on Days 7 and 14 after infection than did BALB/c mice. FACS analyses also revealed significant and similar increases in CD3(+) lung cells in both strains as the infection progressed. The CD4/CD8 ratio was significantly greater in BALB/c mice by 21 d after infection when the majority of these animals, but not B6 mice, had cleared the infection.     

Inhibition of human natural killer cell activity by Pseudomonas aeruginosa alkaline protease and elastase.

The present study was designed to examine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (Ela) on human natural killer (NK) cell activity in vitro. AP and Ela were found to inhibit NK cell function. Addition of alpha interferon and interleukin-2 did not abolish this inhibition of NK cell activity. Adhesion of effector to target cells was studied in a single-cell agarose assay of monocyte-depleted NK-cell-enriched cell populations. AP and Ela were shown to inhibit effector/target cell conjugate formation. Furthermore, AP and Ela inhibited the binding of the monoclonal antibody Leu-11, which reacts with the Fc receptor of NK cells. The inhibition of NK cell binding to the target cell by P. aeruginosa proteases is most likely due to proteolytic cleavage of the surface receptors involved in the binding of the effector cell to the target cell.